Key events in the cell cycle, their regulation and organization

The review surveys the studies of molecular genetic mechanisms of the cell cycle control on various eukaryotic models. The major cell cycle phenomena are considered: (1) checkpoints and their role in preserving DNA integrity and fidelity of mitosis, (2) the cell oscillator model, and (3) the role of cyclins in timing of cell division and coordination of mitotic events. The main classes of regulatory proteins involved in the cell cycle are discussed in detail.     

From growth to cell cycle control.

How does a quiescent cell decide to re-enter the cell cycle and start replicating its DNA? What controls cell proliferation? These are fundamental questions that have to be solved in order to understand the mechanisms of oncogenesis. Some recent data have provided clues about how signal transduction pathways may be connected to the cell cycle. A protein kinase cascade starting from the membrane growth factor receptor is thought to be involved in transducing extracellular stimuli to the master switches of the cell cycle control machinery. The recently identified extracellular-signal regulated kinases (ERKs) appear to play an important role in this pathway. Expression of cyclins, which are regulatory subunits of the universal cell cycle oscillator cdc2, may also be controlled through this kinase cascade. The products of tumor suppressor genes Rb and p53 also play an important role in regulating cell proliferation by interfering with the cell cycle pathway. Here, I will review and discuss the importance of these different new results.     

A General Role of Hedgehog in the Regulation of Proliferation

The Hedgehog (Hh) pathway regulates proliferation in a variety of tissues, however its specific effects on the cell cycle are unclear. During retinal proliferation in particular, the role of Hh has been controversial, with studies variably suggesting a stimulatory or an inhibitory effect on proliferation. Our recent data provide an underlying mechanism, which reconciles these different views. We showed that Hh signaling in the retina accelerates the G1 and G2 phases of the cell cycle and then pushes these rapidly dividing cells out of the cell cycle prematurely. From this and other evidence, we propose that Hh converts quiescent retinal stem cells into fast-cycling transient amplifying progenitors that are closer to cell cycle exit and differentiation. This is, we suggest, likely to be a general role of Hh in the nervous system and other tissues. This function of Hh in cell cycle kinetics and cell cycle exit may have implications for tumorigenesis and brain evolution.     

The role of cell cycle-dependent neuropathy target esterase in cell proliferation

Neuropathy target esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G₁ and lowest at G₂/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.     

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.     

Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation.

Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.     

Atypical regulation of a green lineage-specific B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are the main regulators of cell cycle progression in eukaryotes. The role and regulation of canonical CDKs, such as the yeast (Saccharomyces cerevisiae) Cdc2 or plant CDKA, have been extensively characterized. However, the function of the plant-specific CDKB is not as well understood. Besides being involved in cell cycle control, Arabidopsis (Arabidopsis thaliana) CDKB would integrate developmental processes to cell cycle progression. We investigated the role of CDKB in Ostreococcus (Ostreococcus tauri), a unicellular green algae with a minimal set of cell cycle genes. In this primitive alga, at the basis of the green lineage, CDKB has integrated two levels of regulations: It is regulated by Tyr phosphorylation like cdc2/CDKA and at the level of synthesis-like B-type CDKs. Furthermore, Ostreococcus CDKB/cyclin B accounts for the main peak of mitotic activity, and CDKB is able to rescue a yeast cdc28(ts) mutant. By contrast, Ostreococcus CDKA is not regulated by Tyr phosphorylation, and it exhibits a low and steady-state activity from DNA replication to exit of mitosis. This suggests that from a major role in the control of mitosis in green algae, CDKB has evolved in higher plants to assume other functions outside the cell cycle.     

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.     

