Cellular ras and cyclin D proteins each play critical roles in the control of cellular proliferation. Evidence increasingly indicates an important connection between these two molecules. Extra cellular growth signals control ras activity; while ras activity stimulates cyclin D levels. These two proteins function to regulate cell cycle progression by regulating the activity of cell cycle control proteins in late GI-phase; when the cell makes a commitment to another round of DNA synthesis and cell division. We now report the possible involvement of ras and cyclin D1 in the control of cell cycle progression at an unexpected cell cycle point; late S- and G2-phases. With a cell cycle analytical procedure based upon time lapse analysis of asynchronous cultures coupled with quantitative fluorescence analytical procedures, we report here that cyclin D1 is expressed at high levels late in S-phase and throughout G2-phase. In this study we will test our hypothesis that ras activity and cyclin D expression late in the cell cycle might play a critical role in the control of cell cycle progression. To conclusively demonstrate that ras is active in late S- and G2- phases, ras activity will be analyzed by its association with target peptides.
In Specific Aim 1, the signaling pathway downstream of ras will also be analyzed late in the cell cycle.
In Specific Aim 2, we will determine to what extent ras activity leads to cyclin D1 expression in late cell cycle stages by comparing ras activity to cyclin D1 mRNA levels.
In Specific Aim 3, the biological importance of ras activity and cyclin D1 expression late in the cell cycle will be analyzed. Experiments are designed to demonstrate that cyclin D1 made in G2-phase effects cyclin D1 levels in G1 phase. In addition, we will test the possibility that cyclin D1 produced prior to mitosis influences entry into quiescence and the length of the next G1- phase. These studies have the potential to broaden our understanding of the role of ras activity throughout the cell cycle in the control of cellular proliferation.
