Accumulation of cyclin E is not a prerequisite for passage through the restriction point

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling tells, G(1)-pm (a postm itotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA- synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical fo r passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both pass age through R and entry into S phase. We found that the postmitotic G(1) ce lls that had not yet reached R were negative for cyclin E accumulation, On the other hand, cells that had passed R were found to accumulate cyclin E a t variable times (1 to 8 h) after passage through R and 2 to 5 h before ent ry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the co nverse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated w ithin 1 to 2 h after entry into S.