Expression Of Mitotic Cyclins Research Articles

Progress in understanding the molecular functions of DDX3Y (DBY) in male germ cell development and maintenance.

Human DDX3 paralogs are housed on the X chromosome (DDX3X) as well as in the non- recombining region Yq11 of the Y-chromosome (DDX3Y or DBY). A gene encoding RNA helicase DDX3Y is located in the AZoospermia Factor a (AZFa) region of the Y-chromosome and expressed only in male germ cells. Deletions encompassing the DDX3Y gene lead to azoospermia and cause Sertoli Cell-Only Syndrome (SCOS) in humans. SCOS is characterized by a complete germ cell lack with preservation of somatic Sertoli cells. This review summarizes current advances in the study of DDX3Y functions in maintenance and development of early male germ cells. Data obtained from a mouse xenotransplantation model reveals that DDX3Y expression is enough to drive germ cell differentiation of AZFa-deleted human induced pluripotent stem cells (iPSCs) and for activation of the specific set of germline developmental genes. Results achieved using the testes of Drosophila demonstrate that DDX3Y homolog Belle is required cell-autonomously for mitotic progression and survival of germline stem cells and spermatogonia as the upstream regulator of mitotic cyclin expression.

Expression of mitotic cyclins in higher plants: transcriptional and proteolytic regulation

Cyclins control progression through the specific phases of the cell cycle by binding to and activating cyclin-dependent kinases (CDKs), which are key cell cycle regulators. Most cyclins with mitotic functions are known to be expressed in a cell-cycle-dependent manner, which in turn leads to the cell-cycle-stage-specific functions of CDKs. To achieve oscillating cyclin expression during the cell cycle, both transcriptional regulation and post-transcriptional regulation are required. In higher plants, mitotic cyclins of the CYCA1, CYCB1, and CYCB2 classes, which are expressed in the late G2 phase and during mitosis, have a cis-acting element called MSA in common that acts as a phase-specific enhancer element. A subclass of R1R2R3-Myb transcription factors bind to this element and activate transcription in a phase-dependent manner. At the post-transcriptional level, the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase plays a central role in the proteolytic regulation of mitotic cyclins. As well as the two conserved APC/C activators CDC20 and CCS52, two plant-specific inhibitors of APC/C, GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and ULTRAVIOLET-B-INSENSITIVE4 (UVI4), have recently been identified. Genetic interactions between R1R2R3-Myb and APC/C inhibitors suggest that the balance between positive (transcription) regulation and negative (protein degradation) regulation may determine the cellular concentrations of mitotic cyclins, which is critical not only for cell division, but also for cell growth that is associated with increase of cellular ploidy.

Forced Mitotic Entry of S-Phase Cells as a Therapeutic Strategy Induced by Inhibition of WEE1

Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase-arrested cells directly into mitosis without completing DNA synthesis, resulting in highly abnormal mitoses characterized by dispersed chromosomes and disorganized bipolar spindles, ultimately resulting in mitotic exit with gross micronuclei formation and apoptosis. This mechanism of cell death is shared by CHK1 inhibitors, and combined WEE1 and CHK1 inhibition forces mitotic entry from S-phase in the absence of chemotherapy. We show that p53/p21 inactivation combined with high expression of mitotic cyclins and EZH2 predispose to mitotic entry during S-phase with cells reliant on WEE1 to prevent premature cyclin-dependent kinase (CDK)1 activation. These features are characteristic of aggressive breast, and other, cancers for which WEE1 inhibitor combinations represent a promising targeted therapy.

Translational Repression of Cyclin E Prevents Precocious Mitosis and Embryonic Gene Activation during C. elegans Meiosis

Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, we demonstrate that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. We describe a mechanism that prevents precocious mitosis in germ cells undergoing meiosis, propose that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and provide a paradigm for the possible origin of human teratomas.

Bruno Inhibits the Expression of Mitotic Cyclins during the Prophase I Meiotic Arrest of Drosophila Oocytes

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. The maintenance of the prophase I arrest requires the silencing of Cdk1 activity. Drosophila oocytes inhibit the accumulation of the mitotic cyclins, the activating subunits of Cdk1, via a poorly defined posttranscriptional mechanism. Here, we demonstrate that the translational repressor Bruno binds the 3' UTR and inhibits the translation of the mitotic cyclin Cyclin A during prophase of meiosis I. In the absence of Bruno, ovarian cysts enter meiosis but rapidly accumulate high levels of mitotic cyclins and return to the mitotic cycle. Based on our results, we propose a model in which Bruno and the anaphase-promoting complex/cyclosome act together to restrict the accumulation of the mitotic cyclins, and thus Cdk1 activity, during the prophase I arrest of the Drosophila oocyte.

Progression through Meiosis I and Meiosis II in Arabidopsis Anthers Is Regulated by an A-Type Cyclin Predominately Expressed in Prophase I

Meiosis is often described as a special case of cell division since it differs from mitosis in having two nuclear divisions without an intervening S-phase. It will be of great interest to uncover what molecular mechanisms underlie these special features of meiosis. We previously reported that the tardy asynchronous meiosis (tam) mutant of Arabidopsis (Arabidopsis thaliana) is slower in cell cycle progression in male meiosis. Here we report that TAM encodes the A-type cyclin, CYCA1;2. The point mutation in tam replaced a conserved threonine with an isoleucine in the linker region between the alpha4 and alpha5 helices of the first cyclin fold. By studying the dynamics of a CYCA1;2-green fluorescent protein fusion protein under the control of the CYCA1;2 promoter, we found that the fusion protein was most abundant at pachytene, but was undetectable from late prophase I until telophase II. Nonetheless, cell cycle progression in tam was delayed in both pachytene and meiosis II. We conclude either that the CYCA1;2 produced in prophase I indirectly regulates meiosis II progression, or that a very low level of CYCA1;2 directly regulates meiosis II progression. Either of these scenarios is a deviation from the typical mode of action of mitotic cyclins in mitosis and meiosis I, in which each nuclear division is coupled with a peak of expression of mitotic cyclins.

Cellular aging of mitochondrial DNA-depleted cells

We have reported that mitochondrial DNA-depleted ρ 0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ 0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ 0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ 0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ 0 cells. These results support the mitochondrial theory of aging, and suggest that ρ 0 cells could serve as an in vitro model for aged cells.

Differential expression of cell cycle regulatory molecules and evidence for a “cyclin switch” during progression of prostate cancer

Deregulation of the cell cycle can be viewed as both cause and consequence of cancer. Cyclin expression regulates progression through the cell cycle and although some cyclins have been examined in prostate cancer, the spatial and temporal changes in expression of these molecules during progression of autochthonous disease has not been fully explored. Expression patterns of cyclins and cyclin dependent kinases during the different stages of progression in the spontaneous autochthonous TRAMP model were examined by RNAse protection assay, Western blot analysis, and immunohistochemistry. Differential expression of cell cycle regulatory molecules was observed during prostate cancer progression. Levels of the D-type cyclins decreased during progression while expression of cyclin E increased both at the mRNA and protein levels. The level of cyclin A and cyclin B expression increased beginning in early stage tumors and continued to increase throughout progression. The levels of cyclin dependent kinases did not change substantially during progression of the TRAMP model. The spatial and temporal pattern of mitotic cyclin expression during prostate cancer progression suggests that these molecules represent potential therapeutic targets. The differential expression of D-type cyclins may have implications with respect to androgen receptor mediated gene expression.

Inhibition of mitotic cyclin B and cdc2 kinase activity by selenomethionine in synchronized colon cancer cells.

Selenomethionine (SeMet), an organic selenium compound, has been demonstrated to have significant chemopreventive activity. However, the mechanism of action of SeMet has yet to be identified. Previously, our laboratory found that treatment of cells with SeMet induced apoptosis and altered the cell cycle. These observations have led to further analysis of the cell cycle effects of SeMet in colon cancer cells. Synchronized HCT 116 colon cancer cells treated with 100 microM SeMet for 66 h were found to have a transient delay in G2/M phase of the cell cycle at 18 and 24 h after treatment. With this was observed an inhibition of cell growth. Coincidentally with this delay was a decrease in mitotic cyclin B RNA expression at 18 h after treatment. In addition, the cdc2 kinase activity of HCT 116 cells was decreased at 18 h. Morphological studies indicate an increase in the number of treated cells (45%) undergoing apoptosis at 66 h compared to control cells (27%). These studies demonstrate that modulation of mitotic cyclin expression and cdc2 kinase activity play a role in the ability of SeMet to inhibit tumor cell growth. A consequence of this prolonged arrest is apoptosis.

Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila.

Entry into S phase of the mitotic cell cycle is normally strictly dependent on progression through the preceding M phase. In contrast, during endoreduplication, which accompanies post-mitotic cell growth in many organisms, repeated S phases occur without intervening M phases. Upon transition from mitotic to endoreduplication cycles in Drosophila embryos, expression of the mitotic cyclins A, B and B3 is terminated and Cyclin E expression is changed from a continuous into a periodic mode [1-3]. Here, we address whether these changes in cyclin expression are required for endoreduplication by continuously expressing Cyclin A, B, B3 or E in the salivary glands of Drosophila throughout late embryonic and larval development. With the exception of Cyclin A, expression of which inhibited endoreduplication effectively but only in a few, apparently randomly distributed, cells of the salivary gland, mitotic cyclin expression was found to have no effect. In contrast, Cyclin E expression resulted in a striking inhibition of endoreduplication and growth, preceded initially by an ectopic S phase occurring just after the onset of ectopic Cyclin E expression. This observation is consistent with our previous findings that Cyclin E is required, and pulses of ectopic expression are sufficient, for triggering endoreduplication S phases [4]. Our results indicate that Cyclin E activity, which triggers DNA replication, needs to be down-regulated to allow a subsequent S phase in vivo.

Dynamics of cell cycle regulators: artifact-free analysis by recultivation of cells synchronized by centrifugal elutriation.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

P34(cdc2) and mitotic cyclin expression in the developing quail neuroretina.

After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become postmitotic between E7 and E8. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2 and of two activators of p34(cdc2): cyclin A and cyclin B2 in the developing neuroretina. We report that these three proteins are downregulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also report, using an immunohistochemical approach, that p34(cdc2) downregulation is correlated with the appearance of the microtubule-associated protein tau. These results strongly suggest that inhibition of cdc2 gene expression is closely linked to the achievement of terminal differentiation in neurons. However, we also show that postmitotic ganglion cells precursors begin to synthesize the early neuronal differentiation marker beta3-tubulin while p34(cdc2) is still detectable in these cells, suggesting that p34(cdc2) or a closely related kinase could play a role in some "young" postmitotic neurons.

The effects of radiation on the expression of mitotic cyclins

The effects of radiation on the expression of mitotic cyclins