p53 Regulation of G₂ Checkpoint Is Retinoblastoma Protein Dependent

Abstract

In the present study, we investigated the role of p53 in G 2 checkpoint function by determining the mechanism by which p53 prevents premature exit from G 2 arrest after genotoxic stress. Using three cell model systems, each isogenic, we showed that either ectopic or endogenous p53 sustained a G 2 arrest activated by ionizing radiation or adriamycin. The mechanism was p21 and retinoblastoma protein (pRB) dependent and involved an initial inhibition of cyclin B1-Cdc2 activity and a secondary decrease in cyclin B1 and Cdc2 levels. Abrogation of p21 or pRB function in cells containing wild-type p53 blocked the down-regulation of cyclin B1 and Cdc2 expression and led to an accelerated exit from G 2 after genotoxic stress. Thus, similar to what occurs in p21 and p53 deficiency, pRB loss can uncouple S phase and mitosis after genotoxic stress in tumor cells. These results indicate that similar molecular mechanisms are required for p53 regulation of G 1 and G 2 checkpoints.

In vitro effects of radiation on human retinoblastoma cells

“…Recently, p53 regulation of the G2 cell cycle checkpoint has been shown to be Rb-dependent in colon carcinoma cell lines and RKO cells with functional Rb displayed a sustained G2 arrest following irradiation [27], similar to that seen for the Rb-reconstituted cell lines in the present study. The mechanisms involved in Rbdependent G2 arrest may include p21 activation and regulation of Rb phosphorylation, inhibition of activity of specific cyclins and CDKs, or alterations in binding to E2F transcription factors and subsequent regulation of E2F target genes [26,27]. Further studies are required to more fully understand the multiple pathways and cell cycle regulatory gene products involved in the cell cycle checkpoints observed in Rb following DNA damage.…”

“…The processes involved are complex and remain to be fully elucidated; however, several central mediators, in particular p53, have been identified [48]. Recently, Rb has also been found to play a major role in cell cycle control following DNA damage [26,27]. In vitro studies of Rb-negative fibroblasts (MEFs) [26,49], SAOS-2 tumor cells that normally lack Rb [39], and colon carcinoma cells that express human papillomavirus type 16 E7 protein (RKO-E7) and have abrogated Rb function [27], indicate that Rb has an essential role in G1/S arrest induced by DNA damage.…”

“…Recently, Rb has also been found to play a major role in cell cycle control following DNA damage [26,27]. In vitro studies of Rb-negative fibroblasts (MEFs) [26,49], SAOS-2 tumor cells that normally lack Rb [39], and colon carcinoma cells that express human papillomavirus type 16 E7 protein (RKO-E7) and have abrogated Rb function [27], indicate that Rb has an essential role in G1/S arrest induced by DNA damage. Irradiated Rb-negative primary MEF cultures displayed an obvious G2/M accumulation of cells compared to MEF cells with wildtype Rb that had cells at both G1/S and G2/M checkpoints, consistent with a defect in G1/S arrest in Rb-negative MEFs [26].…”

“…Cyclin-CDK complexes are required to phosphorylate and inactivate Rb protein, allowing cells to progress from G1 to S in the cell cycle; dephosphorylated Rb protein thus blocks cells in G1 [24,25]. Recently, it has been suggested that the Rb protein may be involved in maintaining genomic stability, and an important role for Rb protein in control of the cellular response to DNA damage has been recognized [24,26,27].…”

RB silencing compromises the DNA damage-induced G2/M checkpoint and causes deregulated expression of the ECT2 oncogene

“…In contrast, previous studies have suggested that all three RB family proteins, including p107 and p130, might be required for sustaining DNA damage checkpoint. For instance, induction of human papilloma virus protein E7 in human colorectal carcinoma RKO cells, in which all the RB family proteins are inactivated, resulted in the loss of the p53-dependent G2/M DNA damage checkpoint (Flatt et al, 2000). In the RKO cells expressing E7, both the amount and activity of G2-M regulatory CDKs (cyclin B1/CDK1 and CDK2) were highly elevated compared to the parental cells with functional RB family proteins, which resulted in the escape from G2/M cell cycle arrest caused by doxorubicin treatment.…”