Abstract
When cell cycle studies are performed following cell cycle synchronization, it is possible that critical properties of an actively cycling cell will be overlooked. For this reason past studies have not revealed critical aspects of cell cycle control; such as how a cell determines when to exit the cell cycle, or how rapidly it should cycle. To address these challenging questions we have developed a procedure to quantitate fluorescent stains in a monolayer culture, where nuclear fluorescence and cell cycle history can be assessed with accuracy on a cell by cell basis. The cell cycle position of each cell can be determined by analyzing DNA and BrdU levels. The behavior of cells in a given cell cycle position can then be studied by quantitating up to two other stained markers. When the microinjection of siRNA, neutralizing antibodies, and expression plasmids are coupled with quantitative image analysis, these cell cycle studies can be conducted following alterations in the expression levels of selected cellular targets. With these techniques we have discovered critical aspects of cell cycle control; including how cyclin D1 levels vary through the cell cycle, the molecular mechanisms governing these changes, and the biological implications of changes in cyclin D1 concentration in various cell cycle stages. Our studies with cyclin D1, coupled with similar studies of p27Kip1, form the basis of an entirely new model of cell cycle control proposed here. This model explains how cell cycle progression is terminated, and how the length of the cell cycle is regulated.