Vol. 8, 31 1-318, March 1997 Cell Growth & Differentiation 311 Cyclin A Message Stability Varies with the Cell Cycle1 Amit Maity, W. Gillies McKenna, and Ruth J. Muschel2 Departments of Radiation Oncology [A. M., W. G. M.] and Pathology and Laboratory Medicine [A. J. Mj, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Abstract 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 BI 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 BI message is unstable until later in S phase. Furthermore, although cyclin BI 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 BI and hence in controlling the cell cycle. Introduction The cyclins are a family of proteins essential for progression through the cell cycle, the levels of which were originally found to oscillate with the cleavage cycle in marine inverte- brate embryos (1). Two different cyclins, designated A and Bi , are required for the transition from G2 into M; however, cyclin A is also required for progression through S phase (2-4), and the highest levels of both of these cyclins are Received 8/27/96; revised 12/31/96; accepted 1/8/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdi- cate this fact. 1This work was supported by NIH Grants GM 47439 (to R. J. M.) and CA 64227 (to W. G. M.) and American Cancer Society Grant FRA (to W. G. M.). The flow cytometry facility was supported by the Lucille B. Markey Trust. 2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, John Morgan Building, Room 270, 36th and Hamiiton Walk, Philadelphia, PA 19104. Phone: (215) 898-8401 ; Fax: (215) 898-4227. found in G2-M. The level of cyclin A protein rises earlier in the cell cycle and falls earlier than the level of cyclin Bi (5, 6). These changes are reflected in the cyclical variation in mRNA levels. In HeLa cells, the level of cyclin A message starts to rise in late G1 , continues to rise in S phase, peaks in G2-M, and falls in early G1 (6). However, the level of cyclin Bi message does not rise until mid-to-late S phase, peaks in G2-M, and falls in early G1 (5). These cyclins act by binding to catalytic subunits known as cyclin-dependent kinases, and this binding is obligatory for the activation of these kinases. Thus, the levels of cyclins control the activity of the kinases that drive the cell cycle. In cycling mammalian cells, the levels of the cyclin proteins mirror that of their mRNAs. Thus, regulation of cyclin A and Bi mRNAs is critical to the regulation of cell cycle progression. Evidence suggests that the regulation of cyclin Bi mRNA through the cell cycle is mediated both transcriptionally (7-9) and posttranscnptionally (1 0). We found previously that a construct containing a luciferase reporter gene downstream of the cyclin Bi promoter showed a 3-4-fold variation in luciferase activity through the cell cycle (8), but this is insuf- ficient to explain the marked change (50-1 00-fold) in mRNA levels seen between G1 and G2-M. In another report (1 0), we showed that the cyclin Bi message was 5-1 0-fold more stable in G2-M than in G1 . In the same report, we also found that the stability of the cyclin Bi mRNA decreased after exposure of the cells to ionizing radiation, contributing to the suppression in cyclin Bi mRNA expression in these cells following radiation (1 1 , 12). In the current report, we investigated the contribution that stability of the cyclin A and E messages plays in regulating their variation through the cell cycle. Changes in transcription play a role in the oscillation of cyclins A and E mRNA levels (1 3-16). However, we find that changes in cyclin A mRNA stability occur through the cell cycle that correlate with the variation in steady-state levels, suggesting that this, also, is important in controlling levels. Results Cyclins A, BI, and E mRNA Expression through the Cell Cycle. The pattern of expression of cyclins A, Bi , and E was followed as synchronized HeLa cells progressed through the cell cycle following release from a G1-S block (see “Materials and Methods”). Northern blots hybridized with either cyclins A, Bi , or E probes are shown in Fig. 1 , and the results of densitometric analysis and the flow cytometry are shown in Fig. 2. All blots were also probed for the rpL32 mRNA (17), which encodes a ribosomal protein and is a message, the level of which does not vary through the cell cycle. The hybridization to this probe served as a loading control, and all of the signals were normalized to it. As expected, each of these messages has a specific pattern of expression. The level of cyclin Bi mRNA was low in G1 and peaked in G2-M. The level of cyclin A mRNA was also low in G1 , but it rose