Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G2/M DNA damage checkpoint and efficient checkpoint recovery

Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process

International audienceBACKGROUND:The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.METHODS:We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.RESULTS:Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.CONCLUSIONS:Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer

Improving Performance of Iterative Methods by Lossy Checkponting

Iterative methods are commonly used approaches to solve large, sparse linear systems, which are fundamental operations for many modern scientific simulations. When the large-scale iterative methods are running with a large number of ranks in parallel, they have to checkpoint the dynamic variables periodically in case of unavoidable fail-stop errors, requiring fast I/O systems and large storage space. To this end, significantly reducing the checkpointing overhead is critical to improving the overall performance of iterative methods. Our contribution is fourfold. (1) We propose a novel lossy checkpointing scheme that can significantly improve the checkpointing performance of iterative methods by leveraging lossy compressors. (2) We formulate a lossy checkpointing performance model and derive theoretically an upper bound for the extra number of iterations caused by the distortion of data in lossy checkpoints, in order to guarantee the performance improvement under the lossy checkpointing scheme. (3) We analyze the impact of lossy checkpointing (i.e., extra number of iterations caused by lossy checkpointing files) for multiple types of iterative methods. (4)We evaluate the lossy checkpointing scheme with optimal checkpointing intervals on a high-performance computing environment with 2,048 cores, using a well-known scientific computation package PETSc and a state-of-the-art checkpoint/restart toolkit. Experiments show that our optimized lossy checkpointing scheme can significantly reduce the fault tolerance overhead for iterative methods by 23%~70% compared with traditional checkpointing and 20%~58% compared with lossless-compressed checkpointing, in the presence of system failures.Comment: 14 pages, 10 figures, HPDC'1

MATHEMATICAL MODELING REVEALS THAT G2/M CHECKPOINT OVERRIDE

Cell cycle checkpoints determine whether cells meet requirements to progress through the next stage. In response to DNA damage, how cells activate checkpoints have been well studied, but little is known about checkpoint deactivation (recovery), which directly impacts on cell fate. In tumor cells, the signaling network has been rewired due to epigenetic and genetic alterations, which result in resistance to the cell cycle control, and thus resistance to chemotherapy or radiation therapy. Therefore, it is critical to identify molecules required for checkpoint recovery or adaptation after DNA damage. To achieve this goal, we performed a multidisciplinary study combining reverse phase protein array (RPPA) data, molecular biology and mathematical modeling to systematically identify molecules required for DNA damage checkpoint recovery. The mTOR complex 1 (mTORC1) plays an essential role to regulate mitotic entry after irradiation. Inhibition of the mTOR pathway delayed G2/M checkpoint recovery, while TSC2-null cells with hyperactivity of mTORC1 exhibited the opposite results. Furthermore, our mechanistic study revealed that mTOR signaling pathway controls a transcriptional program of mitotic entry through regulating histone lysine demethylase KDM4B, which is required for the epigenetic regulation of key mitosis-related genes including CCNB1 and PLK1. Given accelerated G2/M checkpoint recovery in TSC2-null cells with mTORC1 hyperactivity, we postulated that further abrogation of the G2/M checkpoint may facilitate mitotic catastrophe and selectively kill cells. As we expected, TSC2-null cells were more sensitive to the WEE1 inhibitor, a negative regulator of mitotic entry, compared to wild-type cells. In summary, we reported a novel mechanism of the mTORC1 signaling in regulating a transcriptional program required for G2/M checkpoint recovery after DNA damage. This mechanism provided a therapeutic strategy for TSC patients with mTORC1 hyperactivity using the WEE1 inhibitor, which has a potential to be translated into clinical trials

DNA damage stress: Cui prodest?

DNA is an entity shielded by mechanisms that maintain genomic stability and are essential for living cells; however, DNA is constantly subject to assaults from the environment throughout the cellular life span, making the genome susceptible to mutation and irreparable damage. Cells are prepared to mend such events through cell death as an extrema ratio to solve those threats from a multicellular perspective. However, in cells under various stress conditions, checkpoint mechanisms are activated to allow cells to have enough time to repair the damaged DNA. In yeast, entry into the cell cycle when damage is not completely repaired represents an adaptive mechanism to cope with stressful conditions. In multicellular organisms, entry into cell cycle with damaged DNA is strictly forbidden. However, in cancer development, individual cells undergo checkpoint adaptation, in which most cells die, but some survive acquiring advantageous mutations and selfishly evolve a conflictual behavior. In this review, we focus on how, in cancer development, cells rely on checkpoint adaptation to escape DNA stress and ultimately to cell death

Regrowth resistance: low-level platinum resistance mediated by rapid recovery from platinum-induced cell-cycle arrest

The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum drug resistance developed from H69 human small cell lung cancer cells with eight 4-day treatments of 200 ng/ml cisplatin or 400 ng/ml oxaliplatin respectively. A recovery period was given between treatments to emulate the cycles of chemotherapy given in the clinic. The resistant cell lines were approximately 2-fold resistant to cisplatin and oxaliplatin and were cross resistant to both drugs. Platinum resistance was not associated with increased cellular glutathione, decreased accumulation of platinum or increased DNA repair capacity. The H69 platinum sensitive cells entered a lengthy 3 week growth arrest in response to low-level cisplatin or oxaliplatin treatment. This is an example of the coordinated response between the cell cycle and DNA repair. In contrast the H69CIS200 and H69OX400 cells have an alteration in the cell cycle allowing them to rapidly proliferate post drug treatment. The resistant cell lines also have many chromosomal rearrangements most of which are not associated with the resistant phenotype, suggesting an increase in genomic instability in the resistant cell lines. We hypothesised that there was a lack of coordination between the cell cycle and DNA repair in the resistant cell lines allowing proliferation in the presence of DNA damage which has created an increase in genomic instability. The H69 cells and resistant cell lines have mutant p53 and consequently decrease the expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed with the platinum induced cell cycle arrest and did not correlate with resistance or altered DNA repair capacity. These changes may in part be mediating and maintaining the cell cycle arrest in place of p21.The rapidly proliferating resistant cells have restored the levels of both these proteins to their levels in untreated cells. We use the term ‘regrowth resistance’ to describe this low-level platinum resistance where cells survive treatment through increased proliferation. Regrowth resistance may play a role in the onset of clinical resistance

Aurora-A expressing tumour cells are deficient for homology-directed DNA double strand-break repair and sensitive to PARP inhibition.

The protein kinase Aurora-A is a major regulator of the cell cycle that orchestrates mitotic entry and is required for the assembly of a functional mitotic spindle. Overexpression of Aurora-A has been strongly linked with oncogenesis and this has led to considerable efforts at therapeutic targeting of the kinase activity of this protein. However, the exact mechanism by which Aurora-A promotes oncogenesis remains unclear. Here, we show that Aurora-A modulates the repair of DNA double-strand breaks (DSBs). Aurora-A expression inhibits RAD51 recruitment to DNA DSBs, decreases DSB repair by homologous recombination and sensitizes cancer cells to PARP inhibition. This impairment of RAD51 function requires inhibition of CHK1 by Polo-like kinase 1 (PLK1). These results identify a novel function of Aurora-A in modulating the response to DNA DSB that likely contributes to carcinogenesis and suggest a novel therapeutic approach to the treatment of cancers overexpressing this protein