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Progression through the cell cycle in somatic eukaryotic cells is regulated by variations in the levels of cyclin proteins. These protein levels are in turn regulated by cyclical oscillations in their mRNA levels. We show here that regulation of RNA stability plays a role in the mechanisms underlying cell cycle progression. Both cyclin A and B1 messages are expressed at high levels in G2-M and at low levels in early G1. The half-lives of their messages mirror this pattern, long in G2-M (>8 h) and short in early G1 (1-2 h). However, there is evidence of specificity to these changes, because the cyclin A message becomes stable at the G1-S boundary, whereas the cyclin B1 message is unstable until later in S phase. Furthermore, although cyclin B1 mRNA levels are lowered after irradiation because of enhanced instability, cyclin A mRNA levels and message stability are unaffected by irradiation. Additional evidence of specificity was found in an analysis of cyclin E mRNA stability, which remains constant through the cell cycle, although the cyclin E message displays cell cycle-dependent changes in expression. These studies suggest that specific alterations in RNA stability are an important component in regulating the expression of cyclins A and B1 and hence in controlling the cell cycle.

Irradiation of tumor cells results in a G2 delay, which has been postulated to allow DNA repair and cell survival. The G2 delay after irradiation is marked in HeLa and other cells by delayed expression of cyclin B1. To test whether this depression of cyclin B1 contributes to the G2 delay, we induced cyclin B1 expression in irradiated HeLa cells using a dexamethasone-inducible promoter. Induction of cyclin B1 after radiation abrogated the G2 delay by approximately doubling the rate at which the cells reentered mitosis, whereas dexamethasone itself had no effect. However, overexpression of cyclin B1 did not eliminate the G2 delay in irradiated cells. In unirradiated cells, overexpression of cyclin B1 had no effect on cell cycle progression. Confirmation that reduction of cyclin B1 levels would prolong G2 was provided using antisense oligonucleotides to cyclin B1. These results demonstrate that cyclin B1 levels control the length of the G2 delay following irradiation in HeLa cells but do not exclude additional mechanisms controlling the mitotic delay after irradiation.

Exposure of cells to DNA damaging agents results in a G2 arrest. Exposure of HeLa cells to camptothecin, etoposide or nitrogen mustard for 1 h in S phase resulted in delayed expression of cyclin B1 mRNA during the G2 arrest. Initially the levels of cyclin B1 protein were low as well; however, with extended time the cells blocked in G2 regained higher levels of cyclin B1 protein. In the case of cells treated with nitrogen mustard the higher levels coincided with cells exiting the G2 block into G1. However, with camptothecin or etoposide treatment, while the accumulation of cyclin B1 protein was delayed, its levels eventually surpassed peak levels seen in control cells, in spite of the fact that cells were still blocked in G2. These cells did not continue to progress through the cell cycle indicating further complexity to the mechanisms underlying the G2 block. Decreased transcription and stability of cyclin B1 mRNA were shown to occur after treatment with these DNA damaging agents. These results indicate that suppression of cyclin B1 mRNA expression is one consequence of DNA damage in HeLa cells.

Cyclin B1 mRNA expression varies through the cell cycle with its peak in G2/M. In cycling mammalian cells, its lowest level is in G1 with a steady increase in S until a level 50-fold greater than that in G1 is reached. In order to characterize the transcriptional component to this variation in expression, we cloned the upstream region 872 base pairs upstream from the start site of the cyclin B1 gene and have demonstrated that it confers cell cycle-dependent regulation onto two reporter genes, both chloramphenicol acetyltransferase and luciferase. Its activity was 25-fold greater in G2/M than in G1 in HeLa cells with intermediate activity in S. This cyclical activity could be seen with sequences encompassing only 90 base pairs upstream from the start site. Protein binding to this region was demonstrated using electrophoretic mobility shift assays, and the binding profiles appeared to vary depending upon the phase of the cycle in which the extracts are made. Thus, transcriptional control plays an important role in determining cyclin B1 mRNA levels, and cell cycle-dependent activity is regulated through interactions with the region 90 bases upstream from the start site.

Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.

The irradiation of cells results in delayed progression through the G2 phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G2-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G2-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2 ng/ml) staurosporine, which releases the radiation-induced G2-phase arrest. In HeLa cells, the G2-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G2-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G2-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury.

Cyclin B1 is required for the G2/M transition. In HeLa cells, the level of both its protein and mRNA varies dramatically through the cell cycle. In this study we examine the expression of cyclin B1 mRNA in Chinese hamster ovary cells and 3.7, a transformed rat embryo fibroblast line. In contrast to HeLa cells in which a single transcript is seen, both of these lines express two distinct transcripts of approximately 1.4 and 2.7 kbp in length which appear to be made using different polyadenylation sites. In CHO cells, the level of the upper band varies modestly through the cell cycle while the lower band remains almost constant. In contrast to HeLa cells, CHO cells in G1 express a significant amount of cyclin B1 mRNA. In synchronized 3.7 cells, the level of both transcripts is very low in G1 and rises in parallel as cells progress through S and G2, but the longer transcript disappears faster as cells reenter G1.

Many studies have demonstrated the effect of oncogene transfection on the radiation sensitivity of primary rat embryo fibroblasts. Our results indicate that transformation by H-ras plus v-myc oncogenes confers radiation resistance to a much greater extent than transformation by either gene alone. We have further shown that the radioresistant phenotype is accompanied by a prolonged G2-phase delay. This is consistent with the hypothesis that the extent of this delay is an important determinant of radiation sensitivity. The study of cyclin expression during the progression of cells through G2 and M phase after irradiation has also revealed several possible mechanisms for induction of G2-phase arrest. Control of cyclin B levels was seen both at the mRNA level as evidenced by decreased cyclin B mRNA expression after irradiation in the S phase, and at the protein level as demonstrated after irradiation in G2 phase. It remains to be determined how these mechanisms might be differentially regulated in radioresistant and sensitive cells.

