Table of Contents

Volume 6, Issue 1

  January 1, 2007

  Pages 3 - 110

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EXTRA VIEWS

Open Access Article

Phosphatase and Feedback Regulation of Raf-1 Signaling

Amardeep S. Dhillon, Alex von Kriegsheim, Joan Grindlay and Walter Kolch
Pages 3 - 7
http://dx.doi.org/10.4161/cc.6.1.3593
Abstract | PDF

The Raf-1 kinase is an effector of Ras GTPases that lies at the apex of the three-tier Raf/MEK/ERK pathway. Raf-1 activation is a complex process that entails two major events – relief of autoinhibition imposed by the regulatory domain and kinase domain activation. Recent studies indicate that the transition of Raf-1 from an active to an inactive state bears similar complexity to the activation process. Both these events require dynamic changes in Raf-1 phosphorylation. Here, we discuss the critical role of phosphatases and feedback phosphorylation during activation and inactivation of Raf-1 signalling.

Open Access Article

Oct-3/4: Not Just a Gatekeeper of Pluripotency for Embryonic Stem Cell, a Cell Fate Instructor through a Gene Dosage Effect

Sonia Stefanovic and Michel Pucéat
Pages 8 - 10
http://dx.doi.org/10.4161/cc.6.1.3633
Abstract | PDF

Oct-3/4 encoded by Pou5f1 has been longwise recognised as a gatekeeper for embryonic stem (ES) cell pluripotency. Recently, it was suggested that Oct-3/4 one of the earliest transcription factor of the embryo might feature some functions in differentiation of cell lineages. We recently uncovered a key role of Oct-3/4 in both ES cell specification toward a cardiac lineage and in mesodermal commitment of the embryonic epiblast. Our findings demonstrated a gene dosage effect of Oct-3/4, a factor at the cross-road between cell self-renewal and cell lineage specification. What is the genetic and/or epigenetic mechanism underlying this process is still questionable. The extraview presents some hypotheses that might account for the pleiotropic dose-dependent functions of Oct-3/4.

Open Access Article

Is Ribosome Synthesis Controlled by Pol I Transcription?

Stéphane Chédin, Arnaud Laferté, Tran Hoang, Denis L.J. Lafontaine, Michel Riva and Christophe Carles
Pages 11 - 15
http://dx.doi.org/10.4161/cc.6.1.3649
Abstract | PDF

Regulation of growth ultimately depends on the control of synthesis of new ribosomes. Ribosome biogenesis is thus a key element of cell biology, which is tightly regulated in response to environmental conditions. In eukaryotic cells, the supply of ribosomal components involves the activities of the three forms of nuclear RNA polymerase (Pol I, Pol II and Pol III). Recently, we demonstrated that upon rapamycin treatment, a partial derepression of Pol I transcription led to a concomitant derepression of Pol II transcription restricted to a small subset of class II genes encompassing the genes encoding all ribosomal proteins, and 19 additional genes ¹. The products of 14 of these 19 genes are principally involved in rDNA structure, ribosome biogenesis or translation, whereas the 5 remaining genes code for hypothetical proteins. We demonstrate that the proteins encoded by these 5 genes are required for optimal pre-rRNA processing. In addition, we show that cells in which regulation of Pol I transcription was specifically impaired are either resistant or hypersensitive to different stresses compared to wild-type cells. These results highlight the critical role of the regulation of Pol I activity for the physiology of the cells.

Open Access Article

An Emerging Role for Ca2+/Calcineurin/NFAT Signaling in Cancerogenesis

Malte Buchholz and Volker Ellenrieder
Pages 16 - 19
http://dx.doi.org/10.4161/cc.6.1.3650
Abstract | PDF

The NFAT proteins are a family of Ca²⁺/calcineurin-responsive transcription factors primarily recognized for their central roles in T lymphocyte activation. Recent studies, however, have demonstrated that the vital role of NFAT signaling is by no means restricted to the immune system. NFAT proteins are potent mediators of Ca²⁺ signals in many mammalian cells and tissues and control, among others, important steps in myogenesis, chondrocyte differentiation and the development of the cardiovascular system. Owing to the broad spectrum of target genes and the fundamental nature of the cellular processes they control, it comes as no surprise that NFAT proteins are increasingly being recognized as central players in the development of a number of very different human malignancies.

Open Access Article

S6-Haploinsufficiency Activates the p53 Tumor Suppressor

Linda Panić, Jacques Montagne, Maja Cokarić and Siniša Volarević
Pages 20 - 24
http://dx.doi.org/10.4161/cc.6.1.3666
Abstract | PDF

The capacity to detect and appropriately respond to many different stresses that interfere with functional homeostasis is essential for survival. Recent evidence suggests that the nucleolus, the site of ribosome biogenesis, plays a critical role in sensing and responding to both external and internal stresses. To understand these processes, we have recently used a genetically defined in vivo mouse model in which ribosome biogenesis could be manipulated during oogenesis and embryo development. In these mice ribosomal biosynthesis is impaired by a conditional deletion of one allele of the gene encoding 40S ribosomal protein S6. Embryos from these animals fail during gastrulation, apparently due to a p53-dependent checkpoint being triggered, rather than a deficit in translational capacity. These findings imply that molecular mechanisms have evolved during mammalian evolution to strongly guard against potential heterozygosity for ribosomal protein genes.

Open Access Article

TOR-in(g) the Nucleus

Chi Kwan Tsang and X.F. Steven Zheng
Pages 25 - 29
http://dx.doi.org/10.4161/cc.6.1.3675
Abstract | PDF

Target of rapamycin (TOR) is a central component of the eukaryotic growth regulatory network. TOR controls the expression of diverse genes by all three RNA polymerases, including ribosomal biogenesis, utilization and transport of nutrients, and stress-related genes. Until recently, TOR was thought to be a classical signaling kinase that regulates transcription factors in the cytoplasm. However, our recent study shows that in yeast, TOR dynamically shuttles between the cytoplasm and nucleus, and binds to 35S ribosomal DNA (rDNA) promoter 1. Importantly, nuclear localization and promoter-binding is crucial for TOR to control RNA polymerase (Pol) I-dependent 35S rDNA transcription. In contrast, either cytoplasmic or nuclear TOR is sufficient to regulate Pol II-dependent transcription. These observations suggest that TOR in the nucleus plays an important role in gene regulation, and that TOR takes a multifaceted approach to control expression of different genes.

Open Access Article

A Mechanosensory System Controls Cell Shape Changes During Mitosis

Janet C. Effler, Pablo A. Iglesias and Douglas N. Robinson
Pages 30 - 35
http://dx.doi.org/10.4161/cc.6.1.3674
Abstract | PDF

Essential life processes are heavily controlled by a variety of positive and negative feedback systems. Cytokinesis failure, ultimately leading to aneuploidy, is appreciated as an early step in tumor formation in mammals and is deleterious for all cells. Further, the growing list of cancer predisposition mutations includes a number of genes whose proteins control mitosis and/or cytokinesis. Cytokinesis shape control is also an important part of pattern formation and cell-type specialization during multi-cellular development. Inherently mechanical, we hypothesized that mechanosensing and mechanical feedback are fundamental for cytokinesis shape regulation. Using mechanical perturbation, we identified a mechanosensory control system that monitors shape progression during cytokinesis. In this review, we summarize these findings and their implications for cytokinesis regulation and for understanding the cytoskeletal system architecture that governs shape control.

