Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis

We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material.     

PTEN is required for the normal progression of gastrulation by repressing cell proliferation after MBT in Xenopus embryos

PTEN phosphatase mediates several developmental cues involving cell proliferation, growth, death, and migration. We investigated the function of the PTEN gene at the transition from the cell proliferation state to morphogenesis around the midblastula transition (MBT) and gastrulation in Xenopus embryos. An immunoblotting analysis indicated that PTEN expresses constantly through embryogenesis. By up- or down-regulating PTEN activity using overexpression of the active form or C terminus of PTEN before MBT, we induced elongation of the cell cycle time just before MBT or maintained its speed even after MBT, respectively. The disruption of the cell cycle time by changing the activity of PTEN delayed gastrulation after MBT. In addition, PTEN began to localize to the plasma membranes and nuclei at MBT. Overexpression of a membrane-localizing mutant of PTEN caused dephosphorylation of Akt, whereas overexpression of the C terminus of PTEN caused phosphorylation of Akt and inhibited the localization of EGFP-PTEN to the plasma membranes and nuclei. These results indicate that an appropriate PTEN activity, probably regulated by its differential localization, is necessary for coordinating cell proliferation and early morphogenesis.     

Regulatory role of CK2 during the progression of cell cycle

The protein kinase casein kinase 2 (CK2) is a ubiquitous eukaryotic serine/threonine protein kinase that plays an important role in cell cycle progression. We find that (1) CK2 interacts with a tumor suppressor protein, adenomatous polyposis coli (APC) that occurs at the highest level in G₂/M, and (2) the C-terminal region of APC, between amino acid residues 2086–2394, has the strongest activity to suppress CK2. Over-expression of this fragment in HEK293 cells or colorectal carcinoma cells that have truncated mutant APC proteins down-regulates cell proliferation rates as well as colony formation on soft agar. These results indicate that the complex formation between CK2 and full-length APC regulates CK2 activity that, in turn, regulates cell cycle progression, whereas truncated APC in colorectal carcinomas are unable to regulate the cell cycle. In the process to look for the downstream target for CK2, we found that eukaryotic translation initiation factor 5 (eIF5) is phosphorylated by CK2 in vivo as well as in vitro These results suggest an important role of CK2 on promotion of cell growth through eIF5.     

Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser(149), Ser(229), and Ser(321) of Cdc25B, and explored the role of Ser(149) in G(2)/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr(15), resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser(149) was phosphorylated at G(1) and S phases, whereas dephosphorylated at G(2) and M phases, and the phosphorylation of Cdc25B-Ser(149) was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G(1) and S phases and translocated to the nucleus at the G(2) phase. Collectively, our findings provide evidence that Ser(149) may be another potential PKA phosphorylation target of Cdc25B in G(2)/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.     

Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.     

Relevance of histone H1 kinase activity to the G2/M transition during the cell cycle ofDictyostelium discoideum

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated usingDictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13^suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells.     

Golgi localization and dynamics of hyaluronan binding protein 1 （HABP1／p32／C1QBP） during the cell cycle

Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitoticnuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process.     

The APC regulator CDH1 is essential for the progression of embryonic cell cycles in Xenopus

The orderly progression of cell cycle depends on timely destruction of key regulators through ubiquitin-mediated proteolysis. The anaphase-promoting complex (APC) is a major component of this degradation machinery and its activation is regulated by CDC20 and CDH1. We demonstrate here that CDH1 mRNA is ubiquitously expressed in Xenopus embryos of all developmental stages. Loss of CDH1 function during early embryonic cell cycles leads to an immediate and prolonged arrest with low cyclin-dependent kinase activity. In contrast, ectopic overexpression of CDH1 induces cell cycle arrest during the first G(1) phase at the midblastula transition. CDH1-dependent degradation of cyclin A is likely involved in this G(1) arrest. Our findings establish the essential roles of CDH1 in embryonic cell cycles.     

