Mammalian Cell Cycle Control Research Articles

A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model, comprehensive details of cell cycle dynamics under normal and DNA damage conditions revealing the role and value of the added new modules and elements, (ii) assess, through a global sensitivity analysis, the most influential sub-systems, modules and parameters on system response, such as G1-S and G2-M transitions, and (iii) probe deeply into the relationship between DNA damage and cell cycle progression and test the biological evidence that G1-S is relatively inefficient in arresting damaged cells compared to G2-M checkpoint. To perform sensitivity analysis, Self-Organizing Map with Correlation Coefficient Analysis (SOMCCA) is developed which shows that Growth Factor and G1-S Checkpoint sub-systems and 13 parameters in the modules within them are crucial for G1-S and G2-M transitions. To study the relative efficiency of DNA damage checkpoints, a Checkpoint Efficiency Evaluator (CEE) is developed based on perturbation studies and statistical Type II error. Accordingly, cell cycle is about 96% efficient in arresting damaged cells with G2-M checkpoint being more efficient than G1-S. Further, both checkpoint systems are near perfect (98.6%) in passing healthy cells. Thus this study has shown the efficacy of the proposed systems approach to gain a better understanding of different aspects of mammalian cell cycle system separately and as an integrated system that will also be useful in investigating targeted therapy in future cancer treatments.

Logical Modeling and Dynamical Analysis of Cellular Networks.

The logical (or logic) formalism is increasingly used to model regulatory and signaling networks. Complementing these applications, several groups contributed various methods and tools to support the definition and analysis of logical models. After an introduction to the logical modeling framework and to several of its variants, we review here a number of recent methodological advances to ease the analysis of large and intricate networks. In particular, we survey approaches to determine model attractors and their reachability properties, to assess the dynamical impact of variations of external signals, and to consistently reduce large models. To illustrate these developments, we further consider several published logical models for two important biological processes, namely the differentiation of T helper cells and the control of mammalian cell cycle.

Genetic Systems to Investigate Regulation of Oncogenes and Tumour Suppressor Genes in Drosophila

Animal growth requires coordination of cell growth and cell cycle progression with developmental signaling. Loss of cell cycle control is extremely detrimental, with reduced cycles leading to impaired organ growth and excessive proliferation, potentially resulting in tissue overgrowth and driving tumour initiation. Due to the high level of conservation between the cell cycle machinery of Drosophila and humans, the appeal of the fly model continues to be the means with which we can use sophisticated genetics to provide novel insights into mammalian growth and cell cycle control. Over the last decade, there have been major additions to the genetic toolbox to study development in Drosophila. Here we discuss some of the approaches available to investigate the potent growth and cell cycle properties of the Drosophila counterparts of prominent cancer genes, with a focus on the c-Myc oncoprotein and the tumour suppressor protein FIR (Hfp in flies), which behaves as a transcriptional repressor of c-Myc.

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments. DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases. In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Modeling the cell cycle: From deterministic models to hybrid systems

Abstract The cell cycle is a complex biological system frequently investigated from a mathematical perspective. In fact, over the past years a huge number of deterministic mathematical models describing the dynamics and the regulation of this process have been proposed. A crucial point concerning the cell cycle modeling is the combination of continuous and discrete dynamics in order to obtain results which are coherent with the biological context. To face with this problem we propose a novel approach to the mathematical modeling of biological processes based on the use of hybrid systems. This new methodology essentially consists in a model reduction (using the modified Prony’s method) which allows to define the crucial features of the dynamical system. The final aim is to implement a corresponding hybrid system which preserves the properties of the starting deterministic model. Thus, we implemented a methodology which allows to describe the cellular system by combining continuous behavior with discrete events by using the hybrid automata technology. In this way we try to overcome some drawbacks of the deterministic approach, especially regarding the possibility to introduce new variables during simulation and the associated variation of parameters in a more efficient way than the continuous method can do. We applied this innovative methodology to the reconstruction of a simplified hybrid model concerning one of the crucial mammalian cell cycle control point. In particular, we investigated the role of the transcription factors E2F in the R-point transition. The resulting hybrid model preserve the properties of the deterministic one and it allows the identification of the parameter which controls the transition from the inactive (quiescent) to the active state (R-point transition) after the mitogenic stimulation. At the best of our knowledge no hybrid model for the R-point transition are available in literature.

