The spindle checkpoint: two transitions, two pathways

The spindle checkpoint is an evolutionarily conserved mitotic regulatory mechanism that ensures that anaphase is not attempted until chromosomes are properly aligned on the spindle. Two different cell-cycle transitions must be inhibited by the spindle checkpoint to arrest cells at metaphase and prevent mitotic exit. The checkpoint proteins interact in ways that are more complex than was originally envisioned. This review summarizes the evidence for two pathways of spindle-checkpoint regulation in budding yeast. We describe how the proteins are involved in these pathways and discuss the ways in which the spindle checkpoint inhibits the cell-cycle machinery.     

运动与P53调节细胞信号转导通路研究进展

P53调节多个细胞信号转导通路,其功能与肿瘤抑制、细胞周期调控、能量代谢调节、促进线粒体生物发生、保持氧化应激平衡等有关,保持P53基因的稳态表达是预防肿瘤和延缓衰老的策略之一.体育锻炼能促进机体新陈代谢、延缓细胞衰老、减少细胞癌变几率,适宜的运动能够通过影响P53调节的多个细胞信号通路延续P53信号稳态.     

Cell-cycle checkpoint kinases: checking in on the cell cycle

In response to DNA damage, cell-cycle checkpoints integrate cell-cycle control with DNA repair. The idea that checkpoint controls are an integral component of normal cell-cycle progression has arisen as a result of studies in Drosophila and mice. In addition, an appreciation that DNA damage arises as a natural consequence of cellular metabolism, including DNA replication itself, has influenced thinking regarding the nature of checkpoint pathways.     

Linking TGF-beta-mediated Cdc25A Inhibition and Cytoskeletal Tegulation through RhoA/p160ROCK Signaling

Transforming growth factor-beta (TGF-b) can mediate G1/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-b-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160ROCK signaling pathway. The activation of TGF-b-mediated p160ROCK rapidly inhibits the Cdc25A phosphatase as a component of the G1/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160ROCK pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-b.     

CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation

The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.     

Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling

Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.     

Checking on DNA damage in S phase

The precise replication of the genome and the continuous surveillance of its integrity are essential for survival and the avoidance of various diseases. Cells respond to DNA damage by activating a complex network of the so-called checkpoint pathways to delay their cell-cycle progression and repair the defects. In this review we integrate findings on the emerging mechanisms of activation, the signalling pathways and the spatio-temporal organization of the intra-S-phase DNA-damage checkpoint and its impact on the cell-cycle machinery, and discuss its biological significance.     

COST1 balances plant growth and stress tolerance via attenuation of autophagy

ABSTRACT

In plants, macroautophagy/autophagy has been reported to function in various biotic and abiotic stress-response pathways, but few direct regulators linking stress and autophagy have yet been identified. Other than the conserved nutrient sensing kinase TOR (Target of Rapamycin), negative regulators that can directly modulate plant autophagy are unknown. We recently identified a mutant, termed cost1 (Constitutively Stressed 1), which has strong drought tolerance with constitutive induction of autophagy and broad expression of normally stress-responsive genes. The COST1 protein negatively regulates autophagy by direct interaction with the key autophagy adaptor ATG8E, thus directly linking autophagy and drought tolerance. Moreover, plant growth and development in a cost1 mutant is greatly retarded, suggesting that COST1 controls the tradeoff between growth and stress tolerance.     

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

Regulation of DNA repair throughout the cell cycle

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.     

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.     

The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation

ATM and ATR are stress-response kinases which respond to a variety of insults including ionizing radiation, replication arrest, ultraviolet radiation and hypoxia/re-oxygenation. Hypoxia occupies a unique niche in the study of both ATR- and ATM-mediated checkpoint pathways. Hypoxia is a physiologically significant stress that occurs in virtually all solid tumors and differs from most other stresses in that it does not induce DNA damage. Previous studies have indicated that hypoxia provides a unique way to induce ATR in response to inhibition of DNA replication. During tumor expansion hypoxia is inevitably followed by periods of re-oxygenation which in vitro has been shown to induce significant levels of DNA damage and an ATM response. Therefore both ATR and ATM have a role to play in hypoxia/re-oxygenation.     

Direct ribosomal binding by a cellular inhibitor of translation

During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES-driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate.     

Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.     

Mammalian G1- and S-phase checkpoints in response to DNA damage

The ability to preserve genomic integrity is a fundamental feature of life. Recent findings regarding the molecular basis of the cell-cycle checkpoint responses of mammalian cells to genotoxic stress have converged into a two-wave concept of the G1 checkpoint, and shed light on the so-far elusive intra-S-phase checkpoint. Rapidly operating cascades that target the Cdc25A phosphatase appear central in both the initiation wave of the G1 checkpoint (preceding the p53-mediated maintenance wave) and the transient intra-S-phase response. Multiple links between defects in the G1/S checkpoints, genomic instability and oncogenesis are emerging, as are new challenges and hopes raised by this knowledge.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

Signaling Pathways that Regulate Cell Division

Cell division requires careful orchestration of three major events: entry into mitosis, chromosomal segregation, and cytokinesis. Signaling within and between the molecules that control these events allows for their coordination via checkpoints, a specific class of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events. Multiple positive- and negative-feedback loops ensure that a cell is fully committed to division and that the events occur in the proper order. Unlike other signaling pathways, which integrate external inputs to decide whether to execute a given process, signaling at cell division is largely dedicated to completing a decision made in G1 phase—to initiate and complete a round of mitotic cell division. Instead of deciding if the events of cell division will take place, these signaling pathways entrain these events to the activation of the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the opportunity for checkpoint proteins to arrest cell division if things go wrong.     

The spindle checkpoint: tension versus attachment

The spindle checkpoint ensures the fidelity of chromosome segregation by preventing cell-cycle progression until all the chromosomes make proper bipolar attachments to the mitotic spindle and come under tension. Despite significant advances in our understanding of spindle checkpoint function, the primary signal that activates the spindle checkpoint remains unclear. Whereas some experiments indicate that the checkpoint recognizes the lack of microtubule attachment to the kinetochore, others indicate that the checkpoint senses the absence of tension generated on the kinetochore by microtubules. The interdependence between tension and microtubule attachment make it difficult to determine whether these signals are separable. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we consider recent evidence that supports and opposes the hypothesis that defects in tension act as the primary checkpoint signal.