Complexity of the Mechanisms of Initiation and Maintenance of DNA Damage-Induced G2-Phase Arrest and Subsequent G1-Phase Arrest: TP53-Dependent and TP53-Independent Roles (original) (raw)
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Experimental Cell Research, 1997
irradiated cells [7], and that these surviving cells have G 1 phase cell cycle arrest after exposure to ionizing a decreased number of chromosomal aberrations in the radiation has been documented in cells with wild-type ensuing mitosis [8]. According to a current mechanistic p53. The temporal location of this arrest within G 1 model, irradiation of cells with X-or grays causes DNA phase, however, has not been determined. We have strand breaks that result in an accumulation of p53 now used flow cytometric analysis of bromodeoxyuri-[9-11], which in turn acts as a transcription factor dine (BrdUrd)-labeled cells to obtain further informafor and increases the expression of p21 cip1 protein. The tion about the location of the G 1 phase radiation check-p21 cip1 then binds to and transiently inhibits cdk2/ point. Human fibroblasts were irradiated with grays cyclin E kinase activity [12], thereby causing a G 1 and treated with colcemid to stop unlabeled G 2 cells phase block by preventing the phosphorylation of retifrom entering the G 1 phase. Analysis of BrdUrd incornoblastoma protein (Rb) [13], and the subsequent reporation revealed that 73% of G 1 phase human lung lease of the E2F transcription factor that is required fibroblasts remain in G 1 phase after exposure to grays , for further cell progression into S phase [14, 15]. thereby placing the G 1 radiation checkpoint near the The above model of how the DNA damage-induced end of G 1 phase. The location of the radiation check-G 1 checkpoint is mediated predicts that only a fraction point correlates with the reported increased expresof G 1 phase cells in exponentially growing populations sion of cyclin E, increased cyclin E/cdk2 kinase activshould arrest in G 1 after irradiation because they are ity, and hyperphosphorylation of pRb in proliferating at a point in G 1 before which they express the active human fibroblasts. ᭧ 1997 Academic Press molecular machinery to escape the block. The model also predicts that cells beyond the checkpoint will temporally lack the checkpoint competency and proceed to
Control of radiation-induced G1 arrest by cell-substratum interactions
Cancer research, 1997
Ionizing radiation has been reported to cause an irreversible, senescence-like G1 arrest in human fibroblasts, which is accompanied by elevated p21CIP1 amounts. In further support of a senescence-like arrest, we show that expression of p53 and cyclin D1 is elevated in gamma-irradiated, arrested fibroblasts. However, we also demonstrate that the arrest is reversible if the irradiated cells are trypsinized and replated, which may implicate cellular-extracellular matrix interactions in cell cycle control after irradiation.
Molecular and cellular biology, 2000
Entry into mitosis is controlled by the cyclin-dependent kinase CDK1 and can be delayed in response to DNA damage. In some systems, such G(2)/M arrest has been shown to reflect the stabilization of inhibitory phosphorylation sites on CDK1. In human cells, full G(2) arrest appears to involve additional mechanisms. We describe here the prolonged (>6 day) downregulation of CDK1 protein and mRNA levels following DNA damage in human cells. This silencing of gene expression is observed in primary human fibroblasts and in two cell lines with functional p53 but not in HeLa cells, where p53 is inactive. Silencing is accompanied by the accumulation of cells in G(2), when CDK1 expression is normally maximal. The response is impaired by mutations in cis-acting elements (CDE and CHR) in the CDK1 promoter, indicating that silencing occurs at the transcriptional level. These elements have previously been implicated in the repression of transcription during G(1) that is normally lifted as cells ...
Permanent cell cycle exit in G2 phase after DNA damage in normal human fibroblasts
The EMBO Journal, 2003
Although the Cdk inhibitor p21 Waf1/Cip1 , one of the transcriptional targets of p53, has been implicated in the maintenance of G 2 arrest after DNA damage, its function at this stage of the cell cycle is not really understood. Here, we show that the exposure of normal human ®broblasts (NHFs) to genotoxic agents provokes permanent cell cycle exit in G 2 phase, whereas mouse embryo ®broblasts and transformed human cells progress through mitosis and arrest in G 1 without intervening cytokinesis. p21 Waf1/Cip1 exerts a key role in driving this G 2 exit both by inhibiting cyclin B1±Cdk1 and cyclin A±Cdk1/2 complexes, which control G 2 /M progression, and by blocking the phosphorylation of pRb family proteins. NHFs with compromised pRb proteins could still ef®ciently arrest in G 2 but were unable to exit the cell cycle, resulting in cell death. Our experiments show that, when under continuous genotoxic stress, normal cells can reverse their commitment to mitotic progression due to passage through the restriction point and that mechanisms involving p21 Waf1/Cip1 and pocket proteins can induce exit in G 2 and G 1 .
Involvement of the MKK6-p38gamma Cascade in gamma -Radiation-Induced Cell Cycle Arrest
Molecular and Cellular Biology, 2000
The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38␥ (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for ␥-irradiation-induced G 2 arrest. Activation of the MKK6-p38␥ cascade is sufficient to induce G 2 arrest in cells, and expression of dominant negative alleles of MKK6 or p38␥ allows cells to escape the DNA damage-induce G 2 delay. Activation of p38␥ is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by ␥ irradiation leads to the activation of MKK6, p38␥, and Cds1 and that activation of both MKK6 and p38␥ is essential for the proper regulation of the G 2 checkpoint in mammalian cells.
Involvement of the MKK6-p38γ Cascade in γ-Radiation-Induced Cell Cycle Arrest
Molecular and Cellular Biology, 2000
The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38γ (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for γ-irradiation-induced G 2 arrest. Activation of the MKK6-p38γ cascade is sufficient to induce G 2 arrest in cells, and expression of dominant negative alleles of MKK6 or p38γ allows cells to escape the DNA damage-induce G 2 delay. Activation of p38γ is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by γ irradiation leads to the activation of MKK6, p38γ, and Cds1 and that activation of both MKK6 and p38γ is essential for the ...
p53 Mediates Permanent Arrest over Multiple Cell Cycles in Response to γ-Irradiation
Cancer Research, 1997
The seminal observation that p53 mediates a G1 cell cycle arrest following y-irradiation was originally proposed to be analogous to the RAD9-mediated G2 DNA damage checkpoint in budding yeast (5). The RAD9 arrest pathway enhances cell survival by delaying pro gression into mitosis to allow for DNA repair (17). Based on this similarity, it was suggested that the p53-dependent G1 arrest is tran 1171