Eukaryotic Cell Cycle Research Articles

Mitogen-Activated Protein Kinase (MAPK): New Insights in Breast Cancer.

The eukaryotic cell cycle via mitogen-activated protein kinase (MAPK) has been considered as an effective signaling cascade pathway for anticancer therapy. In addition to the MAPK signaling pathway, the Rat Sarcoma Virus (Ras)/Rapidly Accelerated Fibrosarcoma (Raf)/MAPK-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is a signaling cascade that has a crucial role to play in cell survival and development of cancer, especially breast cancer. Novel drugs and clinical trials need to be investigated, taking into consideration the already studied genetic defects related to genesis of tumors in certain populations. The present review provides novel insights into current anticancertreatments involving the MAPK pathway.

Allyl-Isothiocyanate and Microcystin-LR Reveal the Protein Phosphatase Mediated Regulation of Metaphase-Anaphase Transition in Vicia faba.

Horseradish allyl isothiocyanate (AITC, a volatile oil) and cyanobacterial microcystin-LR (MCY-LR, a cyclic heptapeptide) affect eukaryotic cell cycle. MCY-LR inhibits protein phosphatases PP1 and PP2A. We aimed to reveal the mechanisms of their cellular effects in a model eukaryote, Vicia faba. We have shown for the first time that AITC had minor effects on PP1 and PP2A activities in vitro, but it inhibited significantly PP1 in vivo. The combination of 10 μM AITC with 10 μM MCY-LR induced metaphase arrest after short-term (12 h) treatments. 10 μM AITC, 0.2–10 μM MCY-LR and their combinations induced histone H3 hyperphosphorylation, associated with the regulation of metaphase-anaphase transition. This hyperphosphorylation event occurred at any treatment which led to the inhibition of PP1 activity. 10 μM AITC + 10 μM MCY-LR increased the frequency of metaphase spindle anomalies, associated with metaphase arrest. We provide new insights into the mechanisms of metaphase-anaphase transition. Metaphase arrest is induced at the concomitant hyperphosphorylation of histone H3, alteration of metaphase spindle assembly and strong inhibition of PP1 + PP2A activity. Near-complete blocking of metaphase-anaphase transition by rapid protein phosphatase inhibition is shown here for the first time in plants, confirming a crucial role of serine-threonine phosphatases in this checkpoint of cell cycle regulation. Tissue-dependent differences in PP1 and PP2A activities induced by AITC and MCY-LR suggest that mainly regulatory subunits are affected. AITC is a potential tool for the study of protein phosphatase function and regulation. We raise the possibility that one of the biochemical events occurring during AITC release upon wounding is the modulation of protein phosphatase dependent signal transduction pathways during the plant defense response.

Genome-wide identification of evolutionarily conserved Small Heat-Shock and eight other proteins bearing α-crystallin domain-like in kinetoplastid protists.

Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including Trypanosoma spp. (23 species of mammals, reptiles and frogs), Leishmania spp. (mammals and lizards), trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized (HSP20, Tryp23A, Tryp23B and ATOM69), and four characterized for the first time in kinetoplastids (TryACDP, TrySGT1, TryDYX1C1 and TryNudC). A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.

Scaling of G1 Duration with Population Doubling Time by a Cyclin in Saccharomyces cerevisiae.

The longer cells stay in particular phases of the cell cycle, the longer it will take these cell populations to increase. However, the above qualitative description has very little predictive value, unless it can be codified mathematically. A quantitative relation that defines the population doubling time (Td) as a function of the time eukaryotic cells spend in specific cell cycle phases would be instrumental for estimating rates of cell proliferation and for evaluating introduced perturbations. Here, we show that in human cells, the length of the G1 phase (TG1) regressed on Td with a slope of ≈0.75, while in the yeast Saccharomyces cerevisiae, the slope was slightly smaller, at ≈0.60. On the other hand, cell size was not strongly associated with Td or TG1 in cell cultures that were proliferating at different rates. Furthermore, we show that levels of the yeast G1 cyclin Cln3p were positively associated with rates of cell proliferation over a broad range, at least in part through translational control mediated by a short upstream ORF (uORF) in the CLN3 transcript. Cln3p was also necessary for the proper scaling between TG1 and Td In contrast, yeast lacking the Whi5p transcriptional repressor maintained the scaling between TG1 and Td These data reveal fundamental scaling relationships between the duration of eukaryotic cell cycle phases and rates of cell proliferation, point to the necessary role of Cln3p in these relationships in yeast, and provide a mechanistic basis linking Cln3p levels to proliferation rates and the scaling of G1 with doubling time.

Fueling the Cycle: CDKs in Carbon and Energy Metabolism.

Cyclin-dependent kinases (CDKs) are the central regulators of the eukaryotic cell cycle, and are conserved across eukaryotes. Their main and well-studied function lies in the regulation and the time-keeping of cell cycle entry and progression. Additionally, more and more non canonical functions of CDKs are being uncovered. One fairly recently discovered role of CDKs is the coordination of carbon and energy metabolism with proliferation. Evidence from different model organisms is accumulating that CDKs can directly and indirectly control fluxes through metabolism, for example by phosphorylating metabolic enzymes. In this mini-review, we summarize the emerging role of CDKs in regulating carbon and energy metabolism and discuss examples in different models from yeast to cancer cells.