E2f1–3 Are Critical for Myeloid Development

Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1-3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1-3 are required as transcriptional repressors for the survival of CD11b(+) myeloid progenitors, and then they are required as activators for the proliferation of CD11b(+) macrophages. In bone marrow macrophages, we show that E2f1-3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1-3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1-3 in a specific signaling cascade that is critical for myeloid development in vivo.

E2f1–3 switch from activators in progenitor cells to repressors in differentiating cells

In the classic paradigm of mammalian cell cycle control, Rb functions to restrict cells from entering S phase by sequestering E2F activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase1, 2. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examine the effects of E2f1, E2f2 and E2f3 triple deficiency in murine ES cells, embryos and small intestines. We show that in normal dividing progenitor cells E2F1-3 function as transcriptional activators, but contrary to current dogma, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells they function in complex with Rb as repressors to silence E2F targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2F1-3 from repressors to activators, leading to the superactivation of E2F responsive targets and ectopic cell divisions, and loss of E2f1-3 completely suppressed these phenotypes. This work contextualizes the activator versus repressor functions of E2F1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles in vivo.

Differential regulation of the cyclin-dependent kinase inhibitors p21 Cip1 and p27 Kip1 by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the γ2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21 Cip and p27 Kip. Furthermore, M cyclin directed cdk2 to phosphorylate p27 Kip1 on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27 Kip1 and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27 Kip1-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21 Cip1 and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.

Modeling and querying biomolecular interaction networks

We introduce a formalism to represent and analyze protein–protein and protein–DNA interaction networks. We illustrate the expressivity of this language, by proposing a formal counterpart of Kohn's compilation on the mammalian cell-cycle control. This effectively turns an otherwise static knowledge into a discrete transition system incorporating a qualitative description of the dynamics. We then propose to use the computation tree logic (CTL) as a query language for querying the possible behaviors of the system. We provide examples of biologically relevant queries expressed in CTL about the mammalian cell-cycle control and show the effectiveness of symbolic model checking tools to evaluate CTL queries in this context.

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 γ, 14-3-3 ε, 14-3-3 σ, and 14-3-3 ζ, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ α-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue.

The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.

Molecular interaction map of the mammalian cell cycle control and DNA repair systems.

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.

Kaposi's Sarcoma Herpesvirus Encoded Cycun Disruption of Mammalian Cell Cycle Control Mechanisms

Kaposi's Sarcoma Herpesvirus Encoded Cycun Disruption of Mammalian Cell Cycle Control Mechanisms

Bioprobes for investigating mammalian cell cycle control.

Bioprobes are low molecular weight compounds which are useful for investigating mammalian cell functions. The use of bioprobes has substantially assisted the investigation of complex biochemical processes of the mammalian cell cycle. In this review, cell cycle inhibitors mainly isolated from the microorganism are described and their possibility as an antitumor agents is considered. Most cancer cells have some abnormality in the control mechanism of cell cycle progression. Cyclin-dependent kinases (Cdk), which are activated by the binding with the cyclin and simultaneously by the phosphorylation/dephosphorylation of itself, play important roles as engines in the cell cycle. Tubulins are considered to be one of the most important proteins of the cell division machinery. Therefore, Cdk inhibitors and tubulin binders are possible anticancer drugs. Since the function of proteins controlling the cell cycle is also regulated by phosphorylation and dephosphorylation, inhibitors of protein kinases and phosphatases are considered as possible an antitumor agents. We expect that some bioprobes will be developed for clinical use.

P21/WAF1 Cyclin-Kinase Inhibitor Expression in Non-Hodgkin's Lymphomas: A Potential Marker of p53 Tumor-Suppressor Gene Function

