The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase''s phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.     

Overcoming inhibition in the spindle checkpoint

Spindle checkpoint silencing is a critical step during mitosis that initiates chromosome segregation, yet surprisingly little is known about its mechanism. Protein phosphatase I (PP1) was shown recently to be a key player in this process, and in this issue of Genes & Deverlopment, Akiyoshi and colleagues (pp. 2887–2899) identify budding yeast Fin1p as a kinetochore-localized regulator of PP1 activity toward checkpoint targets. Here we review recent mechanistic insights and propose a working model for spindle checkpoint silencing.     

The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.     

The highly conserved Ndc80 complex is required for kinetochore assembly,chromosome congression,and spindle checkpoint activity

We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.     

Inhibitors of cyclin-dependent kinase and cancer

Recent research has yielded a dramatic increase in the number of connections between oncogenesis and the proteins which regulate the cell cycle. Three classes of protein which inhibit the activity of cyclin-dependent kinases (CDKs) have emerged as potential targets for oncogenic inactivation. p16 and related proteins inhibit the cyclin/CDK complexes which regulate the transition from G₁ to S phase; numerous studies have revealed that p16 is mutated in most tumor cell lines and in some types of primary tumor. p21/WAF1/Cip1 and the related p27Kip protein inhibit a broader range of cyclin/CDK complexes than p16. Although the absence of p21/WAF1/Cip1 from cyclin/CDK complexes is correlated with cellular transformation, no mutations in this gene have been found in tumors or tumor-derived cell lines. A third class of genes which are potential targets for oncogenic inactivation are the kinases and phosphatases which regulate the activity of cyclin/CDK complexes by phosphorylation and dephosphorylation of the CDK proteins. Disruption of any of these genes would result in loss of normal regulation of cell growth.Abbreviations ALL Acute Lymphoblastic leukemias - CAK CDC2-activating kinase - CDK Cyclin-dependent kinase - CDI CDK inhibitor - GM Glioblastoma multiforme - TGF- Transforming growth factor-     

The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation

The centromere defines where on a chromosome the kinetochores assemble. Kinetochores, large protein structures, mediate chromosome segregation during mitosis and meiosis by performing three key functions. Firstly, kinetochores attach chromosomes to the microtubule spindle apparatus. Secondly, kinetochores co-ordinate microtubule dynamics to allow chromosomes to move along the spindle. Lastly, kinetochores generate the 'wait' signal which prevents anaphase onset until all the chromosomes are correctly aligned on the spindle. This signal forms part of the spindle checkpoint mechanism, a highly conserved cell cycle checkpoint which maintains the accuracy of the chromosome segregation process. This article provides a brief historical overview before focusing on some of the outstanding issues and more recent developments in the field.     

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase

S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.     

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.     

Expression of cyclins,cyclin-dependent kinases and cyclin-dependent kinase inhibitors in oligodendrogliomas in humans

Cyclins are regulatory proteins of the cell cycle which bind and activate kinases. In gliomas, contrary to many malignancies, cyclin D1 is rarely amplified, but together with other cyclins, it increases with anaplasia. In a series of 23 surgical biopsies of grade II and III oligodendroglioma, cyclin D1, E, A, B1, CDK4-6, CDK2, Cdc2 and p27/Kip.1 have been studied by immunohistochemistry and Western blot. Cyclin D1 and A increased with anaplasia, showing a linear correlation with MIB.1 labeling index and an inverse correlation with p27/Kip.1 expression. Cyclin E and B1 and kinases were almost only expressed in grade III tumors. Normal oligodendrocytes and microglia cells of the cortex and white matter showed a clear positivity for cyclin D1, but not for other cyclins or kinases.     

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.     

The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis

Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.     

Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1)

Alveolar cells of the lung are injured and killed when exposed to elevated levels of inspired oxygen. Damaged tissue architecture and pulmonary function is restored during recovery in room air as endothelial and type II epithelial cells proliferate. Although excessive fibroblast proliferation and inflammation occur when abnormal remodeling occurs, genes that regulate repair remain unknown. Our recent observation that hyperoxia inhibits proliferation through induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1), which also facilitates DNA repair, suggested that p21 may participate in remodeling. This hypothesis was tested in p21-wild-type and -deficient mice exposed to 100% FiO(2) and recovered in room air. p21 increased during hyperoxia, remained elevated after 1 day of recovery before returning to unexposed levels. Increased proliferation occurred when p21 expression decreased. In contrast, higher and sustained levels of proliferation, resulting in myofibroblast hyperplasia and monocytic inflammation, occurred in recovered p21-deficient lungs. Cells with DNA strand breaks and expressing p53 were observed in hyperplastic regions suggesting that DNA integrity had not been restored. Normal recovery of endothelial and type II epithelial cells, as assessed by expression of cell-type-specific genes was also delayed in p21-deficient lungs. These results reveal that p21 is required for remodeling the oxygen-injured lung and suggest that failure to limit replication of damaged DNA may lead to cell death, inflammation, and abnormal remodeling. This observation has important implications for therapeutic strategies designed to attenuate long-term chronic lung disease after oxidant injury.     

