Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons--a function that could become impaired in Alzheimer's and other neurodegenerative diseases.     

A tale of terminal differentiation: IKKα, the master keratinocyte regulator

Keratinocyte differentiation is the process of cellular maturation from a mitotic state to a terminally differentiated state during which skin builds up a tough yet soft skin barrier to protect the body. Its irreversibility also allows the shedding of excessive keratinocytes, thereby maintaining skin homeostasis and preventing skin diseases. Although the entire journey of keratinocyte differentiation is intricate and not well understood, it is known that Ras is able to block keratinocyte terminal differentiation and instead induce keratinocyte proliferation and transformation. It appears that uncontrolled proliferation actually interrupts differentiation.

However, it has been unclear whether there are any innate surveillants that would be able to induce terminal differentiation by antagonizing excessive mitotic activities. Inhibitor of nuclear factor κB kinase-α (IKKα, previously known as Chuk) emerges as a master regulator in the coordinative control of keratinocyte differentiation and proliferation and as a major tumor suppressor in human and mouse skin squamous cell carcinomas. IKKα does so largely by integrating into the epidermal growth factor receptor (EGFR)/Ras/extracellular signal-regulated kinase (Erk)/EGFR ligand pathways during mitosis and differentiation. We discuss these findings herein to extend our understanding of how IKKα-mediated terminal differentiation serves as an innate surveillant in skin.     

Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two-dimensional gel electrophoresis to evaluate changes in specific polypeptides that are induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.     

Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier

The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington''s classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28. This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process.     

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.     

Mitotic cycle reactivation in terminally differentiated cells by adenovirus infection

Different cell types (e.g., neurons, skeletal and heart myocytes, adipocytes, keratinocytes) undergo terminal differentiation, in which acquisition of specialized functions entails definitive withdrawal from the cell cycle. Such cells are distinct from quiescent (reversibly growth-arrested) cells, such as contact-inhibited fibroblasts. Terminally differentiated cells can not be induced to proliferate by means of growth factor stimulation or transduction of cellular oncogenes. An important first step toward defining the molecular basis for such unresponsiveness is to find a practical means to overcome the proliferative block. Furthermore, determining whether terminally differentiated, postmitotic cells still retain a potential competence for proliferation that can be reactivated would have important theoretical and practical implications. To address these questions, we exploited the properties of adenoviruses. These viruses can infect postmitotic cells and express E1A, a powerful activator of proliferation in reversibly growth-arrested cells. We infected terminally differentiated skeletal muscle cells and adipocytes with human adenovirus type 5 or 12, obtaining full reentry into the cell cycle, including DNA synthesis, mitosis, cytokinesis, and extended proliferation. Similar results were obtained with established cell lines and primary cells belonging to several species, from quail to humans. Genetic analysis indicated that the smaller splice product of E1A, E1A 12S, is sufficient to induce cell cycle reactivation in otherwise permanently nonmitotic cells. These results demonstrate that terminally differentiated cells retain proliferative potential and establish adenovirus as a convenient and powerful means to force such cells to reenter the cell cycle. © 1995 Wiley-Liss, Inc.     

Expression of Epstein-Barr viral capsid, complement fixing, and nuclear antigens in stationary and exponential phase cultures

Three continuous lymphoblastoid cell lines, 2 productive of nucleocapsids and 1 nonproductive line, were studied for their content of Epstein-Barr viral (EBV) antigens during transition from stationary to logarithmic phase growth. As a preliminary step, viable cells were separated from degenerating ones in discontinuous gradients of serum albumin. Viral capsid antigens were found in both living and dead cells of the 2 producer lines; however, complement fixing (CF) antigens and nuclear antigen were detected only in viable cell subpopulations. The content of antigen detectable in extracts of viable cells by complement fixation remained constant in replicating and resting cultures; further, all viable cells of the 3 lines demonstrated intranuclear antigen by anticomplement immunofluorescence in all stages of growth. In contrast, the proportion of cells with viral capsid antigen in the producer lines increased 7- to 24-fold following entry of resting populations into the phase of exponential growth.The results suggest that expression of viral capsid antigens is discontinuous and is initiated in response to events in log phase, possibly DNA synthesis or mitosis. Expression of the complement fixing and nuclear antigens in continuous in viable cells. These findings emphasize the intimate relationship of the CF and nuclear antigen to the transformed state and suggest that study of this antigen complex will shed light on the mechanisms of lymphocyte transformation by EBV.     

Differences in centromere positioning of cycling and postmitotic human cell types

Centromere positioning in human cell nuclei was traced in non-cycling peripheral blood lymphocytes (G0) and in terminally differentiated monocytes, as well as in cycling phytohemagglutinin-stimulated lymphocytes, diploid lymphoblastoid cells, normal fibroblasts, and neuroblastoma SH-EP cells using immunostaining of kinetochores, confocal microscopy and three-dimensional image analysis. Cell cycle stages were identified for each individual cell by a combination of replication labeling with 5-bromo-2-deoxyuridine and immunostaining of pKi67. We demonstrate that the behavior of centromeres is similar in all cell types studied: a large fraction of centromeres are in the nuclear interior during early G1; in late G1 and early S phase, centromeres shift to the nuclear periphery and fuse in clusters. Peripheral location and clustering of centromeres are most pronounced in non-cycling cells (G0) and terminally differentiated monocytes. In late S and G2, centromeres partially decluster and migrate towards the nuclear interior. In the rather flat nuclei of adherently growing fibroblasts and neuroblastoma cells, kinetochores showed asymmetrical distributions with preferential kinetochore location close either to the bottom side of the nucleus (adjacent to the growth surface) or to the nuclear upper side. This asymmetrical distribution of centromeres is considered to be a consequence of chromosome arrangement in anaphase rosettes.     