Purpose: To evaluate the hypothesis that the radiation induced G2 delay in the cell cycle is associated with radiation induced effects on cyclin B expression in a rodent cell system., Methods and Materials: Two rodent, rat and Chinese hamster, cyclin B cDNAs were cloned and characterized. The two rodent species were 85% and 86% identical, respectively, when compared to the human cyclin B, indicating that they are the rodent homologous of cyclin B. 3.7 cells (rat embryo cells transformed by H-ras and v-myc) were synchronized and then irradiated. Flow cytometry and Northern blots were performed to evaluate the effects of radiation on cyclin expression in relation to phase of the cell cycle., Results: Examination of the rodent cyclin B sequences revealed only two regions with significant divergence to the human sequence, one in the lysine rich region adjacent to the cyclin destruction box, which is the putative site for ubiquitination, and one at the C terminal end. Although many of the amino acids diverged in the lysine rich region, the positions of the lysines themselves were virtually invariant suggesting their potential importance in ubiquitination. Both rodent species were also noted to have a PEST-like sequence which occurs in the human, but not in non-mammalian cyclins cloned to date and could also potentially contribute to rapid destruction. The rat and Chinese hamster mRNAs contain much longer 3' untranslated regions than the published human sequence with multiple AUUUA and AUUU motifs which are seen in other mRNAs with rapid turnover times. This feature has not been previously found in cyclin mRNAs. In addition we have found that in the 3' region of the rodent cDNAs we find two potential polyadenylation sites suggesting that this gene may have several transcripts. Our studies suggest that multiple mechanisms of control of mammalian cyclin B destruction exist, both at the mRNA and protein level. Evidence is also provided that the levels of rat cyclin B mRNA peaks during G2/M. Irradiation is shown to induce a G2 delay in synchronized 3.7 cells, compared to unirradiated controls, and the delay is temporally related to decreased levels of cyclin B mRNA expression. Since the G2 delay induced by ionizing radiation may contribute to the ability of cells to survive irradiation, cyclin B expression may be a key component in the determination of sensitivity or resistance to radiation therapy., Conclusion: The isolation and characterization of two rodent cyclin B's confirm that multiple mechanisms of control of mammalian cyclin B destruction exist. Our studies show that rat cyclin B expression is influenced by radiation and is temporally related to the delay in the G2 phase induced by radiation.

Ionizing radiation induces a G2 delay in eukaryotic cells. Since mitotic cyclins are required to trigger the transition from G2 into and through mitosis, we chose to investigate their expression after irradiation in HeLa cells. In normally cycling HeLa cells, both cyclin A and B mRNA and protein levels rise dramatically in G2/M and rapidly fall coincident with the completion of mitosis. The rise of cyclin A mRNA at the S/G2 boundary slightly precedes that of cyclin B mRNA. Although the peaks of expression of each of these molecules overlap, cyclin A mRNA and protein diminish before cyclin B. After irradiation in S, cyclin A mRNA and protein levels rose with the same kinetics as in the controls, but ultimately exceeded the levels seen in the control population. Cyclin A mRNA and protein levels remained high throughout the G2 delay induced by irradiation. In contrast, cyclin B mRNA and protein levels did not rise as the irradiated cells entered G2/M. Only later, before the irradiated cells exited from G2/M, did levels of cyclin B reach the levels seen in the unirradiated controls. The decreased amount of cyclin B mRNA and protein was inversely proportional to the dose of radiation. These data indicate that irradiation that results in a G2 delay appears to block cells at a point after production of cyclin A but before cyclin B can be fully expressed and that cells do not exit from the delay until cyclin B is again expressed. Thus, cyclin A and cyclin B expression respond differentially to radiation, with cyclin A rising at the same time as the control and to even higher levels than that seen in the controls, whereas cyclin B shows a temporal delay in expression.

The levels of cyclin B mRNA and protein rise rapidly in G2 + M phase, and fall at the end of mitosis. The studies described here were initiated to determine the effects of ionizing radiation on the level of cyclin B bearing in mind that the division delay induced by ionizing radiation might be influenced by the expression of cyclin B. After irradiation in S phase, the cyclin B mRNA in HeLa cells was measured as the cells proceeded through the cell cycle. Instead of the usual rise, after irradiation cyclin B mRNA levels remained low during the G2 delay. After irradiation in G2 phase, cyclin B mRNA was readily detectable although at slightly lower levels than in the controls. However, cyclin B protein was markedly decreased in amount.

After exposure to ionizing radiation, eukaryotic cells undergo a division delay which is reflected by increased time spent in the G2 portion of the cell cycle. Recent information identifies increased levels of mitotic cyclins as key biochemical events initiating mitosis. In HeLa cells cyclin B mRNA and protein levels have been shown to increase in G2 and to decrease after division is completed. Cyclin B protein binds to cdc2, resulting in histone kinase activity which is necessary for the initiation of mitosis. Accordingly, we chose to investigate how cyclin B mRNA and protein levels were perturbed by irradiation in order to gain further understanding of the mechanisms by which ionizing radiation leads to a division delay. Our experiments revealed at least two effects on cyclin B regulation which might contribute to the division delay: (a) when HeLa cells were irradiated in S phase, there was a delay in the accumulation of cyclin B mRNA; (b) when cells were radiated in G2 phase, at a time when mRNA levels were increasing, a division delay was induced which coincided with a markedly lowered level of cyclin B protein despite high levels of the mRNA.