Potassium channels in cell cycle and cell proliferation

Normal cell-cycle progression is a crucial task for every multicellular organism, as it determines body size and shape, tissue renewal and senescence, and is also crucial for reproduction. On the other hand, dysregulation of the cell-cycle progression leading to uncontrolled cell proliferation is the hallmark of cancer. Therefore, it is not surprising that it is a tightly regulated process, with multifaceted and very complex control mechanisms. It is now well established that one of those mechanisms relies on ion channels, and in many cases specifically on potassium channels. Here, we summarize the possible mechanisms underlying the importance of potassium channels in cell-cycle control and briefly review some of the identified channels that illustrate the multiple ways in which this group of proteins can influence cell proliferation and modulate cell-cycle progression.     

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.     

Invariant mRNA and mitotic protein breakdown solves the Russian Doll problem of the cell cycle

It has been proposed that cyclical gene expression occurs at a large number of different times during the cell cycle. The existence of a large number of cycle-specific variations in mRNA and protein during the eukaryotic cell cycle raises the problem of how cell-cycle variations are regulated. This is the “infinite regression” or Russian Doll problem where postulating a cell-cycle specific control element pushes the explanation of cell-cycle variation back one step to the problem of how that control element varies during the cell cycle.PCR studies on unperturbed cells indicate Cyclin mRNA content is invariant during the cell cycle. Furthermore, calculations reveal that variations in mRNA content do not account for observed protein variations.Continuous and constant gene expression during the cell cycle, continuous protein accumulation, and protein breakdown only within the mitotic window solves the Russian Doll problem or infinite regression problem. These results, and theoretical ideas support an alternative view of the cell cycle where many of the proposed control systems do not exist.     

Endoreplication Controls Cell Fate Maintenance

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.     

Cell cycle and differentiation

The development of multicellular organisms relies on the temporal and spatial control of cell proliferation and cell growth. The relationship between cell-cycle progression and development is complex and characterized by mutual dependencies. On the level of the individual cell, this interrelationship has implications for pattern formation and cell morphogenesis. On a supercellular level, this interrelationship affects meristem function and organ growth. Often, developmental signals not only direct cell-cycle progression but also set the frame for cell-cycle regulation by determining cell-type-specific cell-cycle modes. In other cases, however, cell-cycle progression appears to be required for the further differentiation of some cell types. There are also examples in which cell cycle and differentiation seem to be controlled at the same level and progress rather independently from each other or are linked by the same regulator or pathway. Furthermore, different relationships between cell cycle and differentiation can be combined in a succession of events during development, leading to complex developmental programs.     

Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies.

Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.     

Oncogenes and cell cycle genes

The relationships between oncogenes, cell-cycle control genes, and growth-related genes are described. An important generalization from the data is that all the oncogenes and cell-cycle control genes so far characterized appear to be genes for growth factors or for receptors to growth factors, or to be involved in the general metabolism and structure of the cell. It is suggested that the transition in cell growth from quiescence to the proliferative state, in early G₁, may be less specific than previously thought and involve a general priming of the cells for more specific later stimuli.     

Mitogen-activated protein kinases in cell-cycle control

The mitogen-activated protein kinase (MAPK) family of kinases connects extracellular stimuli with diverse cellular responses ranging from activation or suppression of gene expression to the regulation of cell mortality, growth, and differentiation. The MAPK family has been studied extensively; however, the role of these kinases in cell growth and cell-cycle control has become increasingly complex. Patterns have begun to emerge from these studies that show the functions of MAPK subfamilies at different stages of the cell cycle. Their patterns of subcellular localization and movement during the cell cycle are subfamily-specific and have raised many questions about possible cell-cycle functions that have yet to be demonstrated. This article will compare and contrast our current understanding of the functions and localization patterns of the MAPK subfamilies (ERK, BMK, p38, and JNK) in cell-cycle control.     

