Cell Cycle Events Research Articles

Spotlight on Volasertib: Preclinical and Clinical Evaluation of a Promising Plk1 Inhibitor.

Considering the important side effects of conventional microtubule targeting agents, more and more research focuses on regulatory proteins for the development of mitosis-specific agents. Polo-like kinase 1 (Plk1), a master regulator of several cell cycle events, has arisen as an intriguing target in this research field. The observed overexpression of Plk1 in a broad range of human malignancies has given rise to the development of several potent and specific small molecule inhibitors targeting the kinase. In this review, we focus on volasertib (BI6727), the lead agent in category of Plk1 inhibitors at the moment. Numerous preclinical experiments have demonstrated that BI6727 is highly active across a variety of carcinoma cell lines, and the inhibitor has been reported to induce tumor regression in several xenograft models. Moreover, volasertib has shown clinical efficacy in multiple tumor types. As a result, Food and Drug Administration (FDA) has recently awarded volasertib the Breakthrough Therapy status after significant benefit was observed in acute myeloid leukemia (AML) patients treated with the Plk1 inhibitor. Here, we discuss both preclinical and clinical data available for volasertib administered as monotherapy or in combination with other anticancer therapies in a broad range of tumor types.

A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues.

All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation.

Identification of genes that are essential to restrict genome duplication to once per cell division.

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription.

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression.

Effect of ATRA on the expression of HOXA5 gene in K562 cells and its relationship with cell cycle and apoptosis.

Leukemia is the most common malignant disease in children with high incidence and mortality rates, and a poor treatment effect. The aim of the present study was to examine the changes in the expression of homeobox (Hox) A5 gene and its relationship with cell cycle and apoptosis through the intervention of human K562 myeloid leukemia cell line by all-trans retinoic acid (ATRA), to analyze the role of HOXA5 in the pathogenesis and development process of myeloid leukemia. The optimal concentration of ATRA to be used with K562 cells was determined using a cell counting kit-8 (CCK-8). After 24, 72 and 48 h following treatment of K562 cells with 10 µmol/l ATRA, cell cycle events and apoptosis were measured using flow cytometry. HOXA5 mRNA and protein expression in K562 cells was assessed by RT-PCR and western blot analysis, and the relationship between HOXA5 expression and cell cycle and apoptosis was analyzed. The HOXA5 mRNA and protein expression levels were increased following treatment with ATRA in K562 cells. Apoptosis was increased significantly. The cell cycle was inhibited in G0/G1 phase. Cell proliferation was also inhibited. HOXA5 mRNA and protein expression rates positively correlated with cell apoptosis and the increased percentage and cell cycle of the G0/G1 phase. However, HOXA5 negatively correlated with the reduced percentage of S stage. In conclusion, the expression of HOXA5 in cells was increased following treatment with ATRA in K562 cells, in a time-dependent manner. Additionally, ATRA may inhibit the proliferation of K562 cells and promote apoptosis by upregulating the HOXA5 mRNA and protein expression.

Developments in Mirna Gene Signaling Pathways in Pancreatic Cancer

Pancreatic cancer is a devastating malignancy that ranks as the fourth leading cause of cancer-related deaths worldwide. Dismal prognosis is mainly attributable to limited knowledge of the molecular pathogenesis of the disease. miRNAs have been found to be deregulated in pancreatic cancer, affecting several steps of initiation and aggressiveness of the disease by regulating important signaling pathways, such as the KRAS and Notch pathways. Moreover, the effect of miRNAs on regulating cell cycle events and expression of transcription factors has gained a lot of attention. Recent studies have highlighted the application of miRNAs as biomarkers and therapeutic tools. The current review focuses on latest advances with respect to the roles of miRNAs in pancreatic ductal adenocarcinoma associated signaling pathways and miRNA-based therapeutics.

The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice

The ectopic re-activation of cell cycle in neurons is an early event in the pathogenesis of Alzheimer’s disease (AD), which could lead to synaptic failure and ensuing cognitive deficits before frank neuronal death. Cytostatic drugs that act as cyclin-dependent kinase (CDK) inhibitors have been poorly investigated in animal models of AD. In the present study, we examined the effects of flavopiridol, an inhibitor of CDKs currently used as antineoplastic drug, against cell cycle reactivation and memory loss induced by intracerebroventricular injection of Aß1-42 oligomers in CD1 mice. Cycling neurons, scored as NeuN-positive cells expressing cyclin A, were found both in the frontal cortex and in the hippocampus of Aβ-injected mice, paralleling memory deficits. Starting from three days after Aβ injection, flavopiridol (0.5, 1 and 3mg/kg) was intraperitoneally injected daily, for eleven days. Here we show that a treatment with flavopiridol (0.5 and 1mg/kg) was able to rescue the loss of memory induced by Aβ1-42, and to prevent the occurrence of ectopic cell-cycle events in the mouse frontal cortex and hippocampus. This is the first evidence that a cytostatic drug can prevent cognitive deficits in a non-transgenic animal model of AD.

