细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

DNA Damage Checkpoints and Cancer

DNA damage checkpoint is one of the surveillance systems to maintain genomic integrity. Checkpoint systems sense the DNA damage and execute cell cycle arrest through inhibiting the activity of cell cycle regulators. This pathway is essential for the maintenance of genome stability and prevention of tumor development. Recent studies have showed that the cellular responses towards DNA damage, such as cell cycle arrest, DNA repair, chromatin remodeling, and apoptosis are well coordinated. Here we describe the molecular mechanisms of checkpoint activation in response to DNA damage and the correlation between checkpoint gene mutation and genomic instability.     

p53 in a Crosstalk Between DNA Repair and Cell Cycle Checkpoints

No abstract available.     

Bub2 Is a Cell Cycle Regulated Phospho-Protein Controlled by Multiple Checkpoints

During mitotic exit, a small GTPase Tem1 needs to be activated. During most of the cell cycle, Tem1 activity is antagonized by a GTPase activating complex (GAP) composed of Bub2 and Bfa1. Bfa1 protein has cell cycle regulated phosphorylation depending upon the Polo-like kinase Cdc5. This phosphorylation dissociates Bfa1 from Tem1 and thus relieves the inhibition of Tem1 by the GAP complex. Bub2 and Bfa1 are also required to prevent mitotic exit when there is DNA damage, spindle damage or spindle misorientation at G2/M phase. While Cdc5 inhibits Bfa1/Bub2, mutating the Cdc5 phosphorylation sites on Bfa1 does not have a strong activating effect on Bub2/Bfa1, suggesting there must be additional regulation in this pathway. Here we report that Bub2 protein also has cell cycle regulated phosphorylation. This phosphorylation is partially dependent upon the Polo-like kinase Cdc5 and is consistent with negative regulation of the Bub2/Bfa1 GAP complex. Spindle damage or spindle misorientation prevents Bub2 phosphorylation. The spindle damage effect is dependent upon the spindle assembly checkpoint components Mad2 and Mps1. Thus like Bfa1, Bub2 protein is also controlled both during mitotic exit and in response to cell cycle checkpoints. Bub2 phosphorylation is likely to be controlled by a novel kinase.

Key Words:

Bub2, Bfa1, Cdc5, Phosphorylation, Mitotic exit, Cell cycle checkpoints     

Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background

The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

Principal Findings

In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status.

Conclusion

These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.     

DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.     

细胞DNA损伤检控点

细胞周期检控点是维持细胞基因组稳定性的一个重要机制,主要包括。DNA损伤检控点、DNA复制检控点和纺锤体组装检控点。其中DNA损伤检控点能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,为修复损伤提供足够的时间,以保证细胞遗传的稳定性。有关DNA损伤检控点的研究近年来已经取得了突破性进展,现简要介绍近年来在DNA损伤检控点研究中的一些新进展。     

Amino Acids and mTOR Mediate Distinct Metabolic Checkpoints in Mammalian G1 Cell Cycle

Objective

In multicellular organisms, cell division is regulated by growth factors (GFs). In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R) and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains – especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase.

Methods

We used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs), the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [³H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot.

Results

We report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR – the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints.

Conclusion

The data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.     

A Conserved DNA Damage Response Pathway Responsible for Coupling the Cell Division Cycle to the Circadian and Metabolic Cycles

The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a “zeitgeber”, or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.     

Selection and the Cell Cycle: Positive Darwinian Selection in a Well-Known DNA Damage Response Pathway

Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.     

Macrophage Migration Inhibitory Factor Coordinates DNA Damage Response with the Proteasomal Control of the Cell Cycle

Proper repair of DNA damage is critical for protecting genomic stability, cellular viability and suppression of tumorigenesis. Both p53-dependent and p53-independent pathways have evolved to coordinate the cellular response following DNA damage. In this review, we highlight the importance of the ubiquitously expressed protein macrophage migration inhibitory factor (MIF) for an appropriate response to DNA damage. We discuss the mechanisms by which MIF affects the activity of the ubiquitin-proteasome system, and how this impacts on the integrity of the genome and on cancer.     

p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.     

The Contribution of p53 in the Dynamics of Cell Cycle Response to DNA Damage Interpreted by a Mathematical Model

Despite numerous studies on the tumor suppressor p53, a complete picture of its role in cell arrest and killing in G1, S and G2M phases after drug treatment is lacking. We tackled the analysis of the complexity of cell cycle effects combining the time-course measures with different techniques with the aid of a computer program simulating cell cycle progression. This mixed experimental-simulation approach enabled us to decode the dynamics of the cytostatic and cytotoxic responses to cisplatin and doxorubicin treatments in a p53-proficient colon carcinoma cell line (HCT-116) and in its p53-deficient counterpart. We achieved a separate evaluation of the activity of each cell cycle control and we connected these results with measures of p53 level in G1, S and G2M. We confirmed the action of p53 in all cell cycle phases, but also the presence of strong p53-independent cytostatic and cytotoxic activities exerted by both drugs. In G1 phase, p53 was responsible for a medium/long term block, distinct from the short-term block, which was p53-independent. The delay in traversing S phase was reduced by the presence of p53. In G2M phase, despite a strong p53-independent block, there was a weaker but more persistent p53-dependent block. At cytotoxic concentrations, p53-dependent and p53-independent cell death was observed. The former was poorly phase-specific, occurred earlier and exploited the apoptotic mechanism more than p53-independent death.Computer simulation produced a framework where previous partial and sometimes apparently contradictory observations of the p53-mediated effects could be reconciled and explained.     

The E3 Ubiquitin Ligase EDD Regulates S-Phase and G2/M DNA Damage Checkpoints

The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G1/S and intra S phase DNA damage checkpoint activation and for the maintenance of G2/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer.