Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells

Mitotic entry, a critical decision point for maintaining genetic stability, is governed by the cyclin B/Cyclin dependent kinase 1 (Cdc2) complex. In Xenopus oocytes and early embryos, accumulation of cyclin B activates Cdk1, which then phosphorylates and activates the positive regulator Cdc25 in an autocatalytic feedback loop. However, cyclin B levels do not increase as some human cells approach mitosis, and the key factors regulating Cdk1 activation in human cells are unknown. We report here that reducing cyclin A expression by RNA interference (RNAi) in primary human fibroblasts inhibited activation of Cdc25B and Cdc25C and dephosphorylation of Cdk1 on tyrosine (tyr) 15. These results were reproduced in U2-OS cells by inducing the expression of a dominant-negative (dn) mutant of Cdk2, the principal cyclin A binding partner. Cdk2-dn induction could inhibit Cdc25B activity and foster Cdk1 tyr phosphorylation within the S phase, temporally dissociating these events from Cdk1 activation at mitosis. In contrast, reducing Cdk1 expression delayed mitotic entry without markedly impairing Cdc25B or Cdc25C activity. These results suggest that cyclin A/Cdk2 complexes are key regulators of Cdc25 and Cdk1 activation in human cells. This pathway appears to be commonly deregulated in cancer.     

The polo-like kinase Plx1 prevents premature inactivation of the APC(Fizzy)-dependent pathway in the early Xenopus cell cycle

Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).     

Cdc25C phosphorylation on serine 191 by Plk3 promotes its nuclear translocation

Mitosis in human cells is initiated at the end of G2 by activation of the Cdc2/cyclin B complex. Activation occurs by dephosphorylation of the inhibitory residues, threonine 14 (T14) and tyrosine 15 (Y15), on Cdc2 by the Cdc25C phosphatase. Entry into mitosis is regulated by the subcellular relocalization of Cdc2/cyclin B, which is rapidly imported into the nucleus at the end of G2. Here, we show that polo-like kinase 3 (Plk3) is able to phosphorylate Cdc25C primarily on S191, and to a lesser extent on S198 in vitro, both of which are within a nuclear exclusion motif. Following transfection, the S191D Cdc25C mutant leads to an enhanced accumulation of Cdc25C in the nucleus, while the S191A mutant facilitated the Cdc25C nuclear exclusion. Furthermore, translocation of Cdc25C to the nucleus was accompanied by a decrease in Cdc2 phosphorylation on Y15. Plk3-WT overexpression led to a sharp increase in Cdc25C nuclear accumulation, while Plk3-KD overexpression failed to do so. The effect of Plk3 overexpression on Cdc25C was reversed by coexpression of a Plk3 siRNA. These results support a role for the polo kinases in coordinating the translocation and perhaps the timing of both Cdc25C and its target Cdc2/cyclin B to the nucleus upon entry into mitosis.     

Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization

The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.     

Regulation of APC/CCdc20 activity by RASSF1A-APC/CCdc20 circuitry

RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/C(Cdc20) is the master switch and regulator of mitosis. The activity of APC/C(Cdc20) is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/C(Cdc20) during mitosis. Our study showed that RASSF1A and APC/C(Cdc20) form a molecular relay that regulates the APC/C(Cdc20) activity at early mitosis. We found that RASSF1A inhibits APC/C(Cdc20) function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/C(Cdc20) in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/C(Cdc20). Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/C(Cdc20) during the course of mitotic progression. As a result of RASSF1A degradation, APC/C(Cdc20) can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A-APC/C(Cdc20) interaction in ensuring the sequential progression of mitosis.     

Id-1 promotes chromosomal instability through modification of APC/C activity during mitosis in response to microtubule disruption

Id-1 (Inhibitor of DNA binding/differential-1) plays a positive role in tumorigenesis through regulation of multiple signaling pathways. Recently, it is suggested that upregulation of Id-1 in cancer cells promotes chromosomal instability. However, the underlying molecular mechanism is not known. In this study, we report a novel function of Id-1 in regulation of mitosis through physical interaction with Cdc20 (cell division cycle protein 20) and Cdh1 (Cdc20 homolog 1). During early mitosis, Id-1 interacts with Cdc20 and RASSF1A (Ras association domain family 1A), leading to enhanced APC(Cdc20) activity, which in turn promotes cyclin B1/securin degradation and premature mitosis. During late mitosis, Id-1 binds to Cdh1 and disrupts the interaction between Cdh1 and APC, resulting in suppression of APC(Cdh1) activity. On the other hand, overexpression of Cdh1 leads to Id-1 protein degradation, suggesting that Id-1 may also act as a substrate of APC(Cdh1). The negative effect of Id-1 on APC(Cdh1) results in suppression of APC(Cdh1)-induced Aurora A and Cdc20 degradation, leading to failure in cytokinesis. As a result, overexpression of Id-1 in human prostate epithelial cells leads to polyploidy in response to microtubule disruption, and this effect is abolished when Id-1 expression is suppressed using antisense technology. These results demonstrate a novel function of Id-1 in promoting chromosomal instability through modification of APC/C activity during mitosis and provide a novel molecular mechanism accounted for the function of Id-1 as an oncogene.     