Single-Cell Analysis of Growth in Budding Yeast and Bacteria Reveals a Common Size Regulation Strategy

To maintain a constant cell size, dividing cells have to coordinate cell-cycle events with cell growth. This coordination has long been supposed to rely on the existence of size thresholds determining cell-cycle progression [1]. In budding yeast, size is controlled at the G1/S transition [2]. In agreement with this hypothesis, the size at birth influences the time spent in G1: smaller cells have a longer G1 period [3]. Nevertheless, even though cells born smaller have a longer G1, the compensation is imperfect and they still bud at smaller cell sizes. In bacteria, several recent studies have shown that the incremental model of size control, in which size is controlled by addition of a constant volume (in contrast to a size threshold), is able to quantitatively explain the experimental data on four different bacterial species [4-7]. Here, we report on experimental results for the budding yeast Saccharomyces cerevisiae, finding, surprisingly, that cell size control in this organism is very well described by the incremental model, suggesting a common strategy for cell size control with bacteria. Additionally, we argue that for S. cerevisiae the "volume increment" is not added from birth to division, but rather between two budding events.

Measuring Activity and Specificity of Protein Phosphatases.

Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation of specific substrates. The molecular determinants for substrate recognition by many phosphatases that function in cell division are still poorly delineated. To understand phosphatase specificity, it is critical to employ methods that monitor the dephosphorylation of individual phosphorylation sites on physiologically relevant substrates. Here, using the cell cycle phosphatase Cdc14 as an example, we describe two methods for studying phosphatase specificity, one using synthetic phosphopeptide substrates and the other using intact phosphoprotein substrates. These methods are useful for targeted characterization of small substrate sets and are also adaptable to large-scale applications for global specificity studies.

Factors that Control Mitotic Spindle Dynamics.

Mitosis is the last phase of the cell cycle and it leads to the formation of two daughter cells with the same genetic information. This process must occurr in a very precise way and this task is essential to preserve genetic stability and to maintain cell viability. Accurate chromosome segregation during mitosis is brought about by an important cellular organelle: the mitotic spindle. This structure is made of microtubules, polymers of alpha and beta tubulin, and it is highly dynamic during the cell cycle: it emanates from two microtubules organizing centers (Spindle Pole Bodies, SPBs, in yeast) that are essential to build a short bipolar spindle, and it undergoes two steps of elongation during anaphase A and anaphase B in order to separate sister chromatids. Several proteins are involved in the control of mitotic spindle dynamics and their activity is tightly coordinated with other cell cycle events and with cell cycle progression.

The Nim1 kinase Gin4 has distinct domains crucial for septin assembly, phospholipid binding and mitotic exit

ABSTRACTIn fungi, the Nim1 protein kinases, such as Gin4, are important regulators of multiple cell cycle events, including the G2–M transition, septin assembly, polarized growth and cytokinesis. Compelling evidence has linked some key functions of Gin4 with the large C-terminal non-kinase region which, however, is poorly defined. By systematically dissecting and functionally characterizing the non-kinase region of Gin4 in the human fungal pathogen Candida albicans, we report the identification of three new domains with distinct functions: a lipid-binding domain (LBD), a septin-binding domain (SBD) and a nucleolus-associating domain (NAD). The LBD and SBD are indispensable for the function of Gin4, and they alone could sufficiently restore septin ring assembly in GIN4-null mutants. The NAD localizes to the periphery of the nucleolus and physically associates with Cdc14, the ultimate effector of the mitotic exit network. Gin4 mutants that lack the NAD are defective in spindle orientation and exit mitosis prematurely. Furthermore, we show that Gin4 is a substrate of Cdc14. These findings provide novel insights into the roles and mechanisms of Nim1 kinases in the regulation of some crucial cell cycle events.

Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis.

