Accumulation of Cyclin E Is Not a Prerequisite for Passage through the Restriction Point

Abstract

The restriction point (R) is defined as the point in G₁after which cells can complete a division cycle without growth factors and divides G₁ into two physiologically different intervals in cycling cells, G₁-pm (a postmitotic interval with a constant length of 3 to 4 h) and G₁-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G₁ regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G₁ cells 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 at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry 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 converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.

ACKNOWLEDGMENTS

We thank Julio Draetta and Jonathon Pines for kindly providing cyclin A antibodies and Mia Olsson, Yvonne Lindell, Kerstin Nyström, and Martha Henze for excellent technical assistance.

This project was supported by grants to Anders Zetterberg from the Swedish Cancer Society (0046-B99-33XB) and the Stockholm Cancer Society and by U.S. Public Health Service grant CA 78343 to Steven Reed.