p21WAF1 (wild-type p53-activated fragment 1) is involved in the control of mammalian cell cycle through the binding and inhibition of cyclin-dependent kinases (Cdk). Because the product of WAF1 gene is a potent downstream effector of the p53 tumor-suppressor gene function, its pattern of cellular expression might correlate with nuclear accumulation of p53-encoded protein and/or p53 gene mutations occurring in malignant lymphomas. To investigate this issue, we analyzed immunohistochemically the expression of p53 and p21WAF1 proteins in tissue involved by non-Hodgkin's lymphomas (NHLs;253 cases) of various histologic types. In a proportion of them (80 cases), we also investigated the possible presence of p53 gene mutations using single-strand conformation polymorphism analysis and direct DNA sequencing. The absence of both p21WAF1 and p53 proteins was observed in 147 of 217 cases (67.7%) among CD30-NHL and in only 8 of 36 (22.2%) CD30+cases, which were mostly anaplastic large-cell lymphomas. A consistent number (> 10%) of p21WAF1-expressing cells was shown in 48 of 253 (18.9%) NHL cases, with a higher incidence in CD30+cases (25/36 [69.4%]), which mostly (21/36) coexpressed p53. These latter cases were characterized by a germline configuration of the p53 gene. In 50 of 253 NHL samples (19.7%), 47 of which (21.6%) belong to the CD30-group, neoplastic cells were p53+/p21-. In all of these cases, the p53+cells accounted for more than 50% of neoplastic cells, up to 100%. Point mutations of p53 gene were solely observed in all investigated cases with this latter phenotype. Our findings strongly suggest that the combined immunohistochemical evaluation of p53 and p21WAF1 is a valuable means of assessing the functional status of the p53 tumor-suppressor gene product in NHL with potential application in the monitorage and prognostication of individual cases.

Control of meiotic arrest.

Oocytes of many species arrest at specific cell cycle stages during their development. An external signal from a hormone or the fertilizing sperm causes them to resume the cell cycle. The control of meiotic arrest can be usefully formulated in terms of the interaction between cell signalling mechanisms and the protein machinery that controls the cell cycle. Much of what we know about cell messengers, particularly calcium, and the cell cycle control proteins comes from work on oocytes. Recent work on cell signalling pathways in mammalian cells and cell cycle control in yeast has been essential to our understanding of meiotic arrest in oocytes.

Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1.

Mammalian cell-cycle control by antimitogenic signals involves p21Cip1/WAF1 (refs 1-4), p27Kip1 (refs 5, 6) and p57Kip2 (refs 7, 8), a family of proteins that bind to and inhibit cyclin-dependent kinases (CDKs) required for initiation of S phase. The protein p21 also binds to the DNA polymerase delta processivity factor, proliferating-cell nuclear antigen (PCNA), and inhibits in vitro PCNA-dependent DNA replication. The CDK and PCNA inhibitory activities of p21 are shown here to be functionally independent and to reside in separate protein domains. The PCNA binding and inhibitory activities, which are not observed with p27 or p57, reside in the C-terminal domain of p21, whereas the CDK inhibitory activity resides in the conserved N-terminal domains of these proteins. When separately overexpressed in mammalian cells, the CDK and PCNA inhibitory domains prevent DNA replication, demonstrating a dual function of p21 as a cell-cycle inhibitor in vivo.

Peptide growth factors and cell cycle control

Research on mammalian cell cycle control focuses on the points discussed below. Peptide growth factors are multifunctional regulators of growth and differentiation that act by autocrine and paracrine mechanisms. Gene transcription changes are key steps in the control of the G 02G 1⇒S transition of the cell cycle. Both peptide growth factors and classical tropic hormones, are capable of rapidly modulating transcription through the induction of genes ( fosljun) that encode nuclear transregulator proteins.

Regulated expression and phosphorylation of a possible mammalian cell-cycle control protein.

A novel approach to the study of the control of the mammalian cell cycle was opened by the cloning of a human gene by complementation of a fission-yeast cdc2 cell-cycle mutant. We have investigated the behaviour of the RNA and protein products of this human gene, CDC2Hs, and its murine equivalent, CDC2Mm during serum starvation and re-feeding of cultured fibroblasts. In contrast to the pattern of wild-type cdc2+ expression in fission yeast previously described, the mammalian homologue displays variation in both RNA and protein levels during exit from and re-entry into the mitotic cycle. Like its yeast counterpart, however, the mammalian CDC2 protein (p34CDC2) becomes dephosphorylated upon shifting from exponential growth to quiescence, and rephosphorylated late in the G1 phase when cells are stimulated to re-enter the cycle. We propose that phosphorylation of p34CDC2 serves as a regulatory mechanism generally in eukaryotic cells, while transcriptional control of the CDC2 gene in higher eukaryotes may be relevant to long term processes such as senescence and differentiation.