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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.
Mol Biol Cell 2004 Sep
PMID:p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress. 1518 Nov 48

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.
Mol Cell Biol 2004 Jul
PMID:Noncatalytic requirement for cyclin A-cdk2 in p27 turnover. 1519 59

To determine the activity of all human Cdc25 phosphatases, two different methods are described. For assaying phosphatase activities of recombinant Cdc25 proteins produced in Escherichia coli or insect cells, a fluorimetric assay using fluorescein diphosphate (FDP) as a substrate is recommended. To analyze endogenous Cdc25 phosphatase activities of immunoprecipitates from total cellular extracts, the physiological substrate Cdk1/cyclin B1 is most sensitive.
Methods Mol Biol 2004
PMID:Assaying Cdc25 phosphatase activity. 1522 May 27

Described are four widely used procedures to analyze the cell cycle by flow cytometry. The first two are based on univariate analysis of cellular DNA content following cell staining with either propidium iodide (PI) or 4',6'-diamidino-2-phenylindole (DAPI) and deconvolution of the cellular DNA content frequency histograms. This approach reveals distribution of cells in three major phases of the cycle (G1 vs S vs G2/M) and makes it possible to detect apoptotic cells with fractional DNA content. The third approach is based on the bivariate analysis of DNA content and proliferation-associated proteins. The expression of cyclin D, cyclin E, cyclin A, or cyclin B1 vs DNA content is presented as an example. This approach allows one to distinguish, for example, G0 from G1 cells, identify mitotic cells, or relate expression of other intracellular proteins to the cell cycle position. The fourth procedure relies on the detection of 5'-bromo-2'-deoxyuridine (BrdU) incorporation to label the DNA-replicating cells.
Methods Mol Biol 2004
PMID:Analysis of cell cycle by flow cytometry. 1522 May 39

The presence of retinoic acid (RA) during in vitro maturation (IVM) improves bovine oocyte quality and developmental potential. In this work, we investigated the underlying molecular mechanisms. Cumulus-oocyte complexes were meiotically arrested by roscovitine and/or matured in defined medium containing RA, 1% ethanol (vehicle), or no additives. Cumulus-free oocytes were analyzed for poly-(A) mRNA contents and relative mRNA expression of genes involved in cell cycle regulation (cyclin B1 and H1) and antioxidative defence (Mn-superoxide dismutase and glucose-6-phosphate dehydrogenase). Poly-(A) mRNA increased after meiotic inhibition and decreased with IVM completion, both in meiotically arrested and permissively matured oocytes, i.e., matured without previous meiotic arrest. RA dramatically increased poly-(A) mRNA in meiotically arrested oocytes, but more than half of the poly-(A) mRNA disappeared during maturation. Irrespective of oocyte origin, transcripts were detected for all the genes analyzed. IVM, with or without previous meiotic inhibition, increased expression of cyclin B1 and glucose-6-phosphate dehydrogenase, and decreased cyclin H1 and Mn-superoxide dismutase. Except for a decreasing of Mn-superoxide dismutase in meiotically arrested and matured oocytes, RA did not affect mRNA expression. Ethanol led to an abnormal poly-(A) mRNA profile and expression of all the genes analyzed. RA does not modify expression of cyclin B1 and HI genes in the bovine oocyte, and probably does not generate oxidative stress. In addition, RA enhanced mRNA amount as measured by poly-(A) mRNA contents.
Mol Reprod Dev 2004 Sep
PMID:Retinoid-dependent mRNA expression and poly-(A) contents in bovine oocytes meiotically arrested and/or matured in vitro. 1527 10

