 |
 |
|
||||||||||||
| A conditional transgenic approach to investigate the role of Ciz1 in tumorigenesis | ||||||||||||
Many disorders, from cardiovascular disease to cancer, involve impaired regulation of cell growth and proliferation. We have recently identified a novel cell cycle protein, known as Ciz1, that in vitro experiments suggest plays an important role in regulation of cell proliferation. When up-regulated the number of cells that enter S-phase increases, whereas depletion of Ciz1 through use of RNA interference technology prevents the cells from proliferating (Coverley et al 2005). Ciz1 is an alternatively spliced nuclear matrix associated protein that promotes initiation of DNA replication by functional interaction with cyclin A-cdk2 (Copeland et al 2010). The cyclin interacting N-terminal region encodes DNA replication activity, while the C-terminus mediates anchoring of Ciz1 to the nuclear matrix (Ainscough et al 2007). This separation of domain organisation provides a means for spatial and temporal control of replication foci within the nucleus as S-phase progresses. Expression of Ciz1 and its numerous splice variants is disrupted in a wide range of proliferative disorders. Notably, Ciz1 expression has been shown to be oestrogen responsive and up-regulated in breast cancer cells. Consistent with this ectopic expression of Ciz1 in a breast cancer cell line promoted the growth rate and tumorigenic properties of the cells (den Hollander et al 2006). To explore the importance of Ciz1 in tumorigenesis this project will utilise a ‘conditional’ transgenic mouse model that has recently been generated in our laboratory. The model utilises the ‘Tet-off’ system, which enables transgene expression to be switched on and off both in time and in space. We have previously validated use of this system for other genes (eg. Ainscough et al 2009), and have now developed a GFP tagged Ciz1 expression model. Initial experiments have shown that over-expression of Ciz1 in vivo is sufficient to induce proliferation in differentiated cell types that have exited the cell cycle, such as the cardiomyocyte (heart muscle cell). In this project GFP-Ciz1 expression will be induced specifically in mammary epithelial cells through activity of the mouse mammary tumour virus (MMTV) promoter. The resultant double transgenic mice may also be crossed with mice expressing Estrogen Receptor-alpha in the same cells, which has been previously shown to enhance the development of oestrogen responsive tumours. The successful candidate will utilise a range of techniques to thoroughly investigate the transgenic model, ranging from non-invasive imaging to general histology and immunocytochemistry, fluorescence microscopy, in situ hybridisation, molecular biology and biochemistry, and culture and cell cycle analysis of primary cells isolated directly from the transgenic mice. Funding Notes One 4-year studentship is available, funded by Cancer Research UK, to start in October 2010. The studentship is open to UK/EU students and will provide a stipend and full fees in research areas consistent with both the Leeds Cancer Research UK Centre aims, and Cancer Research UK funding activities. Interested applicants should have, or expect to gain, a good first degree (first or upper second class) in a relevant subject. Projects will be advertised until 31st July and short-listing and interviews conducted in August. Ainscough JF-X, Drinkhill MJ, Sedo A, Turner NA, Brooke DA, Balmforth AJ, Ball SG. (2009). Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent hypertrophy and cardiac dysfunction. Cardiovascular Research. 81: 592-600 Ainscough, JF-X., Rahman, FA., Sercombe, H., Sedo, A., Gerlach, B., Coverley, D. (2007). C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix. Journal of Cell Science. 120: 115-124 Copeland NA, Sercombe HE, Ainscough JFX, Coverley D. (2010). Ciz1 cooperates with cyclin A/CDK2 to activate mammalian DNA replication in vitro. Journal of Cell Science. 123: 1108-1115 Coverley, D., Marr, J., Ainscough, JF-X. (2005). Ciz1 promotes mammalian DNA replication. Journal of Cell Science. 118: 101-112 den Hollander, P., Rayala, S., Coverley, D., Kumar, R. (2006). Ciz1, a novel coactivator of the estrogen receptor-alpha, confers hypersensitivity to estrogen action. Cancer Research. 66: 11021-11029 |
||||||||||||
|