Rho-type small GTPases are involved in cytokinesis in various organisms, but their precise roles and regulation remain unclear. Rho proteins function as molecular switches by cycling between the active GTP-bound and inactive GDP-bound states; the GTP-bound proteins in turn interact with their downstream effectors to transmit the signal. Biochemical assays using Rho-binding domains of effector proteins have been used to specifically pull down GTP-bound Rho proteins from cell extracts. Here, we describe the application of such a method in combination with cell-cycle synchronization in the budding yeast Saccharomyces cerevisiae; this approach allows dissection of the activity of Rho1 at different stages of cytokinesis. We also present data showing the importance of caution in interpreting such biochemical data and of comparing to the results obtained with other approaches where possible. The principle of this protocol is also applicable to analyses of other Rho-type GTPases and cell-cycle events.

Role of Computational Modeling in Understanding Cell Cycle Oscillators.

The periodic oscillations in the activity of the cell cycle regulatory program, drives the timely activation of key cell cycle events. Interesting dynamical systems, such as oscillators, have been investigated by various theoretical and computational modeling methods. Thanks to the insights achieved by these modeling efforts we have gained considerable insights about the underlying molecular regulatory networks that can drive cell cycle oscillations. Here we review the basic features and characteristics of biological oscillators, discussing from a computational modeling point of view their specific architectures and the current knowledge about the dynamics that the life evolution selected to drive cell cycle oscillations.

The Quadrato Motor Training model: implications to the treatment of Alzheimer and Parkinson's disease.

The Quadrato Motor Training model: implications to the treatment of Alzheimer and Parkinson's disease.

Aurora-A: an expedition to the pole of the spindle in Xenopus egg extracts.

The aim of this short review is to describe the contribution of Xenopus laevis egg extracts to the discovery and understanding of the regulation and function of the serine/threonine kinase Aurora-A. The power of these extracts to recapitulate cell cycle events makes them a precious tool to decipher complex biological processes at the molecular level, including the mechanisms that affect Aurora-A (post-translational modifications) and mechanisms in which Aurora-A plays a crucial role (bipolar spindle assembly). We focus on the results obtained in cell-free extracts, but we also give an updated overview of Aurora A functions found in other systems.

Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development.

Elutriation for Cell Cycle Synchronization in Fission Yeast.

Cell synchronization is a powerful technique for studying the eukaryotic cell cycle events precisely. The fission yeast is a rod-shaped cell whose growth is coordinated with the cell cycle. Monitoring the cellular growth of fission yeast is a relatively simple way to measure the cell cycle stage of a cell. Here, we describe a detailed method of unperturbed cell synchronization, named centrifugal elutriation, for fission yeast.

In Silico Cell Cycle Predictor for Mammalian Cell Culture Bioreactor Using Agent-Based Modeling Approach

An Agent-based computational modeling approach was used to develop a model to simulate individual mammalian cell behavior and its cycle regulation in response to dynamic bioreactor conditions. The model can be used as an in silico cell cycle predictor when provided with data from ongoing bioreactor runs. Rules were developed to regulate the distinct cell cycle events as well as to apply decision-making at the critical cellular checkpoints. The model was constructed and validated using different sets of experimental cell culture conditions with cell culture parameters measured using a flow cytometer and other instrumentation.

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer.

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.

Create, activate, destroy, repeat: Cdk1 controls proliferation by limiting transcription factor activity.

Progression through the cell cycle is controlled by a network of transcription factors that coordinate gene expression with cell-cycle events. One transcriptional activator in this network in budding yeast is the forkhead protein Hcm1, which controls the expression of genes that are transcribed during S-phase. Hcm1 activity is coordinated with the cell cycle via its regulation by cyclin-dependent kinase (Cdk1), which both activates Hcm1 and targets it for degradation, through phosphorylation of distinct sites. The mechanisms controlling the differential phosphorylation timing of the activating and destabilizing phosphosites are not clear. However, a recent study shows that the phosphatase calcineurin specifically removes activating phosphates from Hcm1 when cells are exposed to environmental stress, thus extinguishing its activity and slowing proliferation under unfavorable growth conditions. This regulatory mechanism, whereby a phosphatase actively alters the distribution of phosphosites on a cell cycle-regulatory transcription factor to elicit a change in cellular proliferation, adds an additional layer of complexity to the regulatory network controlling the cell cycle. Furthermore, this regulatory paradigm is likely to be a conserved mode of phosphoregulation that controls the cell cycle in diverse systems.