Studies in cyclin message stability

Many DNA damaging agents including drugs and ionizing radiation induce a G2 delay in cycling cells. Previous studies showed that S phase irradiation of HeLa cells resulted in delayed expression of cyclin B1 mRNA and protein coincident with the G2 arrest. Cyclin B1 is required for entry into mitosis; therefore, its suppression could contribute to this delay. To determine whether the suppression of cyclin B1 and mRNA following radiation might be due in part to increased degradation, we measured RNA stability. First we measured the half-life of the cyclin B1 message through the cell cycle. It is less than 2 hours in G1, 8-10 hours in S and 13-17 hours in G2/M. These results mirror the cell cycle variation in cyclin B1 mRNA levels which are high in G2/M and low in G1. Furthermore, after S phase irradiation of HeLa cells with 10 Gy, the half-life of the message is reduced to 2-3 hours when measured 14 hours later during the ensuing G2 delay. This effect is specific for cyclin B1 since the half-life of the cyclin A message is not decreased following radiation. Thus, changes in cyclin B1 mRNA stability contribute to its delayed expression following radiation and are important in modulating its levels through the cycle. Exposure of HeLa cells to other DNA damaging agents (camptothecin, etoposide or nitrogen mustard) in S phase also results in delayed expression of cyclin B1 mRNA during the G2 arrest which, in the case of the first two drugs, is partly due to decreased message stability. Changes in stability are also important for the cell cycle dependent variation for both cyclins A and D1, but not E. Changes in cyclin B1 mRNA stability also occur in Chinese hamster ovary (CHO) cells in which the message is much more stable and abundant in G2 cells than in G0/G1 confluence arrested cells. Pretreatment of CHO cells with the protein synthesis inhibitor cycloheximide retards decay of the message suggesting that its degradation is coupled to translation or mediated by a labile protein.