Colocalization of the cyclin B homologue P56 and β-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During motosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti β-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and β-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-β-tubulin antibodies confirm that p56 antigens colocalize with β-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.     

The role of Ca2+ in oocyte activation during In Vitro fertilization: Insights into potential therapies for rescuing failed fertilization

At fertilization the mature mammalian oocyte is activated to begin development by a sperm-induced series of increases in the cytosolic free Ca²⁺ concentration. These so called Ca²⁺ oscillations, or repetitive Ca²⁺ spikes, are also seen after intracytoplasmic sperm injection (ICSI) and are primarily triggered by a sperm protein called phospholipase Czeta (PLCζ). Whilst ICSI is generally an effective way to fertilizing human oocytes, there are cases where oocyte activation fails to occur after sperm injection. Many such cases appear to be associated with a PLCζ deficiency. Some IVF clinics are now attempting to rescue such cases of failed fertilization by using artificial means of oocyte activation such as the application of Ca²⁺ ionophores. This review presents the scientific background for these therapies and also considers ways to improve artificial oocyte activation after failed fertilization.     

Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes

Background information. Activation of MAPKs (mitogen‐activated protein kinases), in particular ERK1/2 (extracellular‐signal‐regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo‐osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso‐osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD. Results. In turbot hepatocytes, Western‐blot analysis shows that a hypo‐osmotic shock from 320 to 240 mOsm·kg^?1 induced a rapid increase in ERK1/2 phosphorylation, whereas a hyper‐osmotic shock from 320 to 400 mOsm·kg^?1 induced no significant change in the phosphorylation of these proteins. The hypo‐osmotic‐induced ERK1/2 phosphorylation was significantly prevented when hypo‐osmotic shock was performed in the presence of the specific MEK (MAPK/ERK kinase) inhibitor PD98059 (100 μM). In these conditions, the RVD process was not altered, suggesting that ERK1/2 did not participate in this process in turbot hepatocytes. Moreover, the hypo‐osmotic‐induced activation of ERK1/2 was significantly prevented by breakdown of extracellular ATP by apyrase (10 units·ml^?1), by inhibition of purinergic P₂ receptors by suramin (100 μM) or by calcium depletion using EGTA (1 mM) and thapsigargin (1 μM). Conclusions. In turbot hepatocytes, hypo‐osmotic swelling but not hyper‐osmotic shrinkage induced the activation of ERK1/2. However, these proteins do not seem to be involved in the RVD process. Their hypo‐osmotic‐induced activation is partially due to cascades of signalling events triggered by the binding of released ATP on purinergic P₂ receptors and requires the presence of calcium.     

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.     

A novel anticancer effect of butein: inhibition of invasion through the ERK1/2 and NF-kappa B signaling pathways in bladder cancer cells

There is increasing evidence that epithelial-mesenchymal transition (EMT) plays a critical role in cancer metastasis. Butein is a polyphenolic compound, which has been found to exhibit anti-proliferation effects on cancer cells. Here, we report that in addition to its function as an anti-proliferation agent, butein can inhibit migration and invasion through the ERK1/2 and NF-kappaB signaling pathways in human bladder cancer cells, and this inhibitory effect may be associated with reversal of EMT. These results were further confirmed by RNAi-mediated suppression of NF-kappaB, which partly reverses EMT and inhibits cell invasive ability in vitro. These results suggest a novel function of butein as an invasion inhibitor in bladder cancer.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Modulation of cyclic CO2 release in response to endogenous changes of metabolism during pupal development of Zophobas rugipes (Coleoptera: Tenebrionidae)

Understanding the mechanisms of gas exchange regulation in insects currently is a hot topic of insect physiology. Endogenous variation of metabolism during pupal development offers a great opportunity to study the regulation of respiratory patterns in insects. Here we show that metabolic rates during pupal development of the tenebrionid beetle Zophobas rugipes reveal a typical U-shaped curve and that, with the exception of 9-day-old pupae, the time between two bursts of CO₂ (interburst phase) was the only parameter of cyclic CO₂ gas exchange patterns that was adjusted to changing metabolic rates. The volume of CO₂ released in a burst was kept constant, suggesting a regulation for accumulation and release of a fixed amount of CO₂ throughout pupal development. We detected a variety of discontinuous and cyclic gas exchange patterns, which were not correlated with any periods of pupal development, suggesting a high among individual variability. An occasional occurrence of continuous CO₂ release patterns at low metabolic rates was very likely caused by single defective non-occluding spiracles.     