The physical association and phosphorylation of Cdc25C protein phosphatase by Prk

prk encodes a protein serine/threonine kinase involved in regulating M phase functions during the cell cycle. We have expressed His6-Prk and His6-Cdc25C proteins using the baculoviral vector expression system. Purified recombinant His6-Prk, but not a kinase-defective mutant His6-PrkK52R, is capable of strongly phosphorylating His6-Cdc25C in vitro. Co-immunoprecipitation and affinity column chromatography experiments demonstrate that GST-Prk and native Cdc25C interact. When co-infected with His6-Prk and His6-Cdc25C recombinant baculoviruses, sf-9 cells produce His6-Cdc25C antigen with an additional slower mobility band on denaturing polyacrylamide gels compared with cells infected with His6-Cdc25C baculovirus alone. In addition, His6-Cdc25C immunoprecipitated from sf-9 cells co-infected with His6-Prk and His6-Cdc25C baculoviruses, but not with His6-PrkK52R and His6-Cdc25C baculoviruses, contains a greatly enhanced kinase activity that phosphorylates His6-Cdc25C in vitro. Moreover, phosphopeptide mapping shows that His6-Prk phosphorylates His6-Cdc25C at two sites in vitro and that the major phosphorylation site co-migrates with the one that is phosphorylated in vivo in asynchonized cells. Further studies reveal that His6-Prk phosphorylates Cdc25C on serine216, a residue also phosphorylated by Chk1 and Chk2. Together, these observations strongly suggest that Prk's role in mitosis is at least partly mediated through direct regulation of Cdc25C.     

Plumbagin induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun N-terminal kinase pathways in human melanoma A375.S2 cells

This study is the first to investigate the anticancer effect of plumbagin in human melanoma A375.S2 cells. Plumbagin exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Further investigation revealed that plumbagin's inhibition of cell growth was also evident in a nude mice model. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Plumbagin triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of ROS is a critical mediator in plumbagin-induced cell growth inhibition. Plumbagin increased the activation of apoptosis signal-regulating kinase 1, JNK and extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38. In addition, antioxidants vitamin C and catalase significantly decreased plumbagin-mediated c-Jun N-terminal kinase (JNK) activation and apoptosis. Moreover, blocking ERK and JNK by specific inhibitors suppressed plumbagin-triggered mitochondrial apoptotic pathway. Taken together, these results imply a critical role for ROS and JNK in the plumbagin's anticancer activity.     

Up-regulation of cdc2 protein during paclitaxel-induced apoptosis

Microtubule damages induced by paclitaxel inhibit proteasome-dependent degradation of cyclin B, resulting in a sustained activation of cyclin B/cdc2 kinase and a cell cycle arrest in mitosis. It has been previously shown that this kinase activity is also required for paclitaxel-induced apoptosis. We found here that paclitaxel increased cdc2 mRNA and protein levels and led to an accumulation of cdc2 in the active dephosphorylated form in NIH-OVCAR-3 cells. The addition of cycloheximide inhibited the paclitaxel-induced increase in cdc2 protein level, further indicating that paclitaxel stimulates cdc2 synthesis. This increase in cdc2 synthesis is a consequence of paclitaxel-induced arrest in mitosis. Indeed, dual analysis of DNA and cdc2 protein contents indicated that cdc2 up-regulation occurred in cells arrested with a G2/M DNA content. Furthermore, no up-regulation of cdc2 protein was observed when paclitaxel-treated cells were prevented from entering mitosis by treatment with purvalanol A, a cyclin-dependent kinase (CDK) inhibitor, or stimulated to exit mitosis with 2-AP, a non-specific kinase inhibitor. In addition, when paclitaxel-induced apoptosis was inhibited by Bcl-2 over-expression, cdc2 up-regulation did not occur, leading to a lower level of activation of the cyclin B/cdc2 complex. Taken together, these results indicated that paclitaxel-induced cdc2 protein synthesis participates in a positive feedback loop designed to increase the activity of cyclin B/cdc2 kinase and thus may play a role in paclitaxel-induced apoptosis.     

Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50-100 microg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2-M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2-M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3beta. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2-M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins-CDKs-CDKIs for G1 arrest, and the Chk2-Cdc25C-Cdc2/cyclin B1 pathway for G2-M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.     

The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1

The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK''s degradation by promoting the association of MCAK with APC/C^Cdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.     

Cdc25C interacts with PCNA at G2/M transition

Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo. Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.     

Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1

The cyclin-dependent kinase 1 (Cdc2)/cyclin B1 complex performs cardinal roles for eukaryotic mitotic progression. Phosphorylation of four serine residues within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at the G(2)/M transition. Still, the role of individual phosphorylation sites and their corresponding kinases remain to be elucidated. Polo-like kinase 1 (Plk1) shows a spatial and temporal distribution which makes it a candidate kinase for the phosphorylation of cyclin B1. We could demonstrate the interaction of both proteins in mammalian cells. Plk1 phosphorylated wild-type cyclin B1 expressed in bacteria and in mammalian cells. Ser-133 within the cytoplasmic retention signal (CRS) of cyclin B1, which regulates the nuclear entry of the heterodimeric complex during prophase, is a target of Plk1. In contrast, MAPK (Erk2) and MPF phosphorylate Ser-126 and Ser-128 within the CRS. Phosphorylation of CRS by MAPK (Erk2) prior to Plk1 treatment induced enhanced phosphorylation of cyclin B1 by Plk 1 suggesting a synergistic action of both enzymes towards cyclin B1. In addition, pretreatment of cyclin B1 by MAPK (Erk2) altered the phosphorylation pattern of Plk 1. Mutation of Ser-133 to Ala decreased the phosphorylation of cyclin B1 in vivo. An immunofluorescence study revealed that a mutation of Ser-133 reduced the nuclear import rate of cyclin B1. Still, multiple serine mutations are required to prevent nuclear translocation completely indicating that orchestrated phosphorylation within the CRS triggers rapid import of cyclin B1.     

Butyrolactone I induces cyclin B1 and causes G2/M arrest and skipping of mitosis in human prostate cell lines.

Several naturally occurring cyclin-dependent kinase (CDK) inhibitors have been isolated from different lower organisms. In this report, we examined the effect of one of the CDK inhibitors, butyrolactone I (BL), on the expression of cyclins D2, A and B1 in three human prostatic cancer cell lines (DU145, PC-3, LNCaP) using two colored flow cytometric analysis. The percentage of DU145 cells in the 4C phase of the cell cycle were increased significantly at both 70 microM and 100 microM BL. Furthermore, an additional 8C peak was observed which had double the DNA content of the 4C phase at these concentrations of BL. The appearance of the 8C peak increased gradually and was more evident in DU145 and PC-3 than LNCaP. Cells in the 8C peak had either two nuclei or abnormal nuclei as observed by Papanicolaou stain. BL also increased the amount of cyclin B1 positive cells in the 4C phase. This increase was apparent on day 1 and returned to normal by day 3. Since BL selectively inhibits cyclin-dependent kinase, cyclin B1 might accumulate without being degraded. Other cyclins were not significantly changed by BL. The data demonstrate that BL inhibited Cdc2 of unsynchronized cultured prostate cancer cells, and interrupted the cell cycle progression toward cell division. The BL inhibition of Cdc2 led to the accumulation of cells in the 4C phase without mitosis resulting in an accumulation of cyclin B1. The appearance of cells in the 8C phase may be due to the progression of cells in the 4C phase through the cell cycle skipping mitosis. Cyclin B1 decreased in correlation with the progression through a new cell cycle. These results suggest that BL does not cause a complete arrest of the cell cycle in G2/M but that BL occasionally allows for the skipping of mitosis and subsequent progression through the cell cycle to occur.     

Alterations of anaphase-promoting complex genes in human colon cancer cells

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.     

The Plk3-Cdc25 circuit

Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis. They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD). Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals. Polo, Cdc5, and Plo1 are essential for viability. The Plks can be separated into two groups according to their functions. The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle. Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation. This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle.     

Diallyl trisulfide‐induced apoptosis in human cancer cells is linked to checkpoint kinase 1‐mediated mitotic arrest

Growth suppressive effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, against cultured human cancer cells correlates with checkpoint kinase 1 (Chk1)‐mediated mitotic arrest, but the fate of the cells arrested in mitosis remains elusive. Using LNCaP and HCT‐116 human cancer cells as a model, we now demonstrate that the Chk1‐mediated mitotic arrest resulting from DATS exposure leads to apoptosis. The DATS exposure resulted in G₂ phase and mitotic arrest in both LNCaP and HCT‐116 cell lines. The G₂ arrest was accompanied by downregulation of cyclin‐dependent kinase 1 (Cdk1), cell division cycle (Cdc) 25B, and Cdc25C leading to Tyr15 phosphorylation of Cdk1 (inactivation). The DATS‐mediated mitotic arrest correlated with inactivation of anaphase‐promoting complex/cyclosome as evidenced by accumulation of its substrates cyclinB1 and securin. The DATS treatment increased activating phosphorylation of Chk1 (Ser317) and transient transfection with Chk1‐targeted siRNA conferred significant protection against DATS‐induced mitotic arrest in both cell lines. The Chk1 protein knockdown also afforded partial yet statistically significant protection against apoptotic DNA fragmentation and caspase‐3 activation resulting from DATS exposure in both LNCaP and HCT‐116 cells. Even though DATS treatment resulted in stabilization and Ser15 phosphorylation of p53, the knockdown of p53 protein failed to rescue DATS‐induced mitotic arrest. In conclusion, the results of the present study indicate that Chk1 dependence of DATS‐induced mitotic arrest in human cancer cells is not influenced by the p53 status and cells arrested in mitosis upon DATS exposure are driven to apoptotic DNA fragmentation. © 2009 Wiley‐Liss, Inc.     

Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis

In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.     

Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ phosphatase

Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.