Transcriptional timing and noise of yeast cell cycle regulators\u2014a single cell and single molecule approach

Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response necessitates expression reprogramming and cell cycle arrest. While previous studies are mostly based on bulk experiments influenced by synchronization effects or lack temporal distribution, time-resolved methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. Using smFISH, microscopy and morphological markers, we monitored mRNA abundances over cell cycle phases and calculated transcriptional noise for SIC1, CLN2, and CLB5, the main G1/S transition regulators in budding yeast. We employed mathematical modeling for in silico synchronization and for derivation of time-courses from single cell data. This approach disclosed detailed quantitative insights into transcriptional regulation with and without stress, not available from bulk experiments before. First, besides the main peak in G1 we found an upshift of CLN2 and CLB5 expression in late mitosis. Second, all three genes showed basal expression throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. Finally, exposing cells to osmotic stress revealed different periods of transcriptional inhibition for CLN2 and CLB5 and the impact of stress on cell cycle phase duration. Combining experimental and computational approaches allowed us to precisely assess cell cycle progression timing, as well as gene expression dynamics.

Design, synthesis and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells

Cyclin-dependent kinase 2 (CDK2) plays a key role in eukaryotic cell cycle progression which could facilitate the transition from G1 to S phase. The dysregulation of CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied kinase targets in oncology. In this article, 24 benzamide derivatives were designed, synthesized and investigated for the inhibition activity against CDK2. Our results revealed that the compound 25 is a potent CDK2 inhibitor exhibiting a broad spectrum anti-proliferative activity against several human breast cancer cells. Additionally, compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in MDA-MB-468 cells. These findings highlight a rationale for further development of CDK2 inhibitors to treat human breast cancer.

Pervasive Protein Thermal Stability Variation during the Cell Cycle

SummaryQuantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Colibactin: More Than a New Bacterial Toxin.

Cyclomodulins are bacterial toxins that interfere with the eukaryotic cell cycle. A new cyclomodulin called colibactin, which is synthetized by the pks genomic island, was discovered in 2006. Despite many efforts, colibactin has not yet been purified, and its structure remains elusive. Interestingly, the pks island is found in members of the family Enterobacteriaceae (mainly Escherichia coli and Klebsiella pneumoniae) isolated from different origins, including from intestinal microbiota, septicaemia, newborn meningitis, and urinary tract infections. Colibactin-producing bacteria induce chromosomal instability and DNA damage in eukaryotic cells, which leads to senescence of epithelial cells and apoptosis of immune cells. The pks island is mainly observed in B2 phylogroup E. coli strains, which include extra-intestinal pathogenic E. coli strains, and pks E. coli are over-represented in biopsies isolated from colorectal cancer. In addition, pks E. coli bacteria increase the number of tumours in diverse colorectal cancer mouse models. Thus, colibactin could have a major impact on human health. In the present review, we will focus on the biological effects of colibactin, the distribution of the pks island, and summarize what is currently known about its synthesis and its structure.

Systems-level feedback regulation of cell cycle transitions in Ostreococcus tauri

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes.

The concepts of Ludwik Fleck and their application to the eukaryotic cell cycle

The concepts of Ludwik Fleck (1896–1961), a microbiologist, historian, and philosopher of medicine, can be used to analyze the conservative nature of scientific ideas. This is discussed and applied to ideas dominant in the understanding of the eukaryotic cell cycle. These are (a) the G1-phase restriction point as a regulatory element of the mammalian cell cycle, (b) the Rate Change Point proposed to exist in fission yeast, and (c) the proposal that a large number of genes are expressed in a cell-cycle-dependent manner. Fleck proposed that scientific ideas become fixed and difficult to change because criticisms of current and dominant models are either ignored or turned to support of the current model. The idea of a thought-collective leading to the stability of scientific ideas is a central theme of the theory of Ludwik Fleck.

Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress

Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.

Whi7 is an unstable cell-cycle repressor of the Start transcriptional program

Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCFGrr1 ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.

DNA methylation profiles in cancer diagnosis and therapeutics.

Cancer initiation and proliferation is regulated by both epigenetic and genetic events with epigenetic modifications being increasingly identified as important targets for cancer research. DNA methylation catalyzed by DNA methyltransferases (DNMTs) is one of the essential epigenetic mechanisms that control cell proliferation, apoptosis, differentiation, cell cycle, and transformation in eukaryotes. Recent progress in epigenetics revealed a deeper understanding of the mechanisms of tumorigenesis and provided biomarkers for early detection, diagnosis, and prognosis in cancer patients. Although DNA methylation biomarker possesses potential contributing to precision medicine, there are still limitations to be overcome before it reaches clinical setting. Hence, the current status of DNA methylation biomarkers was reviewed and the future use in clinic was also predicted.

