The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex

Neuronal precursors remain in the proliferative zone of the developing mammalian neocortex until after they have undergone neuronal differentiation and cell cycle arrest. The newborn neurons then migrate away from the proliferative zone and enter the cortical plate. The molecules that coordinate migration with neuronal differentiation have been unclear. We have proposed in this study that the cdk inhibitors p57 and p27 play a role in this coordination. We have found that p57 and p27 mRNA increase upon neuronal differentiation of neocortical neuroepithelial cells. Knockdown of p57 by RNA interference resulted in a significant delay in the migration of neurons that entered the cortical plate but did not affect neuronal differentiation. Knockdown of p27 also inhibits neuronal migration in the intermediate zone as well as in the cortical plate, as reported by others. We have also found that knockdown of p27 increases p57 mRNA levels. These results suggest that both p57 and p27 play essential roles in neuronal migration and may, in concert, coordinate the timing of neuronal differentiation, migration, and possibly cell cycle arrest in neocortical development.     

pRb and the cdks in apoptosis and the cell cycle

Apoptosis is a fundamental biological process present in metazoan cells. Linking apoptosis to the cell cycle machinery provides a mechanism to maintain proper control of cell proliferation in a multicellular organism. pRb and the cyclin-dependent kinases may have dual roles as integral components of the cell cycle and regulators of apoptosis. In many instances manipulation of the cell cycle through these molecules can induce or inhibit apoptosis. Recent studies also identify pRb as a substrate for an apoptotic protease; however, other cell cycle components are not known substrates. While it is clear that many common molecules can affect cell proliferation and cell death, the universality of any one cell cycle molecule in apoptosis has yet to be determined.     

Coupling Cell Cycle Exit,Neuronal Differentiation and Migration in Cortical Neurogenesis

The generation of new neurons in the cerebral cortex requires that progenitor cells leave the cell cycle and activate specific programs of differentiation and migration. Genetic studies have identified some of the molecules controlling these cellular events, but how the different aspects of neurogenesis are integrated into a coherent developmental program remains unclear. One possible mechanism implicates multifunctional proteins that regulate, both cell cycle exit and cell differentiation 1. A prime example is the cyclin-dependent kinase inhibitor p27Kip1, which has recently been shown to function beyond cell cycle regulation and promote both neuronal differentiation and migration of newborn cortical neurons, through distinct and separable mechanisms. p27Kip1 is therefore part of a machinery that couples the multiple events of neurogenesis in the cerebral cortex.     

BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.     

Molecular movements in oocyte patterning and pole cell differentiation.

Central to the differentiation and patterning of the Drosophila oocyte is the asymmetric intracellular localization of numerous mRNA and protein molecules involved in developmental signalling. Recent advances have identified some of the molecules mediating oocyte differentiation, specification of the anterior pole of the embryo, and determination of the embryonic germ line. This work is considered in the context of the classical model of the germ plasm as a cytoplasmic determinant for germ cell formation.     

Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: control of stem cell differentiation by incubation temperatures

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

Inhibition of the cell cycle with chemical inhibitors: a targeted approach

Genetic links between deregulation of the cell cycle and cancer are well established. There have been significant recent developments both in our understanding of the molecular mechanisms that control cell cycle progression and in methods for protein structure determination at atomic resolution. These advances have allowed the rational design of small molecules that modulate the cell cycle by competing for sites of protein-protein or protein-ATP interactions. There is considerable optimism that these compounds, a selection of which are here reviewed, will become clinically significant drugs.     

Potential morphogens involved in pattern formation during Dictyostelium differentiation.

Upon starvation, Dictyostelium amoebae aggregate together and then differentiate into either the stalk or spore cells that, respectively, form the stalk and sorus of the fruiting body. During differentiation, the prestalk and prespore cells become spatially segregated in a clearly defined developmental pattern. Several low molecular weight molecules that influence cell type determination during in vitro differentiation have been identified. The possible role of these molecules as morphogens, responsible for the formation of the developmental pattern, is discussed.     

p53 arrests growth and induces differentiation of v-Myb-transformed monoblasts

Abstract The p53 protein can control cell cycle progression, programmed cell death, and differentiation of many cell types. Ectopic expression of p53 can resume capability of cell cycle arrest, differentiation, and apoptosis in various leukemic cell lines. In this work, we expressed human p53 protein in v-Myb-transformed chicken monoblasts. We found that even this protein possessing only 53% amino acid homology to its avian counterpart can significantly alter morphology and physiology of these cells causing the G2-phase cell cycle arrest and early monocytic differentiation. Our results document that the species-specific differences of the p53 molecules, promoters/enhancers, and co-factors in avian and human cells do not interfere with differentiation- and cell cycle arrest promoting capabilites of the p53 tumor suppressor even in the presence of functional v-Myb oncoprotein. The p53-induced differentiation and cell cycle arrest of v-Myb-transformed monoblasts are not associated with apoptosis suggesting that the p53-driven pathways controlling apoptosis and differentiation/proliferation are independent.     

High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival

Disentangling the complex interactions that govern stem cell fate choices of self-renewal, differentiation, or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632, HA-1077, and H-8 all strongly inhibit the kinases ROCK and PRK2, highlighting the important role of these kinases in EC cell survival. Two molecules, GF109203x and rottlerin, induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells, caused the cell cycle arrest, and repressed the expression of pluripotency-associated genes.     

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.     

Cell cycle and cell-fate determination in Drosophila neural cell lineages

"Normal" development requires a finely tuned equilibrium between cell differentiation and cell proliferation. Important issues in development include whether the cell cycle controls the cell-fate determination and whether cell identity in turn regulates cell-cycle progression. Although, these issues are of general biological relevance, stereotyped Drosophila neural lineages are particularly suited to address these questions and have provided insights into the links between cell-cycle progression and cell-fate specification.     

PMS‐1077, a PAF antagonist,induced differentiation of HL‐60 cells with its novel activity

The cell differentiation‐inducing effect of 2‐N,N‐diethylaminocarbonyloxymethyl‐1 ‐diphenylmethyl‐4‐(3,4,5‐trimethoxybenzoyl) piperazine, hydrochloride (PMS‐1077) was determined in human leukaemic HL‐60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL‐60 cells. After treatment with PMS‐1077, HL‐60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀‐/G₁‐phase. Flow cytometric analysis showed CD11b and CD14 were up‐regulated, whereas CD15 was unaffected. Together with the finding that PMS‐1077‐treated HL‐60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation‐inducing role were determined for PMS‐1077 in HL‐60 cells.     

Cyclosporin A Induces Cardiac Differentiation but Inhibits Hemato-Endothelial Differentiation of P19 Cells

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.     

Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms?

Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.