RB Inhibition of Cellular Proliferation during S-phase and Origin Density Determination through Alterations in Nucleotide Pools

Abstract

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of the cell cycle that functions to prevent cells from entering S-phase when conditions are not appropriate for proliferation. It is thought that RB works to inhibit cellular proliferation in late Gi phase of the cell cycle. Several recent discoveries indicate that RB may actually inhibit proliferation in S-phase rather than Gi. One of the biggest of these discoveries is that Cyclin E, an initiator of DNA replication, is produced in the presence of active RB. Cyclin E is responsible for the binding of cyclin dependent kinases at the end of Gi and moving the cell into S-phase. At this point cyclin A, which is inhibited by active RB, takes over and continues the initiation of DNA replication. Because cyclin E is still produced in the presence of active RB, the cell cycle may not be inhibited until the initial stages of S-phase, where the lack of cyclin A prevents DNA replication. A second function of RB is to prevent cellular proliferation not only by cyclin A inhibition, but also through the depletion of dNTP pools. When nucleotide levels decrease below a certain threshold, proliferation can no longer proceed. In conjunction with this idea, it was recently discovered that the number of utilized origins changes with increases or decreases in nucleotide levels, but the cell doubling time and length of Sphase remain the same. Here I began preliminary testing on these two functions of active RB. First, RB actually inhibits cellular proliferation very early in the S-phase of the cell cycle, after primer formation but before processive elongation of replication forks. Second, alterations made in nucleotide pools in the DHFR locus will decrease or increase origin utilization.