Checkpoint adaptation and recovery: back with Polo after the break

S. cerevisiae cells that are unable to repair a double strand break ultimately escape the DNA damage checkpoint arrest and enter mitosis. This process called 'adaptation' depends on functional Cdc5, a Polo-like kinase, and was long thought to be limited to single-cell organisms. However, the recent finding that Xenopus extracts can adapt to a long-lasting stall in DNA replication indicates that checkpoint adaptation does also occur in multicellular organisms. Interestingly, the Xenopus Polo-like kinase (Plx1) plays an important role in this adaptation. To add to this, data from our laboratory have shown that the human Polo-like kinase (Plk1) is also required for cell cycle re-entry following a DNA damage-induced arrest. But here, Plk1 was shown to be required for bona-fide checkpoint recovery, rather than adaptation. That is, Plk1 is required to restart the cell cycle once all of the damage is repaired and checkpoint signaling is turned off. While the target of Plx1 during adaptation is a component of the checkpoint machinery (Claspin), the target of Plk1 during recovery turns out to be a mitotic regulator (Wee1). Here, we discuss some of the remarkable similarities and subtle differences in the molecular mechanisms that control checkpoint adaptation and recovery, and the role of Polo-like kinases therein

Getting in and out of mitosis with Polo-like kinase-1

Research in different species has shown that Polo-like kinases are essential for successful cell division. In human cells, Polo-like kinase-1 (Plk1) has been implicated in the regulation of different processes, including mitotic entry, spindle formation and cytokinesis. Recently, a range of new downstream targets of Plk1 has been identified, as well as a molecular mechanism that explains recruitment of Plk1 to potential substrate proteins through its polo-box domain. On the basis of these reports, we discuss possible mechanisms by which Polo-like kinases can exert their multiple functions during mitosis. Polo-like kinases also function in DNA damage checkpoints. Plk1 has been shown to be a target of the G2 DNA damage checkpoint, while Cdc5, the Polo-like kinase in Saccharomyces cerevisiae, has long been known to be required for adaptation to persistent DNA damage. Just recently, a similar requirement for Polo-like kinases during checkpoint adaptation was demonstrated in multicellular organisms. Moreover, Plk1 was also shown to be required for checkpoint recovery following checkpoint inactivation, that is, in cells where the damage is completely repaired. Thus, Plk1 appears to play a role at multiple points during a restart of the cell cycle following DNA damage. Based on these novel observations, we discuss possible consequences of using Plk1 as a target in anticancer strategies

Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Less is known about the mechanisms that allow resumption of the cell cycle once checkpoint signaling is silenced. Here we show that while in undamaged cells several redundant pathways can promote the onset of mitosis, this redundancy is lost in cells recovering from a DNA damage-induced arrest. We demonstrate that Plk1 is crucial for mitotic entry following recovery from DNA damage. However, Plk1 is no longer required in cells depleted of Wee1, and we could show that Plk1 is involved in the degradation of Wee1 at the onset of mitosis. Thus, our data show that the cell cycle machinery is reset in response to DNA damage and that cells become critically dependent on Plk1-mediated degradation of Wee1 for their recovery

Polo-like kinase-1: activity measurement and RNAi-mediated knockdown

Polo-like kinase-1 (Plk-1) is an important cell cycle regulatory kinase that has been implicated in a multitude of cell cycle events. In this chapter we review those multiple functions of Plk-1 and describe the methods routinely used in our laboratory to purify Plk-1 from cellular lysates and measure Plk-1 kinase activity in vitro. In addition, we describe a method to analyze cell cycle progression after depletion of Plk-1 by RNA-interference in tissue culture cells

Restarting the cell cycle when the checkpoint comes to a halt

The DNA damage checkpoint coordinates a block in cell proliferation with the DNA repair process that follows when lesions are inflicted on the genome. However, we do not know exactly how cell division can recommence following a DNA damage–induced arrest. Recent work from our lab has identified Polo-like kinase-1 and Cdc25B as two essential components of the machinery that sets the cell division process back in motion when the checkpoint response is abrogated. Here, we discuss these novel insights and discuss their possible implications for the treatment of cancer

Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Less is known about the mechanisms that allow resumption of the cell cycle once checkpoint signaling is silenced. Here we show that while in undamaged cells several redundant pathways can promote the onset of mitosis, this redundancy is lost in cells recovering from a DNA damage-induced arrest. We demonstrate that Plk1 is crucial for mitotic entry following recovery from DNA damage. However, Plk1 is no longer required in cells depleted of Wee1, and we could show that Plk1 is involved in the degradation of Wee1 at the onset of mitosis. Thus, our data show that the cell cycle machinery is reset in response to DNA damage and that cells become critically dependent on Plk1-mediated degradation of Wee1 for their recovery

CLIP-170 facilitates the formation of kinetochore-microtubule attachments

CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore–microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore–microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore–microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore–microtubule attachments, possibly through direct capture of microtubules at the kinetochore

Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis

Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to separate their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addition, co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degradation of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis

A randomized phase II study investigating the addition of the specific COX-2 inhibitor celecoxib to docetaxel plus carboplatin as first-line chemotherapy for stage IC to IV epithelial ovarian cancer, Fallopian tube or primary peritoneal carcinomas: the DoCaCel study

In ovarian cancer, cyclooxygenase-2 (COX-2) overexpression is prognostic for poor survival. We investigated the efficacy of celecoxib (C), a selective COX-2 inhibitor, added to docetaxel (Taxotere)/carboplatin (DC) in advanced ovarian cancer. In a phase II, randomized study, 400 mg celecoxib b.i.d. was added to first-line DC treatment (DCC). Celecoxib was to be continued after DC termination up to 3 years. Study end points were tolerability, progression-free survival (PFS) and overall survival (OS). 151 of 196 eligible patients were diagnosed with stage IIIC/IV disease. Median follow-up for patients alive was 32.3 months. Celecoxib was used during a mean of 8.5 months. Twenty-three of 97 DCC patients stopped celecoxib prematurely, mainly due to skin reactions. Complete biochemical response was achieved in 51/78 DC patients (65%) versus 57/78 DCC patients (75%, not significant). In both study arms, median PFS was 14.3 months and median OS 34 months. COX-2 was expressed in 82% of 120 tumor samples retrospectively recovered. The PFS and OS of patients with intermediate/high COX-2 expression were similar to that in the other patients. Celecoxib did not influence PFS and OS, but interpretation of results is hampered by premature celecoxib discontinuation