Vol. 3, 637-644, September 1992 Cell Growth & Differentiation 637 Down-Regulation of Cell Cycle Control Genes by Ionizing Radiation’ Rakesh Datta, Ralf Hass, Hisato Gunji, Ralph Weichselbaum, and Donald kufe2 Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [R. D., R. H., H. G., D. K.J, and Department of Radiation and Cellular Oncology, University of Chicago and Pritzker School of Medicine, Chicago, Illinois 60637 ER. W.] Abstrad The cellular responses to ionizing radiation include growth arrest, DNA repair, and lethality. However, little is known about the signaling events responsible for these responses. The present studies have examined the effects of ionizing radiation on the expression of genes involved in cell cycle control. The results demonstrate that the treatment of asynchronous cells with 20 Gy ionizing radiation is associated with transient down-regulation of the cdc2, cyclin A, cyclin B, and cdc25 genes. This effed was associated with transient indudion of the c-jun gene. RNA stability studies demonstrate that the down-regulation of gene expression following ionizing radiation exposure is at least in part due to a decrease in transcript half-life. Other studies were performed with elutriated cells enriched for populations in G1 and S phases. Treatment of C1 enriched cell populations with 10 Gy resulted in a selective decrease in cyclin B mRNA levels, whereas this effed on cyclin B expression was less pronounced at 5 Gy and undetedable at 1 Gy. Similar results were obtained with S phase enriched cells. Taken together with clonogenic survival studies, these findings indicate that down-regulation of cell cycle control gene expression is associated with lethality, whereas lower doses of ionizing radiation have little, if any, effed on the expression of these genes. The findings also suggest that DNA damage may adivate signaling events which regulate expression of cell cycle control genes. Introdudion The responses of eukaryotic cells to ionizing radiation include growth arrest, DNA repair, and lethality (1). How- ever, the molecular mechanisms responsible for these effects are unknown. Recent studies have demonstrated that the cellular response to ionizing radiation involves the induction of immediate early response gene expres- sion (2-4). Immediate early response genes that encode Received 4/25/92. 1 This work was supported by USPHS Grants CA55241 and CA41068, awarded by the National Cancer Institute, Department of Health and Human Services, by a Fellowship from the Deutsche Forschungemein- schaft (R. H.) and by a Burroughs Wellcome Award in Clinical Pharma- cology (D. K.). 2 To whom requests for reprints should be addressed at Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. transcription factors include the jun (c-jun. jun-B, and jun-D; Refs. 5-9), fos (c-fos, fos-B, fra-i; Refs. 10-12), and EGR (EGR-i, EGR-2; Refs. 13-15) families. Treatment of diverse cell types with x-rays is associated with increases in c-jun. jun-B, and c-fos mRNA levels (2, 3). These effects are mediated at least in part by activation at the transcrip- tional level (2). Similar findings have been obtained for the EGR-i gene (3). Moreover, recent results have dem- onstrated that ionizing radiation activates DNA binding activity of the nuclear factor kB (NF-icB) and increases expression of this gene (4). The products of these differ- ent classes of early response genes function as trans- ducers of nuclear signals and therefore may couple early biochemical signals induced by X-rays to longer term changes in gene expression. Several checkpoints in cell cycle progression control growth in response to diverse positive and negative sig- nals (16). Ionizing radiation, for example, slows growth by inducing delays in both S and G2 phases of the cell cycle. The available evidence indicates that G2 arrest is necessary for repair of DNA damage before entry into mitosis. Indeed, treatment of cells with caffeine or the protein kinase inhibitor 2-aminopurine overcomes G2 delay following DNA damage and increases chromo- somal fragmentation (17, 18). Genetic studies in yeast have identified certain genes that are important in the control of G2 and entry into mitosis. For example, muta- tions in the rad9 gene of Saccharomyces cerevisiae have demonstrated that the RAD9 protein controls G2 arrest induced by DNA damage (19, 20). Mutants at the rad9 locus are unable to delay entry into mitosis following exposure to DNA damaging agents. The replication of damaged chromosomes is associated with mutations and lethality. In contrast to RAD9, which is not required for cell cycle progression, the onset of mitosis is controlled by a complex of the serine/threonine protein kinase p34cdc2 and cyclin B (21 -23). p34 k2 or related kinases appear to be essential for G1-S and G2-M transitions in both yeast and mammalian cells (21 -25). Transformed cells may bypass the requirement for cdc2 activity in G1- S transition (26), although it is not known whether cells can enter mitosis without this kinase. Studies in fission yeast have also demonstrated that the cdc25 gene prod- uct, p8O 25, regulates mitosis by dephosphorylation of p34cdc2 and thereby activation of the cdc2/cyclin B com- plex (27-29). Although these findings in eukaryotic cells have pro- vided certain insights into the regulation of G2 delay, little is known about the control of genes coding for these cell cycle regulatory proteins, particularly in response to DNA damage. The present studies have examined the effects of ionizing radiation on cdc2, cyclin A, cyclin B, and cdc2S gene expression. The results demonstrate that X-ray exposure is associated with down-regulation of these genes and that this effect occurs at least in part by posttranscriptional mechanisms.