Open Access Article

Involvement of Cellular Membrane Traffic Proteins in Poliovirus Replication

George A. Belov and Ellie Ehrenfeld
Pages 36 - 38
http://dx.doi.org/10.4161/cc.6.1.3683
Abstract | PDF

Expression of viral genes results in the modulation of cellular metabolism to support virus replication. Poliovirus infection of HeLa cells induces a dramatic rearrangement of intracellular membrane structures: virtually all cellular organelles except mitochondria are converted into virus replication vesicles. Although this phenomenon is known for almost half a century, very few mechanistic details explaining this transformation are understood. Recently we found that small GTPases, Arf, key components of the secretory pathway, translocate to sites of poliovirus RNA replication in infected cells, and that two poliovirus proteins, 3A and 3CD, can independently induce such translocation in vitro. Our most recent work shows the recruitment to viral replication complexes of activators of Arf, specific guanine nucleotide exchange factors (GEFs). Diversion of these GEFs from their normal activities in the secretory pathway provides a plausible explanation for the inhibition of protein secretion in poliovirus-infected cells as well as for the sensitivity of poliovirus infection to brefeldin A, a drug known to inhibit the secretory pathway. Identification of these cellular components defines a new class of host factors involved in virus replication complex formation and allows us to propose a hypothesis for remodeling of endoplasmic reticulum membranes into poliovirus replication complexes.

Open Access Article

Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases

R. Öllinger, K. Yamashita, Martin Bilban, A. Erat, P. Kogler, M. Thomas, E. Csizmadia, A. Usheva, R. Margreiter and Fritz H. Bach
Pages 39 - 43
http://dx.doi.org/10.4161/cc.6.1.3700
Abstract | PDF

We have recently shown that the natural bile pigment bilirubin has antiproliferative effects on vascular smooth muscle cells (VSMCs). Bilirubin is the end product of heme catabolism mediated by heme oxygenases and has for decades been considered a toxic waste product of our bodies. However, 14 separate studies and a meta-analysis have documented an inverse correlation between atherosclerosis and the levels of bilirubin in normal individuals. Having high normal or supranormal levels of bilirubin is associated with less atherosclerotic-type disease as compared with that in individuals with low normal levels of bilirubin. This combined with experimental data showing anti-atherosclerotic properties of the enzyme heme oxygenase-1 encouraged us to hypothesize that bilirubin and its precursor biliverdin, would act to ameliorate components of atherosclerosis, in a manner similar to what has been shown with HO 1. Both did so in an animal model of restenosis in which vascular smooth muscle cell proliferation leads to intimal proliferation and causes narrowing of the vessels. We also analyzed the antiproliferative effects of the bile pigments in an in vitro system where bilirubin/biliverdin caused p53 dependent cell cycle arrest by hypophosphorylation of the retinoblastoma tumor suppressor protein in growth factor stimulated VSMCs.




PERSPECTIVES

Open Access Article

Polyclonal Tumors in the Mammalian Intestine: Are Interactions Among Multiple Initiated Clones Necessary for Tumor Initiation, Growth, and Progression?

Richard B. Halberg and William F. Dove
Pages 44 - 51
http://dx.doi.org/10.4161/cc.6.1.3651
Abstract | PDF

Studies in both man and mouse indicate that the majority of familial intestinal tumors are polyclonal being composed of cells from at least two distinct progenitors. The formation of polyclonal tumors in the mouse can be explained by short-range interactions between multiple initiated clones within one or two crypt diameters of each other. These clonal interactions might be critical, if not necessary, for initiation, growth, progression, or all three stages of tumorigenesis. This view is diametrically opposed to the widely held view that intestinal tumors are monoclonal and progress by clonal expansion. The data supporting the latter are neither extensive nor definitive. In addition, the results from a recent study indicate that earlier studies of tumor clonality were heavily biased because lineage patches in the intestinal epithelium of humans resulting from X-inactivation are relatively large. Consequently, hundreds of tumors from familial and sporadic cases need to be analyzed to accurately assess tumor clonality. Investigators must keep an open mind regarding the clonality of tumors in the mammalian intestine as new experimental approaches are developed which will eventually provide a definitive answer to this fundamental question in the field of cancer biology.

