A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

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.     

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.     

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 diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage

In the Drosophila bristle lineage, five differentiated cells arise from a precursor cell after a rapid sequence of asymmetric cell divisions (one every 2 hours). We show that, in mitotic cells, this rapid cadence of cell divisions is associated with cell cycles essentially devoid of the G1-phase. This feature is due to the expression of Cyclin E that precedes each cell division, and the differential expression of the S-transition negative regulator, Dacapo. Thus, apart from endocycles (G/S), which occurred in two out of five terminal cells, two other cell cycles coexist in this lineage: (1) an atypical cell cycle (S/G2/M), in which the S-phase is initiated during the preceding telophase; and (2) a canonical cell cycle (G1/S/G2/M) with a brief G1 phase. These two types of cell cycle result from either the absence or very transient expression of Dap, respectively. Finally, we show that the fate determinant factor, Tramtrack, downregulates Cyclin E expression and is probably involved in the exit of the cells from the cell cycle.     

Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.     

Synthesis, cytotoxic activities and cell cycle arrest profiles of half-sandwich N-sulfonamide based dithio-o-carborane metal complexes

Two half-sandwich cobalt and rhodium complexes 2a and 2b with combination of carborane and N-Sulfonamide were synthesized and fully characterized by NMR spectroscopy, mass spectrometry, elemental analysis as well as X-ray crystallography. In an in vitro cytotoxicity assay toward the non-small cell lung cancer cell lines of A549 and NCI-H460, 2b showed the stronger activity than 2a, which was confirmed by the morphological test. Mechanistic studies for 2b showed that inhibition of NSCLC cell growth was mediated by G0/G1 cell cycle arrested without the significant apoptosis induction. Furthermore, 2b altered the mRNA levels of CCND1, CCNE1 and PCNA, which were known to control G0/G1 phase of the cell cycle. Our western blot analysis also showed that 2b-induced G0/G1 cell cycle arrest was mediated through the decreased expression of cyclin D1, cyclin E1 and PCNA.     

Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina

The laminar arrays of distinct cell types in the vertebrate retina are built by a histogenic process in which cell fate is correlated with birth order. To explore this co-ordination mechanistically, we altered the relative timing of cell cycle exit in the developing Xenopus retina and asked whether this affected the activity of neural determinants. We found that Xath5, a bHLH proneural gene that promotes retinal ganglion cell (RGC) fate, ( Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997) Neuron 19, 981-994), does not cause these cells to be born prematurely. To drive cells out of the cell cycle early, therefore, we misexpressed the cyclin kinase inhibitor, p27Xic1. We found that early cell cycle exit potentiates the ability of Xath5 to promote RGC fate. Conversely, the cell cycle activator, cyclin E1, which inhibits cell cycle exit, biases Xath5-expressing cells toward later neuronal fates. We found that Notch activation in this system caused cells to exit the cell cycle prematurely, and when it is misexpressed with Xath5, it also potentiates the induction of RGCs. The potentiation is counteracted by co-expression of cyclin E1. These results suggest a model of histogenesis in which the activity of factors that promote early cell cycle exit enhances the activity of factors that promote early cellular fates.     

p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina

A precise balance between proliferation and differentiation must be maintained during retinal development to obtain the correct proportion of each of the seven cell types found in the adult tissue. Cyclin kinase inhibitors can regulate cell cycle exit coincident with induction of differentiation programs during development. We have found that the p57(Kip2) cyclin kinase inhibitor is upregulated during G(1)/G(0) in a subset of retinal progenitor cells exiting the cell cycle between embryonic day 14.5 and 16.5 of mouse development. Retroviral mediated overexpression of p57(Kip2) in embryonic retinal progenitor cells led to premature cell cycle exit. Retinae from mice lacking p57(Kip2) exhibited inappropriate S-phase entry and apoptotic nuclei were found in the region where p57(Kip2) is normally expressed. Apoptosis precisely compensated for the inappropriate proliferation in the p57(Kip2)-deficient retinae to preserve the correct proportion of the major retinal cell types. Postnatally, p57(Kip2) was found to be expressed in a novel subpopulation of amacrine interneurons. At this stage, p57(Kip2 )did not regulate proliferation. However, perhaps reflecting its role during this late stage of development, animals lacking p57(Kip2) showed an alteration in amacrine subpopulations. p57(Kip2) is the first gene to be implicated as a regulator of amacrine subtype/subpopulation development. Consequently, we propose that p57(Kip2) has two roles during retinal development, acting first as a cyclin kinase inhibitor in mitotic progenitor cells, and then playing a distinct role in neuronal differentiation.     

Rac1 inhibits myogenic differentiation by preventing the complete withdrawal of myoblasts from the cell cycle

The small GTPase protein Rac1 is involved in a wide range of biological processes, yet its role in cell differentiation is mostly unknown. Here we show that Rac1 activity is high in proliferating myoblasts and decreases during the differentiation process. To analyze the involvement of Rac1 in muscle differentiation, different forms of the protein were expressed in muscle cells. A constitutively activated form of Rac1 (Rac1Q61L) inhibited the activity of MyoD in promoting muscle differentiation, whereas a dominant negative form of Rac1 (Rac1T17N) induced the activity of MyoD in promoting muscle differentiation. Expression of Rac1T17N imposed myogenic differentiation on myoblasts growing under mitogenic conditions. In inquiring whether Rac1 affected the withdrawal of myoblasts from the cell cycle, we analyzed the expression of cyclin D1 and p21(WAF1) and the phosphorylation state of the retinoblastoma protein. According to these markers and bromodeoxyuridine incorporation, C2 myoblasts expressing Rac1T17N exited the cell cycle earlier than control C2 cells. Myoblasts expressing Rac1Q61L did not permanently withdraw from the cell cycle. An indication of the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in Rac1-mediated myoblast proliferation was obtained by the use of MAPK kinase inhibitors U0126 and PD098059. These inhibitors arrested C2-Rac1Q61L cell cycling. Taken together, our results show that Rac1 activation interferes with myoblast exit from the cell cycle via or in concert with the MAPK pathway.     

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. We examined the process of cell cycle exit in Drosophila wings and eyes and discovered that cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation.