Biomedical subjects

René H Medema

Publications and source records attributed to René H Medema.

At least 19 recordsLinked to original sources

PubMed2006

Claspin: timing the cell cycle arrest when the genome is damaged.

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for check-point recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The case for Survivin as mitotic regulator.

Survivin has been proposed to inhibit apoptosis and to regulate cell division. However, controversy still exists as to whether Survivin can indeed execute these distinct functions and if Survivin somehow coordinates apoptosis and (abnormal) cell division. Recent evidence has demonstrated that Survivin acts as a subunit of the chromosomal passenger complex, which is essential for proper chromosome segregation and cytokinesis. Within this complex, the mitotic kinase Aurora B acts as the enzymatic core, whereas Survivin dictates chromosomal passenger complex localization. This function of Survivin appears to be conserved throughout evolution. Although these findings do not exclude a role for Survivin as apoptosis inhibitor, they make a very strong case for Survivin as mitotic regulator.

Animals↗

PubMed2006

FoxM1: at the crossroads of ageing and cancer.

Forkhead transcription factors are intimately involved in the regulation of organismal development, cell differentiation and proliferation. Here we review the current knowledge of the role played by FoxM1 in these various processes. This particular member of the Forkhead family is broadly expressed in actively dividing cells and is crucial for cell cycle-dependent gene expression in the G2 phase of the cell cycle. FoxM1 plays a crucial role in insuring the fidelity of the cell division process, as inhibition of FoxM1 activity results in serious aberrancies during mitosis, such as frequent chromosome missegregation, defects in cytokinesis and overt aneuploidy. FoxM1 expression also appears to be tightly correlated with the proliferative rate of a cell. For example, FoxM1 is one of the most significantly down-regulated genes in prematurely aged human fibroblasts (Progeria syndrome), while elevated expression of FoxM1 is seen in most human carcinomas. These observations suggest that interference with FoxM1 activity may contribute to the increase in mitotic errors seen in human diseases such as cancer and early onset of ageing diseases. In this review, several aspects of FoxM1 function will be discussed, as well as their implication in tumorigenesis.

Aging↗

PubMed2006

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery.

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The chromosomal passenger complex: guiding Aurora-B through mitosis.

During mitosis, the chromosomal passenger complex (CPC) orchestrates highly different processes, such as chromosome alignment, histone modification, and cytokinesis. Proper and timely localization of this complex is the key to precise control over the enzymatic core of the CPC, the Aurora-B kinase. We discuss the molecular mechanisms by which the CPC members direct the dynamic localization of the complex throughout cell division. Also, we summarize posttranslational modifications that occur on the CPC and discuss their roles in regulating localization and function of this mitotic complex.

Animals↗

PubMed2006

Polo-like kinases: a team in control of the division.

Polo, the founding member of the family of polo-like kinases (Plks) was identified in a Drosophila screen for mutants affecting spindle pole behavior.(1) Several mutants showed defects at their spindle poles and were hence named after the magnetic poles of the earth or geo-magnetic phenomena associated with them, like Polo and Aurora. Currently, the conserved family of Plks consists of many members throughout various species. Multiple Plks are present in mammalian cells (Plk1, Plk2/Snk, Plk3/Fnk/Prk, and Plk4/Sak) and Xenopus (Plx1-3), whereas in other species only one member has been identified, like Polo in Drosophila, Cdc5 in budding yeast and Plo1 in fission yeast. Plks are now viewed as important regulators of multiple functions before and during the mitotic cell division. In this review, we will focus our attention on human Plk1 and its family members Plk2-4 and the many roles they play during mitosis. Furthermore, we will describe the currently knowledge of the regulation of these functions.

Amino Acid Motifs↗

PubMed2006

Uncoupling the central spindle-associated function of the chromosomal passenger complex from its role at centromeres.

Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, we complemented Survivin-depleted cells with a variety of point- and deletion-mutants of Survivin. We show that an intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localized normally to the central spindle, but their localization to inner centromeres was severely perturbed. Consequently, these mutants failed to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they had retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, our data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres.

Aurora Kinase B↗

PubMed2006

Exploiting the compromised spindle assembly checkpoint function of tumor cells: dawn on the horizon?

