Mechanisms of G2 Arrest in Response to Overexpression of p53

Abstract

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 and cyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions Ϫ287 to Ϫ123 of the cyclin B1 promoter and Ϫ104 to Ϫ74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest. INTRODUCTIONThe p53 tumor suppressor helps to protect mammals from developing neoplasia by blocking cell cycle progression or by inducing cell death in response to stress Levine, 1997;Agarwal et al., 1998b). p53-mediated arrest at the G1-S boundary prevents the replication of DNA damaged by ionizing or UV radiation or by chemical mutagens (Kastan et al., 1991;Gujuluva et al., 1994;Ceraline et al., 1998). G1 arrest depends on the ability of p53 to activate the transcription of specific genes (Dulic et al., 1994;Pietenpol et al., 1994). An important target, p21/waf1, inhibits cyclin-dependent kinases (CDKs) 1 2, 4, and 6, which are required to enter S phase (El-Deiry et al., 1993;Harper et al., 1993;Xiong et al., 1993). p53 also inhibits S-phase entry in response to damage to the mitotic spindle, preventing the rereplication of DNA (Cross et al., 1995), possibly by a transcription-independent mechanism (Notterman et al., 1998). p53 mediates G1 arrest in response to nucleotide deprivation, preventing DNA synthesis under conditions that would generate damaged DNA (Chernova et al., 1995;Linke et al., 1996). p53 also protects cells arrested within S phase by a lack of pyrimidine nucleotides, preventing the replication from unbalanced pools of deoxynucleoside triphosphates and consequent DNA damage (Agarwal et al., 1998a). A variety of stimuli that trigger p53-dependent cellular responses increase the ability of p53 to bind to DNA and induce the accumulation of the protein, attributable in large part to an increase in its stability (Maltzman and Czyzyk, 1984;Fritsche et al., 1993;Hupp et al., 1995). Responses to p53 vary in different types of cells and depend on the level of p53 expression. For example, thymocytes undergo p53-dependent apoptosis more readily than do fibroblasts (Lowe et al., 1993;Di Leonardo et al., 1994). In some cells, high levels of p53 induce apoptosis, whereas lower levels induce cell cycle arrest (Chen et al., 1996).p53 plays an important role in regulating the G2-M transition. ...

Alteration of subcellular and cellular expression patterns of cyclin B1 in renal cell carcinoma is significantly related to clinical progression and survival of patients

“…17 A possible mechanism that could explain this observation is that cytoplasmic accumulation of cyclin B1 in tumor cells favors the premature entry into mitosis by overriding a p53-mediated G2/M checkpoint, thus promoting uncontrolled cellular proliferation and disease progression. 41 Conclusively, the association of altered protein levels and an altered subcellular expression pattern with disease progression support a substantial role of cyclin B1 in the carcinogenesis of RCC. Moreover, it has been described that tumors retaining cyclin B1 in the cytoplasm tend to be resistant to DNA damage-induced apoptosis.…”

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

“…Increasing evidence indicates that deregulation and overexpression of cyclin B1 is involved in neoplastic transformation in many tumor types (Soria et al, 2000;Hassan et al, 2002;Yuan et al, 2006). Constitutive activation of cyclin B1 and associated cdc2 kinase can override the G2 DNA damage checkpoint, which leads to an accumulation of genomic defect found in malignant tumors (Kao et al, 1997;Taylor et al, 1999;Park et al, 2000). Recent reports that forced RNAi-mediated reduction of cyclin B1-induced continuous apoptosis in tumor cells (Yuan et al, 2004(Yuan et al, , 2006 are in accordance with this observation.…”

Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

“…35,37,38 Well-known p53-responsive genes include CDC2, CCNB1, and TOP2a. [39][40][41] Despite the transcription factors E2F and NF-Y being enriched in both groups, cell cycle-dependent element (CDE) was significantly enriched in the target group and upstream stimulating factor (USF) was enriched in the non-target group. Previous analyses of CDC2 and CCNB1 promoters suggested that p53 interacted with NF-Y to mediate trans-repression, 42 although a subsequent study suggested that p53 trans-repressed through interaction with SP1.…”