scholarly journals p21-Mediated Nuclear Retention of Cyclin B1-Cdk1 in Response to Genotoxic Stress

2004 ◽  
Vol 15 (9) ◽  
pp. 3965-3976 ◽  
Author(s):  
Fabienne Baus Charrier-Savournin ◽  
Marie-Thérèse Château ◽  
Véronique Gire ◽  
John Sedivy ◽  
Jacques Piette ◽  
...  
Keyword(s):  
Dna Damage ◽  
Current Model ◽  
Human Fibroblasts ◽  
Genotoxic Stress ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
G2 Arrest ◽  
Nuclear Retention ◽  
Mitotic Cyclin ◽  
Normal Human

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest

1999 ◽  
Vol 91 (9) ◽  
pp. 665-674 ◽  
Author(s):  
Florence Vincent ◽  
Gaël Deplanque ◽  
Jocelyn Ceraline ◽  
Brigitte Duclos ◽  
Jean-Pierre Bergerat
Keyword(s):  
Human Fibroblasts ◽  
Cyclin B1 ◽  
G2 Arrest ◽  
Normal Human

scholarly journals DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex

2001 ◽  
Vol 21 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Olga K. Mirzoeva ◽  
John H. J. Petrini
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Human Fibroblasts ◽  
Strand Break ◽  
Immunofluorescent Localization ◽  
Nuclear Retention ◽  
Atm Kinase ◽  
Mre11 Complex ◽  
Previous Demonstration

ABSTRACT The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.

scholarly journals Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.

1991 ◽  
Vol 115 (1) ◽  
pp. 1-17 ◽  
Author(s):  
J Pines ◽  
T Hunter
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Nuclear Lamina ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cell Fractionation ◽  
Mitotic Apparatus ◽  
Epithelial Tumor ◽  
Mitotic Cyclin ◽  
Cycle Dependent

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.

scholarly journals Repression of CDK1 and Other Genes with CDE and CHR Promoter Elements during DNA Damage-Induced G2/M Arrest in Human Cells

2000 ◽  
Vol 20 (7) ◽  
pp. 2358-2366 ◽  
Author(s):  
Christophe Badie ◽  
Jane E. Itzhaki ◽  
Matthew J. Sullivan ◽  
Adam J. Carpenter ◽  
Andrew C. G. Porter
Keyword(s):  
Dna Damage ◽  
Transcriptional Repression ◽  
Human Fibroblasts ◽  
Cyclin B1 ◽  
Human Cells ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Cyclin Dependent Kinase ◽  
Promoter Regions ◽  
Topoisomerase Iiα

ABSTRACT Entry into mitosis is controlled by the cyclin-dependent kinase CDK1 and can be delayed in response to DNA damage. In some systems, such G2/M arrest has been shown to reflect the stabilization of inhibitory phosphorylation sites on CDK1. In human cells, full G2 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 G2, 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 G1that is normally lifted as cells progress into S and G2. Interestingly, we find that other genes, including those for CDC25C, cyclin A2, cyclin B1, CENP-A, and topoisomerase IIα, that are normally expressed preferentially in G2 and whose promoter regions include putative CDE and CHR elements are also downregulated in response to DNA damage. These data, together with those of other groups, support the existence of a p53-dependent, DNA damage-activated pathway leading to CHR- and CDE-mediated transcriptional repression of various G2-specific genes. This pathway may be required for sustained periods of G2 arrest following DNA damage.

The relationship between cellular radiosensitivity and DNA damage in primary normal human fibroblasts

1993 ◽  
Vol 29 ◽  
pp. S216
Author(s):  
R. Wurm ◽  
N.G. Burnet ◽  
N. Duggal ◽  
Y.R. Yarnold ◽  
J.H. Peacock
Keyword(s):  
Dna Damage ◽  
Human Fibroblasts ◽  
Cellular Radiosensitivity ◽  
Normal Human ◽  
The Relationship

scholarly journals An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage

2002 ◽  
Vol 22 (24) ◽  
pp. 8552-8561 ◽  
Author(s):  
Timothy P. Heffernan ◽  
Dennis A. Simpson ◽  
Alexandra R. Frank ◽  
Alexandra N. Heinloth ◽  
Richard S. Paules ◽  
...  
Keyword(s):  
Dna Damage ◽  
Uv Light ◽  
Human Fibroblasts ◽  
Ectopic Expression ◽  
Nijmegen Breakage Syndrome ◽  
Velocity Sedimentation ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Damage Response ◽  
Normal Human

ABSTRACT Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m2). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.

scholarly journals DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation

2005 ◽  
Vol 25 (21) ◽  
pp. 9608-9620 ◽  
Author(s):  
Koji Okamoto ◽  
Kenji Kashima ◽  
Yaron Pereg ◽  
Michiko Ishida ◽  
Satomi Yamazaki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Human Fibroblasts ◽  
Consensus Sequence ◽  
Cancer Genetic ◽  
Cellular Defense ◽  
P53 Activation ◽  
Normal Human ◽  
Biochemical Mechanisms ◽  
Low Levels ◽  
Binding Sequence

ABSTRACT Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.

Sign in / Sign up

Export Citation Format

Share Document