The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment

The metastasis of cancer cells from the site of the primary tumor to distant sites in the body represents the most deadly manifestation of cancer. In order for metastasis to occur, cancer cells need to evade anoikis, which is defined as apoptosis caused by loss of attachment to extracellular matrix (ECM). Signaling from ErbB2 has previously been linked to the evasion of anoikis in breast cancer cells but the precise molecular mechanisms by which ErbB2 blocks anoikis have yet to be unveiled. In this study, we have identified a novel mechanism by which anoikis is inhibited in ErbB2-expressing cells: multicellular aggregation during ECM-detachment. Our data demonstrate that disruption of aggregation in ErbB2-positive cells is sufficient to induce anoikis and that this anoikis inhibition is a result of aggregation-induced stabilization of EGFR and consequent ERK/MAPK survival signaling. Furthermore, these data suggest that ECM-detached ErbB2-expressing cells may be uniquely susceptible to targeted therapy against EGFR and that this sensitivity could be exploited for specific elimination of ECM-detached cancer cells.     

The role of microfilaments in the organization and orientation of microtubules during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells

Summary During cell cycle transition from M to G₁ phase, micro-tubules (MTs), organized on the perinuclear region, reached the cell cortex. Microfilaments (MFs) were not involved in this process, however, MFs accumulated to form a ring-like structure in the division plane and from there they elongated toward the distal end in the cell cortex. Subsequently, when MTs elongated along the long axis of the cells, towards the distal end, the MTs ran into and then associated with the predeveloped MFs in the cell cortex, suggesting the involvement of MFs in organizing the parallel oriented MTs in the cell cortex. When cortical MTs were formed in the direction transverse to the long axis of cells, the two structures were again closely associated. Therefore, with regards to the determination of the direction of organizing MTs, predeveloped MFs may have guided the orientation of MTs at the initial stage. Disorganization of MFs in this period, by cytochalasins, prevented the organization of cortical MTs, and resulted in the appearance of abnormal MT configurations. We thus demonstrate the involvement of MFs in determining the orientation and organization of cortical MTs, and discuss the possible role of MFs during this process.Abbreviations CB cytochalasin B - CD cytochalasin D - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - EF-1 elongation factor 1 - MF microfilament - MT microtubule     

The origin and organization of cortical microtubules during the transition between M and G1 phases of the cell cycle as observed in highly synchronized cells of tobacco BY-2

The organization of microtubules (MTs) during the transition from the M phase to the G₁ phase of the cell cycle was followed in highly synchronized suspension-cultured cells ofNicotiana tabacum L. (tobacco BY-2) by sequential treatment of cells with aphidicolin and propyzamide. Short MTs were first formed in the perinuclear regions at the expense of phragmoplasts, but when these short MTs elongated to reach the cell cortex, they grew parallel to the long axis and towards the distal end of the cells. As soon as, or shortly before the tips of elongated MTs reached the distal end, transverse cortical MTs were formed in the region proximal to the division plane. Thereafter, almost all cells retained cortical MTs which were transversely orientated to the long axis of cells and could be observed in the G₁ phase. Thus, in the organization of cortical MTs, there are two steps that have been overlooked thus far. This novel observation provides a new scheme for the organization of cortical MTs, which could unify two contrasting hypotheses, i.e. organization in the perinuclear regions versus that in the cell cortex. These observations are discussed in relation to the microtubule-organizing center of plant cells.