Unique requirement for Rb/E2F3 in neuronal migration: evidence for cell cycle-independent functions

The cell cycle regulatory retinoblastoma (Rb) protein is a key regulator of neural precursor proliferation; however, its role has been expanded to include a novel cell-autonomous role in mediating neuronal migration. We sought to determine the Rb-interacting factors that mediate both the cell cycle and migration defects. E2F1 and E2F3 are likely Rb-interacting candidates that we have shown to be deregulated in the absence of Rb. Using mice with compound null mutations of Rb and E2F1 or E2F3, we asked to what extent either E2F1 or E2F3 interacts with Rb in neurogenesis. Here, we report that E2F1 and E2F3 are both functionally relevant targets in neural precursor proliferation, cell cycle exit, and laminar patterning. Each also partially mediates the Rb requirement for neuronal survival. Neuronal migration, however, is specifically mediated through E2F3, beyond its role in cell cycle regulation. This study not only outlines overlapping and distinct functions for E2Fs in neurogenesis but also is the first to establish a physiologically relevant role for the Rb/E2F pathway beyond cell cycle regulation in vivo.     

Clusterin and DNA repair: a new function in cancer for a key player in apoptosis and cell cycle control

The glycoprotein clusterin (CLU), has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, while a secretory form (sCLU) is pro-survival. Both forms are implicated in various cell functions, including DNA repair, cell cycle regulation, and apoptotic cell death. CLU expression has been associated with tumorigenesis and the progression of various malignancies. In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms, while simultaneously stimulating energy-expensive cell cycle checkpoints that delay the cell cycle progression to allow more time for DNA repair. This review summarizes our current understanding of the role of clusterin in DNA repair, apoptosis, and cell cycle control and the relevance.     

BCL2 family in DNA damage and cell cycle control

Individual BCL2 family members couple apoptosis regulation and cell cycle control in unique ways. Antiapoptotic BCL2 and BCL-x(L) are antiproliferative by facilitating G0. BAX is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the multi-domain BCL2 family members (MCL-1) and suggest that survival is maintained at the expense of proliferation. The role of BH3-only molecules in cell cycle is more variable. BAD antagonizes both the cell cycle and antiapoptotic functions of BCL2 and BCL-x(L) through BH3 binding. BID has biochemically separable functions in apoptosis and S-phase checkpoint, determined by post-translational modification. p53-induced PUMA is known only to have apoptotic function. Inhibition of apoptosis is oncogenic, whereas promotion of cell cycle arrest is tumor suppressive. Paradoxically, selected BCL2 family members can be both oncogenic and tumor suppressive. Which of the dual functions predominates is lineage specific and context dependent.     

Mathematical modeling of fission yeast Schizosaccharomyces pombe cell cycle: exploring the role of multiple phosphatases

Cell cycle is the central process that regulates growth and division in all eukaryotes. Based on the environmental condition sensed, the cell lies in a resting phase G0 or proceeds through the cyclic cell division process (G1??S??G2??M). These series of events and phase transitions are governed mainly by the highly conserved Cyclin dependent kinases (Cdks) and its positive and negative regulators. The cell cycle regulation of fission yeast Schizosaccharomyces pombe is modeled in this study. The study exploits a detailed molecular interaction map compiled based on the published model and experimental data. There are accumulating evidences about the prominent regulatory role of specific phosphatases in cell cycle regulations. The current study emphasizes the possible role of multiple phosphatases that governs the cell cycle regulation in fission yeast S. pombe. The ability of the model to reproduce the reported regulatory profile for the wild-type and various mutants was verified though simulations.     

Plant cell cycle transitions

Three decades have passed since the first recognition of restriction checkpoints in the plant cell cycle. Although many core cell cycle genes have been cloned, the mechanisms that control the G1-->S and G2-->M transitions in plants have only recently started to be understood. The cyclin-dependent kinases (CDKs) play a central role in the regulation of the cell cycle, and the activity of these kinases is steered by regulatory subunits, the cyclins. The activities of CDK-cyclin complexes are further controlled by an intricate panoply of monitoring mechanisms, which result in oscillating CDK activity during the division cycle. These fluctuations trigger transitions between the different stages of the cell cycle.     