Statin expression associated with terminally differentiating and postreplicative lens epithelial cells

The expression of a nuclear (57 kDa) protein statin has been previously characterized as a specific marker of quiescent or senescent aging human fibroblasts in vitro. In these studies we have shown that the expression of statin is associated specifically with the postreplicative and terminally differentiating lens epithelial cell. By monitoring the synthesis of specific lens crystallin proteins, and the morphological and cellular changes associated with this differentiated system, we have demonstrated a close correlation between statin expression and cell commitment to the G0 nonreplicative cell cycle state.     

Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant

Heterokaryons are the product of cell fusion without subsequent nuclear or chromosome loss. Decades of research using Sendai-virus or polyethylene glycol (PEG)-mediated fusion in tissue culture showed that the terminally differentiated state of a cell could be altered. But whether stable non-dividing heterokaryons could occur in animals has remained unclear. Here, we show that green fluorescent protein (GFP)-positive bone-marrow-derived cells (BMDCs) contribute to adult mouse Purkinje neurons through cell fusion. The formation of heterokaryons increases in a linear manner over 1.5 years and seems to be stable. The dominant Purkinje neurons caused the BMDC nuclei within the resulting heterokaryons to enlarge, exhibit dispersed chromatin and activate a Purkinje neuron-specific transgene, L7-GFP. The observed reprogrammed heterokaryons that form in brain may provide insights into gene regulation associated with cell-fate plasticity.     

Reconstitution of Cyclin D1-Associated Kinase Activity Drives Terminally Differentiated Cells into the Cell Cycle

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.     

Nonterminally differentiated cells express decreased growth factor responsiveness

In 3T3 T mesenchymal stem cells, at least four types of biological states exist that can mediate the control of cell differentiation and/or proliferation. These include the predifferentiation growth arrest state, the nonterminal differentiation state, the terminal differentiation state, and a growth arrest state induced by growth factor/serum deficiency. The current studies were performed to investigate the relative mitogenic responsiveness of cells at these four states and specifically to determine if nonterminally differentiated cells show decreased responsiveness to specific mitogens. Twenty-five different serum, plasma, and growth factor combinations were evaluated. The results show that undifferentiated, growth-arrested cells are highly responsive to numerous mitogens and that by definition terminally differentiated cells are not responsive to any mitogens. In contrast, nonterminally differentiated cells demonstrate a unique pattern of mitogenic responsiveness. Whereas nonterminally differentiated cells can be stimulated to proliferate by high concentrations of serum or plasma supplemented with growth factors, they cannot be stimulated to proliferate by combinations of multiple purified growth factors. These results suggest that the process of nonterminal differentiation is associated with a significant change in factors/cofactors required to stimulate cell proliferation and that these factors/cofactors are present in plasma.     

Adult sertoli cells are not terminally differentiated in the Djungarian hamster: effect of FSH on proliferation and junction protein organization

Sertoli cell number is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the seasonal breeding adult Djungarian hamster (Phodopus sungorus) model challenged this assertion by demonstrating a decrease in Sertoli cell number after gonadotropin depletion and a return to control levels following 7 days of FSH replacement. The present study aimed to determine whether adult Sertoli cells are terminally differentiated using known characteristics of cellular differentiation, including proliferation, junction protein localization, and expression of particular maturational markers, in the Djungarian hamster model. Adult long-day (LD) photoperiod (16L:8D) hamsters were exposed to short-day (SD) photoperiod (8L:16D) for 11 wk to suppress gonadotropins and then received exogenous FSH for up to 10 days. Sertoli cell proliferation was assessed by immunofluorescence by the colocalization of GATA4 and proliferating cell nuclear antigen and quantified by stereology. Markers of Sertoli cell maturation (immature, cytokeratin 18 [KRT18]; mature, GATA1) and junction proteins (actin, espin, claudin 11 [CLDN11], and tight junction protein 1 [TJP1, also known as ZO-1]) also were localized using confocal immunofluorescence. In response to FSH treatment, proliferation was upregulated within 2 days compared with SD controls (90% vs. 0.2%, P < 0.001) and declined gradually thereafter. In LD hamsters, junction proteins colocalized at the basal aspect of Sertoli cells, consistent with inter-Sertoli cell junctions, and were disordered within the Sertoli cell cytoplasm in SD animals. Exogenous FSH treatment promptly restored localization of these junction markers to the LD phenotype. Protein markers of maturity remain consistent with those of adult Sertoli cells. It is concluded that adult Sertoli cells are not terminally differentiated in the Djungarian hamster and that FSH plays an important role in governing the differentiation process. It is proposed that Sertoli cells can enter a transitional state, exhibiting features common to both undifferentiated and differentiated Sertoli cells.     

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line

A permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotube-like giant cells neither incorporated 3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.     

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.