Aneuploidy is a frequent property of cancer cells that arises as a consequence of chromosomal instability (CIN). A major safeguard mechanism protecting cells from CIN is the spindle assembly checkpoint. This checkpoint surveys proper attachment of chromosomes to the mitotic spindle and effectively suppresses erroneous chromosome segregation by delaying mitotic progression until proper spindle-chromosome interactions have been established. Several lines of evidence suggest that the development of aneuploidy may be a gradual process that in many cases could result from subtle spindle checkpoint defects that occur during tumorgenesis and steadily weaken spindle checkpoint function. Here we discuss the evidence for this concept and address the question whether normal somatic cells and tumor cells could perhaps exhibit differences in spindle checkpoint regulation that allow the design of more specific anti-tumor strategies that effectively kill tumor cells but spare normal cells.

Animals↗

PubMed2006

Survivin mediates targeting of the chromosomal passenger complex to the centromere and midbody.

The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, thereby allowing it to act at the right place at the right time. Here, we addressed the individual contributions of INCENP, Survivin and Borealin to the proper functioning of this complex. We show that INCENP has an important role in stabilizing the complex, and that Borealin acts to promote binding of Survivin to INCENP. Importantly, when Survivin is directly fused to INCENP, this hybrid can restore CPC function at the centromeres and midbody, even in the absence of Borealin and the centromere-targeting domain of INCENP. Thus, Survivin is an important mediator of centromere and midbody docking of Aurora-B during mitosis.

Animals↗

PubMed2005

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.

Cells, Cultured↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Cell Cycle Proteins↗

PubMed2005

Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells.

In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively phosphorylated in the Bcr-Abl-positive BV173 and BaF3/Bcr-Abl cells. Inhibition of Bcr-Abl kinase by STI571 results in FoxO3a activation, induction of Bim expression and apoptosis. Using reporter gene assays, we demonstrate that STI571 and FoxO3a activate Bim transcription through a FoxO-binding site (FHRE) located within the promoter. This was verified by DNA pull-down and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to induction of Bim expression and apoptosis. Conversely, silencing of FoxO3a in Bcr-Abl-expressing cells abolishes STI571-mediated Bim induction and apoptosis. Together, the results presented clearly confirm FoxO3a as a key regulator of apoptosis induced by STI571, and show that Bim is a direct transcriptional target of FoxO3a that mediates the STI571-induced apoptosis. Thus, STI571 induces an accumulation of FoxO3a activity which in turn binds directly to an FHRE in the promoter to activate Bim expression and apoptosis.

Animals↗

PubMed2005

FoxM1 is required for execution of the mitotic programme and chromosome stability.

Transcriptional induction of cell-cycle regulatory proteins ensures proper timing of subsequent cell-cycle events. Here we show that the Forkhead transcription factor FoxM1 regulates expression of many G2-specific genes and is essential for chromosome stability. Loss of FoxM1 leads to pleiotropic cell-cycle defects, including a delay in G2, chromosome mis-segregation and frequent failure of cytokinesis. We show that transcriptional activation of cyclin B by FoxM1 is essential for timely mitotic entry, whereas CENP-F, another direct target of FoxM1 identified here, is essential for precise functioning of the mitotic spindle checkpoint. Thus, our data uncover a transcriptional cluster regulated by FoxM1 that is essential for proper mitotic progression.

Animals↗

PubMed2005

Mitogen requirement for cell cycle progression in the absence of pocket protein activity.

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.

Animals↗

PubMed2005

Ablation of the spindle assembly checkpoint by a compound targeting Mps1.

The spindle assembly checkpoint ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are properly attached to the mitotic spindle. Here, we show that the previously reported c-Jun amino-terminal kinase (JNK) inhibitor SP600125 effectively disrupts spindle checkpoint function in a JNK-independent fashion. SP600125 potently inhibits activity of the mitotic checkpoint kinase monopolar spindle 1 (Mps1) in vitro and triggers efficient progression through a mitotic arrest imposed by spindle poisons. Importantly, expression of an Mps1 mutant protein refractory to SP600125-mediated inhibition restores spindle checkpoint function in the presence of SP600125, showing that its mitotic phenotype is induced by Mps1 inhibition in vivo. Remarkably, primary human cells are largely resistant to the checkpoint-inactivating action of SP600125, suggesting the existence of Mps1-independent checkpoint pathways that are compromised in tumour cells.

Amino Acid Sequence↗

PubMed2005

Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1.

Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

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.

Animals↗