Is Down-Regulated by Ionizing Radiation in a p53-dependent Manner1

The mammalian cellular response to ionizing radiation results in delays in progression through the cell cycle at several checkpoints and includes alterations in the activity of cyclin-dependent kinases. The product of the CDC2 gene is a key kinase involved in cell cycle progression. The signaling events that regulate its expression after exposure to DNA-damaging agents are not known. We show that cdc2 mRNA and protein are down-regulated after irradiation of normal human and mouse fibroblasts with doses as low as 0.5 Gy. This down-regulation is preceded by induction of p53 and p21W8fl proteins. In human cells in which p53 was nonfunctional and in p53/ or p21 / mouse embryo fibroblasts, no effect of ionizing radiation on p34 expression levels was observed. These findings indicate that CDC2 down-regulation after irradiation is p53-dependent and involves the cyclin-dependent kinase inhibitor p2lw as a negative factor in the control of CDC2 expression. Correspondence between the delay in initiation of DNA synthesis in irradiated cells and the down-regulation of CDC2 is described. Introduction Reversible, dose-dependent delays at one or more points in the progression of irradiated cells through the cell cycle have been found in a wide variety of mammalian cells (reviewed in Ref. 1). Such arrests have been hypothesized to provide more time for repair of radiation-induced DNA damage. Failure to repair such damage before replication of DNA and mttosis may result in cell death or segregation of DNA lesions that can initiate or contribute to the development of cancer cells. Knowledge of how the activity of genes involved in cell cycle control is regulated by exposure to lR3 is critical to understandReceived 2/1 1/97; revised 7/29/97; accepted 9/5/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Research Grant CA-47542 and Center Grant ES00002. 5. M. d. T. was supported by the CANDU Owners Group. 2 To whom requests for reprints should be addressed, at Laboratory of Radiobiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 021 15. Phone: (617 432-1 184; Fax: (617) 432-0107; E-mail: [email protected]. 3 The abbreviations used are: IR, ionizing radiation; RT-PCR, reverse ing the molecular mechanisms underlying radiation-induced cell cycle delays. Previous investigations have described alterations in irradiated cells of the expression patterns and activity of several key cell cycle regulators (2-4). In mammalian cells, the accumulation of the tumor suppressor p53 protein, predominantly by posttranslational mechanisms, in response to DNA damage has been shown to be critical for the regulation of the G1 checkpoint and apoptosis (5-7). Irradiated human dipbid fibroblasts with mutated or abrogated p53 function and irradiated MEFs in which the p53 or p21 genes were deleted showed neither the G1 arrest nor the sensitivity to radiationinduced reproductive failure as measured by clonogenic survival (8-1 1). The p53-dependent G1 arrest caused by IR has been correlated with inactivation of cyclin E-Cdk2 and cyclin A-cdk2 kinases (3), accumulation of hypophosphorylated retinoblastoma protein (3, 12), and suppression of E2F activity (13). The inactivation of cyclin-cdk kinase complexes that control entry into S phase is mediated, at least in part, by binding of the p53-inducible inhibitor p21Wafl to the complex (reviewed in Ref. 14). The cdc2 protein kinase is thought to be an essential cell cycle regulator of all eukaryotic cells (15). In yeasts, p34cdc2/cdc28 through the association with distinct cyclins, activates entry into both S phase and mitosis (16). In mammalian cells, the cdc2 protein complexes with cyclin B and is essential for the G2-Mphase transition. Whereas 34cdc2 is present at a steady-state level during the cycle of continuously proliferating cells such as HeLa (1 7), it is induced with different kinetics during the stimulation of various quiescent cell types (18-20). After exposure of human tumor and diploid cells to IR, changes in cdc2 activity have been observed and were found to be associated with altered patterns of phosphorylation ofthe cdc2 protein (21-26). Recently, studies with human pre-B leukemia cells have shown that IR promotes the physical interactions between p34 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of the irradiated cells, leading to tyrosine phosphorylation of p34cdc2 (27). Also, radiation-induced binding of p34 to p56/ p53 Yn kinase that was associated with inhibition of cdc2 activity has been reported in nuclear lysates of HL6O cells (28). The involvement of p34cdc2 in the control of G1-S-phase transition is unclear and is a matter of debate (18, 29-31). Also, the regulation of cdc2 mRNA and protein levels after exposure of normal human cells to IA is unknown. To better understand the biochemical events involved in DNA damage checkpoints, we examined in the present investigation the relationship between p53 expression and cdc2 regulation in irradiated normal human diploid fibroblasts synchronized in G0-G1 by density inhibition of growth and in human fibroblasts in which p53 is mutated or its expression is abrogated Li-Fraumeni (1264) A AG1521