Minimal Models for Cell-Cycle Control Based on Competitive Inhibition and?Multisite Phosphorylations of Cdk Substrates

The eukaryotic cell cycle is characterized by alternating oscillations in the activities of cyclin-dependent kinase (Cdk) and the anaphase-promoting complex (APC). Successful completion of the cell cycle is dependent on the precise, temporally ordered appearance of these activities. A modest level of Cdk activity is sufficient to initiate DNA replication, but mitosis and APC activation require an elevated Cdk activity. In present-day eukaryotes, this temporal order is provided by a complex network of regulatory proteins that control both Cdk and APC activities via sharp thresholds, bistability, and time delays. Using simple computational models, we show here that these dynamical features of cell-cycle organization could emerge in a control system driven by a single Cdk/cyclin complex and APC wired in a negative-feedback loop. We show that ordered phosphorylation of cellular proteins could be explained by multisite phosphorylation/dephosphorylation and competition of substrates for interconverting kinase (Cdk) and phosphatase. In addition, the competition of APC substrates for ubiquitylation can create and maintain sustained oscillations in cyclin levels. We propose a sequence of models that gets closer and closer to a realistic model of cell-cycle control in yeast. Since these models lack the elaborate control mechanisms characteristic of modern eukaryotes, they suggest that bistability and time delay may have characterized eukaryotic cell divisions before the current cell-cycle control network evolved in all its complexity.     

Role of cell-cycle regulatory proteins in gynecological cancer

Human malignant tumors are characterized by abnormal proliferation resulting from alterations in cell-cycle regulatory mechanisms. This review summarizes the current knowledge about these aberrations in malignant tumors of the ovary, endometrium, cervix uteri, and vulva. The data indicate that analysis of single cell cycle stimulating or inhibiting proteins partly produces unexpected, apparently paradoxical results, and cell-cycle regulatory pathways should be regarded as a whole in order to identify the molecular mechanisms leading to abnormal tumor cell proliferation. For the papillomavirus (HPV)- associated cervical and vulvar carcinomas, the manifold effects of the viral oncogenes E6 and E7 on cell-cycle control are described.     

The structural and mechanical complexity of cell-growth control

Tight control of cell proliferation is required to ensure normal tissue patterning and prevent cancer formation. The analysis of cultured cells has led to an explosion in our understanding of the molecules that trigger growth and mediate cell-cycle progression. However, the mechanism by which the local growth differentials that drive morphogenesis are established and maintained still remains unknown. Here we review recent work that reveals the importance of cell binding to the extracellular matrix, and associated changes in cell shape and cytoskeletal tension, to the spatial control of cell-cycle progression. These findings change the paradigm of cell-growth control, by placing our understanding of molecular signalling cascades in the context of the structural and mechanical complexity of living tissues.     

Four-dimensional control of the cell cycle

The cell-division cycle has to be regulated in both time and space. In the time dimension, the cell ensures that mitosis does not begin until DNA replication is completed and any damaged DNA is repaired, and that DNA replication normally follows mitosis. This is achieved by the synthesis and destruction of specific cell-cycle regulators at the right time in the cell cycle. In the spatial dimension, the cell coordinates dramatic reorganizations of the subcellular architecture at the entrance to and exit from mitosis, largely through the actions of protein kinases and phosphatases that are often localized to specific subcellular structures. Evidence is now accumulating to suggest that the spatial organization of cell-cycle regulators is also important in the temporal control of the cell cycle. Here I will focus on how the locations of the main components of the cell-cycle machinery are regulated as part of the mechanism by which the cell controls when and how it replicates and divides.     

Genetic framework of cyclin-dependent kinase function in Arabidopsis

Cyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.     

Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.     

Cyclin-dependent kinases and cell-cycle transitions: does one fit all?

Cell-cycle transitions in higher eukaryotes are regulated by different cyclin-dependent kinases (CDKs) and their activating cyclin subunits. Based on pioneering findings that a dominant-negative mutation of CDK1 blocks the cell cycle at G2-M phase, whereas dominant-negative CDK2 inhibits the transition into S phase, a model of cell-cycle control has emerged in which each transition is regulated by a specific subset of CDKs and cyclins. Recent work with gene-targeted mice has led to a revision of this model. We discuss cell-cycle control in light of overlapping and essential functions of the different CDKs and cyclins.