Cyclic adenosine monophosphate (cAMP) keeps oocytes in meiotic arrest, thereby preventing activation of the key regulators of meiosis, p34cdc2/cyclin B1, (known as maturation-promoting factor (MPF)) and Erk 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. The activity of MAPK in oocytes is upregulated by Mos. We previously demonstrated that Mos translation in rat oocytes is negatively regulated by a PKA-mediated cAMP action, which inhibits c-mos mRNA polyadenylation and is associated with the suppression of p34 cdc2 kinase. The goal of the present study was to provide definitive evidence that Mos translation is subjected to MPF regulation. In order to inhibit MPF activity, we employed the double-stranded (ds) RNA interference (RNAi) of gene expression. We demonstrated that the introduction of cyclin B1 dsRNA into rat oocytes selectively depleted the corresponding mRNA, further ablating its protein product. These oocytes, which exhibit low MPF activity, failed to elongate the c-mos mRNA poly(A) tail, did not accumulate Mos and were unable to activate MAPK. We conclude that an active MPF in rat oocytes is necessary for c-mos mRNA polyadenylation and Mos translation.
J Mol Endocrinol 2004 Aug
PMID:Selective degradation of cyclin B1 mRNA in rat oocytes by RNA interference (RNAi). 1529 44

Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes.
Mol Reprod Dev 2004 Dec
PMID:Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activity. 1545 12

Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.
Mol Cancer Ther 2004 Oct
PMID:G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. 1548 90

Anticancer effects of the dietary isothiocyanate sulforaphane were investigated in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Sulforaphane-treated cells accumulated in metaphase as determined by flow cytometry [4C DNA content, cyclin A(-), cyclin B1(+), and phospho-histone H3 (Ser(10))(+)]. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-8, loss of mitochondrial membrane potential, and loss of plasma membrane integrity. The initial detection of caspase-3 cleavage occurring in G(2)-M arrest was independent of a change in phospho-cdc2 (Tyr(15)) protein; consequently, sulforaphane treatment combined with UCN-01 had no significant impact on cellular toxicity. Incubations at higher sulforaphane doses (>10 micromol/L) resulted in cleavage of caspase-3 in the G(1) subpopulation, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Cellular toxicity in MIA PaCa-2, and to a greater extent in PANC-1, was positively correlated with a decrease in cellular glutathione levels, whereas sustained increases in glutathione observed in MIA PaCa-2 cells or the simultaneous incubation with N-acetyl-L-cysteine in PANC-1 cells were associated with resistance to sulforaphane-induced apoptosis. Daily sulforaphane i.p. injections (375 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with PANC-1 s.c. tumors resulted in a decrease of mean tumor volume by 40% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have activity in established pancreatic cancer.
Mol Cancer Ther 2004 Oct
PMID:The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. 1548 91

It is well accepted that homeostasis of continuously renewing adult tissues, such as the epidermis, is maintained by somatic stem cells. These are undifferentiated, self-renewing cells, which also produce daughter transit amplifying (TA) cells that make up the majority of the proliferative cell population in the tissues. Although still proliferative in nature, it is thought that TA cells can undergo only a finite number of cell divisions before they commit to leave the proliferative compartment and move toward terminal differentiation. Stem cells, on the other hand, have been assumed to persist throughout the lifetime of the organism. We directly demonstrated the presence of putative stem cells in the proliferative compartment of murine epithelia in 1981 when we identified a small population of label-retaining cells (LRCs) in mouse stratified squamous epithelia. Since then, we have developed the method described here to isolate this population of epidermal stem cells (EpiSC). We showed that EpiSC are all keratin 14+ and thus of keratinocyte origin and not of mesenchymal or hematopoietic origin. We have also demonstrated that EpiSC can regenerate the epidermis, that they can permanently express a recombinant gene in the regenerated tissue, and that while the majority of EpiSC reside in the G1 phase of the cell cycle, they are not held out of the cell cycle, that they express proliferating genes and the mitotic cyclin B1 protein. Recently, we have shown that EpiSC have the capacity to alter their cell fate in vivo if placed into stress environments, i.e. after irradiation or wounding or when injected into a developing blastocyst environment. Thus being able to isolate EpiSC is critical for testing their use in cell and gene therapy.
Methods Mol Biol 2005
PMID:Isolation, characterization, and culture of epithelial stem cells. 1550 74


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