De novo synthesis of glutathione is required for both entry into and progression through the cell cycle

To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH-3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose-dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO-induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit. © 1995 Wiley-Liss, Inc.     

Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein

Hyperoxia induces growth arrest, apoptosis, necrosis, and morphological changes (spreading and adhesion) in various types of cells. The mechanism of hyperoxia-induced cell growth arrest has not been well elucidated, especially in macrophages. One possible mechanism is a role of cell adhesion in hyperoxia-induced cell cycle arrest. To evaluate this finding, macrophages were cultured in normoxia (21% O2) or hyperoxia (95% O2) in adhesion or low adhesion conditions. Incubation of macrophages in hyperoxia induced cell cycle arrest. The hyperoxia-induced cell cycle arrest was prevented by low adhesion conditions. To evaluate pathways potentially involved in hyperoxia-induced growth arrest, we measured extracellular regulated kinase and retinoblastoma protein activation and p21Cip1 and p53 accumulation. Hyperoxia strongly induced activation of extracellular regulated kinase and retinoblastoma protein as well as up-regulation of p21Cip1. These effects of hyperoxia were attenuated under low adhesion conditions, suggesting a role for integrin-dependent signaling. The induction of p21Cip1 and activation of retinoblastoma protein occurred via a p53-independent mechanism. These results suggest that adhesion-dependent pathways are required for hyperoxia-induced cell cycle arrest in macrophages.     

CELL CYCLE REGULATORS IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell cycle in many dinoflagellates, and may play a rate limiting role in bloom formation. Diel phasing of the cell cycle in the Florida red tide dinoflagellate, Gymnodinium breve Davis was previously described in our laboratory. In cultures grown on a 16:8 light:dark cycle, S-phase began 6–8 h into the light phase, and mitosis followed 12–14 h later. The dark/light "dawn" transition was found to provide the diel cue that serves to entrain the G. breve cell cycle. However the cell cycle mechanisms and regulators acted upon by this cue are poorly understood in dinoflagellates. The cell cycle regulatory complex, CDK1-cyclinB, is therefore currently being investigated. Cyclin dependent kinase (CDK) was first identified in G. breve using two approaches: (1) identification of a 34 kDa protein immunoreactive to an antibody raised against a conserved amino acid sequence unique to the CDK protein family (PSTAIR) and (2) inhibition of the cell cycle by olomoucine, a selective CDK inhibitor. Several approaches are currently being employed in order to describe its partner, cyclin B: (1) PCR on genomic DNA with primers deduced from known cyclin box sequences, (2) G. breve expression library screening with an antibody raised against the fission yeast cyclin B (3) western blot analysis on whole protein extracts and cyclin B immunoprecipitated proteins. Current work focuses on the differential expression of the cyclin B homologue in G. breve during its cell cycle and its relation to diel cycle control.     

From START to FINISH: The Influence of Osmotic Stress on the Cell Cycle

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes.     

ESTIMATION OF THE MEAN AND VARIANCE OF CYCLE TIMES IN CINEMICROGRAPHICALLY RECORDED CELL POPULATIONS DURING BALANCED EXPONENTIAL GROWTH

The mathematical and statistical problem of estimating the mean and variance of cell cycle times from cinemicrographically observed durations until mitosis (cytokinesis at late anaphase), disintegration, collision (cells superimposing each other) or emigration (moving out of the field of vision) of randomly chosen individual cells in a population in balanced exponential growth is treated. The resulting formulae are simple and considerably reduce the average cell observation time. They are applied to two normal human foetal cell lines and their SV40-transformed counterparts. One result is that the latter seem to have longer cycle times in spite of their shorter doubling times. Another result is that the cell mobility seems somewhat increased in the transformed populations. This corroborates earlier findings, indicating that the extent of cell loss, rather than the length of cycle times, may play a decisive role for the (net) doubling time of cultivated cell populations.