Nicotine increases sleep spindle activity

Studies have shown that both nicotine and sleep spindles are associated with enhanced memorisation. Further, a few recent studies have shown how cholinergic input through nicotinic and muscarinic receptors can trigger or modulate sleep processes in general, and sleep spindles in particular. To better understand the interaction between nicotine and sleep spindles, we compared in a single blind randomised study the characteristics of sleep spindles in 10 healthy participants recorded for 2 nights, one with a nicotine patch and one with a sham patch. We investigated differences in sleep spindle duration, amplitude, intra‐spindle oscillation frequency and density (i.e. spindles per min). We found that under nicotine, spindles are more numerous (average increase: 0.057 spindles per min; 95% confidence interval: [0.025–0.089]; p = .0004), have higher amplitude (average amplification: 0.260 μV; confidence interval: [0.119–0.402]; p = .0032) and last longer (average lengthening: 0.025 s; confidence interval: [0.017–0.032]; p = 2.7e‐11). These results suggest that nicotine can increase spindle activity by acting on nicotinic acetylcholine receptors, and offer an attractive hypothesis for common mechanisms that may support memorisation improvements previously reported to be associated with nicotine and sleep spindles.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer

Cytogenetic analyses have revealed that many aneuploid breast cancers have cell-to-cell variations of chromosome copy numbers, suggesting that these neoplasms have instability of chromosome numbers. To directly test for possible chromosomal instability in this disease, we used fluorescent in situ hybridization to monitor copy numbers of multiple chromosomes in cultures of replicating breast cancer-derived cell lines and nonmalignant breast epithelial cells. While most (7 of 9) breast cancer cell lines tested are highly unstable with regard to chromosome copy numbers, others (2 of 9 cell lines) have a moderate level of instability that is higher than the "background" level of normal mammary epithelial cells and MCF-10A cells, but significantly less than that seen in the highly unstable breast cancer cell lines. To evaluate the potential role of a defective mitotic spindle checkpoint as a cause of this chromosomal instability, we used flow cytometry to monitor the response of cells to nocodazole-induced mitotic spindle damage. All cell lines with high levels of chromosomal instability have defective mitotic spindle checkpoints, whereas the cell lines with moderate levels of chromosomal instability (and the stable normal mammary cells and MCF10A cells) arrest in G(2) when challenged with nocodazole. Notably, the extent of mitotic spindle checkpoint deficiency and chromosome numerical instability in these cells is unrelated to the presence or absence of p53 mutations. Our results provide direct evidence for chromosomal instability in breast cancer and show that this instability occurs at variable levels among cells from different cancers, perhaps reflecting different functional classes of chromosomal instability. High levels of chromosomal instability are likely related to defective mitotic checkpoints but not to p53 mutations.     

The role of cyclin-dependent kinase inhibitor p27 in squamous cell carcinoma of the esophagus

The level of p27 expression decreases during tumor development and progression. Loss of p27 protein provides independent prognostic information in breast, prostate, colon, stomach and lung carcinomas. We generated a new polyclonal antibody against p27 and carried out immunohistochemical analysis of p27 expression in 61 specimens of esophageal squamous cell carcinoma, 10 carcinoma in situ specimens, and 5 squamous epithelial dysplasia specimens of the esophagus. We examined the correlation of p27 expression with various clinicopathologic features and prognosis. In squamous epithelial dysplasia, the p27-positive cells were located in the superficial normal-appearing cells, not in the atypical cells. In carcinoma in situ, the expression of p27 was markedly lower than in normal esophageal epithelium. In advanced squamous cell carcinomas, high p27 correlated significantly with the degree of differentiation (p = 0.0002) and the depth of tumor invasion (p = 0.001) was statistically significant. The high p27 expression group had a better prognosis than did the low or negative p27 expression groups, but these differences were not statistically significant. These observations may imply that a decrease in p27 expression occurs early on in the carcinogenesis of esophageal carcinoma. In addition, p27 expression may correlate with the histologic differentiation of squamous cell carcinoma of the esophagus.     

Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint

A conditional allele of type 1 protein phosphatase (glc7-129) in Saccharomyces cerevisiae causes first cycle arrest in G2/M, characterized by cells with a short spindle and high H1 kinase activity. Point-of-execution experiments indicate Glc7p function is required in G2/M just before anaphase for the completion of mitosis. Loss of the spindle/kinetochore checkpoint in glc7-129 cells abolishes the G2/M cell cycle arrest with a concomitant increase in chromosome loss and reduced viability. These results support a role for Glc7p in regulating kinetochore attachment to the spindle, an event monitored by the spindle/kinetochore checkpoint.