This dissertation, "Investigating the Role of FoxM1 in Cell Cycle Progression by Inducible RNA Interference" by Man-sze, Cheung, 張敏思, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Investigating the Role of FoxM1 in Cell Cycle Progression by Inducible RNA Interference Submitted by Cheung Man Sze for the degree of Master of Philosophy at The University of Hong Kong in August 2004 FOXM1 is a forkhead box transcription factor ubiquitously expressed in proliferating cells. Over the years, extensive studies have been carried out, demonstrating the involvement of FOXM1 in the cell cycle. Knockout of FoxM1 in mice results in postnatal death, with cardiomyocytes and hepatocytes exhibiting extensive DNA polyploidy. This suggests that FoxM1 is required to prevent DNA re-replication. On the other hand, overexpression of FoxM1 in regenerating hepatocytes enhances both DNA replication and mitosis, indicating that FOXM1 is likely to be facilitating S phase progression and mitotic entry. There seems to be some apparent contradictions in the current perspective on FOXM1's function. Having realized that the existing animal models used are unable to address FOXM1's role in regulating the overall cell cycle mechanics and its functions in distinct cell cycle phases, I employ in this study a cell-based system to further study and refine FOXM1's action in the cell cycle. FoxM1 expression in NIH3T3 cells is knocked down using the doxycycline (dox)- inducible-RNA interference (RNAi) system. I have cloned shRNA sequences targeting FoxM1 into the pTER plasmid which contains a dox-sensitve form of the H1 promoter that drives the expression of shRNAs. By electroporating the cloned pTER plasmids into tetracycline repressor (TetR)-expressing NIH3T3 cells, I have generated a dox-inducible cell line C3. Western blot analysis shows that addition of dox to C3 cells effectively reduces the endogenous FOXM1 protein level by more than 75%. Upon knockdown of FoxM1 expression, a consistent increase in G2/M cell population is observed, indicating the tendency of FOXM1-deficient cells to attain a tetraploid state. In view of the fact that FoxM1 knockout mice display severe DNA polyploidy, I also test the effect of long term suppression of FoxM1. Unexpectedly, prolonged reduction in endogenous FOXM1 does not result in DNA polyploidy, though an unusually high G2/M cell population is observed. Considering that significant decrease in FoxM1 level in aging cells has been reported in a number of recent studies, I speculate that a low FoxM1 level is implicated in the aging process, and that the inducible C3 cell line, upon addition of dox, exhibits some early signs of aging. To resolve the functional window of FOXM1 within the cell cycle, I synchronize the cells at early G1. Flow cytometric analysis is performed to follow the cell cycle progression of cells with and without dox-induced knockdown of FOXM1 expression. Contrary to the prevailing model of FOXM1's function in facilitating the G1/S checkpoint transition, I observe that knockdown of FOXM1 does not affect the cell's entry into S phase. Surprisingly, I find that reduction in FOXM1 level results in a prolonged S phase, suggesting possible defects in replication mechanics. Based on the present data, I propose a model for the function of FOXM1, highlighting that FOXM1 may be involved in the completion of DNA replication during late S phase and, by inducing an array of its gene targets, promotes entry into mitosis. (482 words) DOI: 10.5353/th_b3039640