Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

Premature expression of cyclin B sensitizes human HT1080 cells to caffeine-induced premature mitosis

Eukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S-phase arrested hamster cells undergo mitosis-specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine-induced PCC and the level of cyclin B-associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S-phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S-phase arrested HT1080 cells. The transfected cell population displayed a 5-fold increase in the frequency of caffeine-induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope-tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically-induced uncoupling of mitosis from the completion of DNA replication. © 1995 Wiley-Liss, Inc.     

Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells

Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.     

Histone deacetylation is required for progression through mitosis in tobacco cells

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.     

Patched1 interacts with cyclin B1 to regulate cell cycle progression

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, we identified a novel interaction between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacted specifically with constitutively phosphorylated cyclin B1 derivatives and was able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells was inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to ALA: In addition, we demonstrate that endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, we propose that ptc1 participates in a G(2)/M checkpoint by regulating the localization of MPF.     

The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells

We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.     

Dose-dependent and sequence-sensitive effects of amyloid-beta peptide on neurosteroidogenesis in human neuroblastoma cells

Interactions between neurosteroidogenesis and proteins involved in age-related diseases are unknown. High concentrations of amyloid-beta (A beta) peptides induce plaques in Alzheimer's disease but several studies demonstrated that physiological or non-toxic doses are neuroprotective. We compared the effects of non-toxic and toxic concentrations of A beta 1-42 and A beta 25-35 on neurosteroidogenesis in human neuroblastoma SH-SY5Y cells. Viability assays revealed that nanomolar doses of A beta are devoid of cytotoxicity while 12 microM induced cell death. Pulse-chase, high-performance liquid chromatography and flow-scintillation analyses showed that non-toxic A beta 1-42 concentrations, acting selectively, decreased [3H]progesterone but increased [3H]estradiol production from the precursor [3H]pregnenolone. Non-toxic A beta 25-35 doses reduced [3H]progesterone formation but had no effect on [3H]estradiol biosynthesis. At 12 microM, both A beta 1-42 and A beta 25-35 inhibited [3H]progesterone formation but only A beta 1-42 reduced [3H]estradiol production. The results demonstrate a selective and amino-acid sequence-dependent action of A beta on neurosteroidogenesis. The fact that non-toxic A beta 1-42 doses stimulated neuroprotective-neurosteroid estradiol synthesis, which is inhibited by high A beta 1-42 doses, may explain A beta 1-42 ability to exert either protective or deleterious effects on nerve cells.     

A novel role for Cdk1/cyclin B in regulating B-raf activation at mitosis

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase-dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S(144)PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.     

Dose-dependent effects of endotoxin on human sleep

The role of the central nervous system in the host response to infection and inflammation and modulation of these responses by the hypothalamic-pituitary-adrenal system are well established. In animals, activation of host defense mechanisms increases non-rapid eye movement (NREM) sleep amount and intensity, which, in turn, are thought to support host defense, or the body's ability to defend itself against challenges to its immune system. In humans, the evidence is conflicting. Therefore, we investigated the effects of three placebo-controlled doses of endotoxin on host response, including nocturnal sleep in healthy volunteers. Administered before nocturnal sleep onset, endotoxin dose dependently increased rectal temperature, heart rate, and the plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-1 receptor antagonist, IL-6, and cortisol. The lowest dose reliably increased circulating levels of cytokines and soluble cytokine receptors, but it did not affect rectal temperature, heart rate, or cortisol. This subtle host defense activation increased deep NREM sleep amount, often referred to as slow-wave sleep (stages 3 and 4), and intensity (delta power). Conversely, the highest dose of endotoxin disrupted sleep. Whereas it is well established that the endocrine and thermoregulatory systems are very sensitive to endotoxin, this study shows that human sleep-wake behavior is even more sensitive to activation of host defense mechanisms.     

Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.     

Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction.

Previous work has established that destruction of cyclin B is necessary for exit from mitosis and entry into the next interphase. Sea urchin cyclin B lacking an N-terminal domain is stable, permanently activates cdc2 kinase, resulting in mitotic arrest, and permanently activates the destruction pathway acting on full length cyclin B. Here we have compared the properties of clam cyclins A and B lacking related N-terminal domains. Both cyclin A delta 60 and B delta 97 bind to cdc2 kinase, keep it hyperactivated and block the completion of mitosis. By adding purified delta cyclin proteins to a cell-free system at different cell cycle times, we find that when the cell-free system reaches the cyclin destruction point in the presence of either A delta 60 or B delta 97, the cyclin destruction pathway acting on full length cyclins fails to be turned off. However, the two cyclins differ dramatically in their ability to turn on cyclin destruction. When added to emetine-arrested interphase lysates devoid of endogenous cyclins, only cyclin B delta 97 activates the cyclin destruction system; cyclin A delta 60 does not. This functional difference between the two cyclin types, the first to be described, provides strong support for the idea that the two cyclins have different roles in the cell cycle and suggests that one specialized role of the cyclin B-cdc2 complex is to activate the cyclin destruction pathway and drive cells into interphase of the next cell cycle.     

Chromosome damage and progression into and through mitosis in vertebrates

How cells behave as they divide in the presence of chromosome (DNA) damage is only just beginning to be explored. It appears to depend on the cell type and organism, the stage of development, how extensive the damage is and when it occurs. The existing data support the conclusion that vertebrate somatic cells lack a conventional DNA damage checkpoint during mitosis, and that when damaged DNA does prolong mitosis it is mediated by the spindle assembly checkpoint. As a rule, in the presence of DNA damage cells ultimately undergo an aberrant mitosis and enter the ensuing G1. They then either die, via apoptosis or mitotic catastrophe, or survive with an altered genome. To avoid these outcomes, cells with DNA damage are normally prevented from entering mitosis by a number of G2 checkpoint control pathways.     

Cyclic AMP delays G2 progression and prevents efficient accumulation of cyclin B1 proteins in mouse macrophage cells

In mouse macrophage cells, the increase of the intracellular cAMP level activates protein kinase A (PKA) and results in inhibition of cell cycle progression in both G1 and G2/M phases. G1 arrest is mediated by a cdk inhibitor, p27Kip1, which prevents G1 cyclin/cdk complexes from being activated in response to colony stimulating factor-1, whereas inhibition of G2/M progression has not been fully elucidated. In this report we analyzed the effect of cAMP on G2/M progression in a mouse macrophage cell line, BAC1.2F5A. Flow cytometric analysis and mitotic index measurement using both synchronized and asynchronized cells revealed that addition of cAMP-elevating agents (8-bromoadenosine 3':5'-cyclic monophosphate and 3-isobutyl-methyl-xanthine), although they did not affect S phase progression or M/G1 transition, temporarily arrested cells in G2 but eventually the cells proceeded to M phase, resulting in about 4 hours delay of G2 progression. Timing of cyclin B1/Cdc2 kinase activation was also retarded by about 4 hours, which was accompanied by inhibition of efficient accumulation of cyclin B1 proteins. Initial induction and accumulation of cyclin B1 mRNA were not hampered, but the half life of cyclin B1 proteins was significantly shorter during G2 phase in the presence of cAMP-elevating agents compared with that of the cells blocked from progressing through M phase by nocodazole. These results imply that the cAMP/PKA pathway regulates G2 phase progression by altering the stability of a crucial cell cycle regulator.     

Mechanisms governing maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human cytomegalovirus

Previous work has demonstrated dysregulation of key cell cycle components in human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. L. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). The activation of the mitotic kinase Cdk1/cyclin B, which was detected as early as 8 h postinfection (p.i.) and maintained throughout the time course, was particularly interesting. To understand the mechanisms underlying the induction of this kinase activity, we have examined the pathways that regulate the activation of Cdk1/cyclin B1 complexes. The accumulation of the cyclin B1 subunit in HCMV-infected cells is the result of increased synthesis and reduced degradation of the protein. In addition, the catalytic subunit, Cdk1, accumulates in its active form in virus-infected cells. The decreased level of the Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due in part to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1 via the proteasome also accounts for its absence at 24 h p.i. At late times, we observed accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Interestingly, biochemical fractionation studies revealed that the active form of Cdk1, a fraction of total cyclin B1, and the Cdc25 phosphatases reside predominantly in the cytoplasm of infected cells. Collectively, these data suggest that the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected cells can be explained by three mechanisms: the accumulation of cyclin B1, the inactivation of negative regulatory pathways for Cdk1, and the accumulation of positive factors that promote Cdk1 activity.     

A novel RING finger protein,human enhancer of invasion 10, alters mitotic progression through regulation of cyclin B levels

The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle. The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation. Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins. Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G(2)/M transition. In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts. Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast. HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site. Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro. These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G(2)/M.     

Cyclin E overexpression impairs progression through mitosis by inhibiting APC(Cdh1)

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APC(Cdh1)) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APC(Cdh1) substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E-mediated mitotic phenotype.     

Diethylstilbestrol induces arrest of rat vascular smooth muscle cell cycle progression through downregulation of cyclin D1 and cyclin E

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.