Bistability analysis in mathematical model of M-phase control in Xenopus oocyte extracts

To gain more insight into the molecular mechanism that regulates eukaryotic cell cycle, we propose a modification to an existing Mathematical model of mitosis-promoting factor control in Xenopus oocyte extract. To achieve this, we redirect how cell exits from mitosis using ubiquitin ligase enzyme APCP:Cdc20 which is acting as a modifier in cyclin degradation. We present a modified wiring map using combination of mass action kinetics and Michaelis–Menten kinetics to represent the biochemical processes. This results in a system of non-linear ordinary differential equations. The equilibria of the system were analyzed analytically. Using MPF as a bifurcation parameter, we analytically derived conditions under which bistability can occur in the MPF activation module. We find that for bistability to occur, it is necessary that the rate of phosphorylation of Wee1 must be greater than the rate of its dephosphorylation and that the concentration of MPF must lie in certain range of parameter values. Numerical simulations conducted show that the time taken to exit the cell cycle is shorter than those published in some reports and the sensitivity analysis results suggest that the rate of synthesis of cyclin is the most sensitive parameter in the dynamics of cell cycle. Through numerical experiments, we found new parameter sets that will give rise to bistability in the MPF activation module.

The cyclin dependent kinase subunit Cks1 is required for infection-associated development of the rice blast fungus Magnaporthe oryzae.

Cell cycle regulation is pivotal for proper cell division and cellular differentiation in eukaryotic cells. The central regulators that govern eukaryotic cell cycle progression are cyclin-dependent kinases (CDKs) and their partners. Here, we report that Magnaporthe oryzae CKS1 encodes a cyclin-dependent kinase subunit, which plays a significant role in regulation of plant infection. We demonstrate that CKS1 is a functional homolog of CKS1/SUC1 and can physically interact with the CDK protein Cdc28, and Som1, a downstream regulator of the cyclic AMP-dependent Protein Kinase A pathway. CKS1 deletion mutants are severely impaired in hyphal growth, sexual reproduction, melanin pigmentation and conidiogenesis. Cks1 mutants are able to form appressoria from hyphal tips, but these are unable to re-polarize, and rice infection is impaired. CKS1 also affects chitin and glucan synthase activity during cell wall differentiation and fungal hydrophobin function. CKS1, therefore, encodes a conserved CDK-binding partner, essential for appressorium-mediated plant infection by the rice blast fungus.

Cell Cycle-Dependent Expression Dynamics of G1/S Specific Cyclin, Cellulose Synthase and Cellulase in the Dinoflagellate Prorocentrum donghaiense.

Dinoflagellates undergo a typical eukaryotic cell cycle consisting of G1, S, G2, and M phases and some of the typical cell cycle related genes have been computationally identified. However, very few of these genes have been experimentally linked to the cell cycle phases. Besides, although thecate dinoflagellates are known to possess theca composed of cellulose, information on cellulose synthesis and degradation associated with the cell cycle is also limited. In this study, we isolated G1/S cyclin, cellulose synthase and cellulase encoding genes in dinoflagellate Prorocentrum donghaiense. Further, using reverse transcription quantitative PCR (RT-qPCR), we characterized the expression profiles of the three genes throughout the cell cycle. All three showed clear expression dynamics throughout the cell cycle, with fold changes of 26, 2.4 and 9.3 for G1/S cyclin, cellulose synthase and cellulase gene, respectively. The transcript abundance of G1/S cyclin increased in late G1 phase and dropped in early S phase, indicating that this protein is involved in the G1/S transition. Throughout the cell cycle, the average transcript level of cellulose synthase was 4.5-fold higher than that of cellulase. Cellulose synthase and cellulase gene expressions showed peak transcript abundances at middle G1 phase and G2M phase, respectively, indicating the respective roles of these enzymes in the growth of newly divided cells and in cytokinesis. Our results suggest that G1/S cyclin, cellulase, and cellulose synthase genes associated with G1/S transition, G2M, and G1 phases of the cell cycle and are candidates of biomarkers for assessing growth status of P. donghaiense.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

ABSTRACTAll well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections.

Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

TORC1 coordinates the conversion of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1

Eukaryotic cell cycle progression through G1–S is driven by hormonal and growth-related signals that are transmitted by the target of rapamycin complex 1 (TORC1) pathway. In yeast, inactivation of TORC1 restricts G1–S transition due to the rapid clearance of G1 cyclins (Cln) and the stabilization of the B-type cyclin (Clb) cyclin-dependent kinase (CDK) inhibitor Sic1. The latter mechanism remains mysterious but requires the phosphorylation of Sic1-Thr173 by Mpk1 and inactivation of the Sic1-pThr173-targeting phosphatase (PP2ACdc55) through greatwall kinase-activated endosulfines. Here we show that the Sic1-pThr173 residue serves as a specific docking site for the CDK phospho-acceptor subunit Cks1 that sequesters, together with a C-terminal Clb5-binding motif in Sic1, Clb5-CDK-Cks1 complexes, thereby preventing them from flagging Sic1 for ubiquitin-dependent proteolysis. Interestingly, this functional switch of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1 also operates in proliferating cells and is coordinated by the greatwall kinase, which responds to both Cln-CDK-dependent cell-cycle and TORC1-mediated nutritional cues.