Open Access Article

Tumor Suppressor Dosage Regulates Stem Cell Dynamics during Aging

Catherine Gatza, Lynette Moore, Melissa Dumble and Lawrence A. Donehower
Pages 52 - 55
http://dx.doi.org/10.4161/cc.6.1.3667
Abstract | PDF

The ability of tissues to maintain homeostasis is dependent in part on the function of adult tissue stem cells, which have the capability to self-renew and differentiate into multiple lineages. It has been hypothesized that the ability of stem cells to maintain tissue homeostasis declines functionally with age and that this decline may account for many of the biological phenotypes associated with aging. Recently, tumor suppressors such as p53 have been implicated in both aging and the regulation of stem cell dynamics. Our recent findings suggest that p53 may impact hematopoietic stem cell (HSC) dynamics during mammalian aging.25 Utilizing mouse models of varying levels of p53 dosage, we have shown that alteration of p53 activity affects stem cell number, proliferation, and functionality with age. Several other recent studies have implicated other tumor suppressors in potential age-related regulation of HSC dynamics as well.30,37 These data support a model in which aging is caused in part by a decline in tissue stem cell regenerative function, regulated in part by tumor suppressors.

Open Access Article

The Role of VHL in the Regulation of E-Cadherin: A New Connection in an Old Pathway

Ryan C. Russell and Michael Ohh
Pages 56 - 59
http://dx.doi.org/10.4161/cc.6.1.3668
Abstract | PDF

The von Hippel-Lindau (VHL) protein is a critical regulator of the ubiquitous oxygen-sensing pathway. VHL is a component of an E3 ubiquitin ligase that targets the α subunit of hypoxia-inducible factor (HIFα) for ubiquitin-mediated destruction under normal oxygen tension. As a consequence of HIFα stabilization upon the functional loss of VHL, the cell constitutively upregulates the hypoxic program resulting in the inappropriate expression of genes responsible for global changes in angiogenesis, energy metabolism, and proliferation. The emerging evidence suggests that the inactivation of VHL-HIF pathway is critical for the development of clear-cell renal cell carcinoma (CC-RCC). Until recently, the downstream effector(s) of HIF responsible for the oncogenic transformation of renal epithelial cells remained largely unknown. This review highlights recent discoveries uncovering the transcriptional regulation of E-cadherin, a homophilic cell adhesion molecule with anti-invasive properties in numerous epithelial-derived cancers, via the VHL-HIF pathway.

Open Access Article

Cysteine Cathepsins and the Cutting Edge of Cancer Invasion

Vasilena Gocheva and Johanna A. Joyce
Pages 60 - 64
http://dx.doi.org/10.4161/cc.6.1.3669
Abstract | PDF

Cysteine cathepsins are a family of lysosomal proteases that are often upregulated in various human cancers, and have been implicated in distinct tumorigenic processes such as angiogenesis, proliferation, apoptosis and invasion. During cancer progression, cathepsins are often translocated to the cell surface of tumor cells or are secreted into the extracellular milieu, where they can promote tumor invasion through several possible mechanisms. First, they can directly cleave components of the extracellular matrix and basement membrane, essentially clearing a path for the migration of tumor cells away from the primary tumor mass. Second, at the cell membrane, cathepsins can direct a proteolytic cascade in which they activate other proteases such as matrix metalloproteinases and urokinase plasminogen activator, which in turn promote invasion. Finally, cleavage of the cell adhesion protein, E-cadherin, at the cell surface can disrupt adherens junctions and thus facilitate cancer cell migration and invasion. Therefore, cathepsins are now emerging as major players in tumor progression, making them potential drug targets for a wide range of human cancers.

Open Access Article

Controlling Gene Expression through RNA Regulons: The Role of the Eukaryotic Translation Initiation Factor eIF4E

Biljana Culjkovic, Ivan Topisirovic and Katherine L.B. Borden
Pages 65 - 69
http://dx.doi.org/10.4161/cc.6.1.3688
Abstract | PDF

The eukaryotic translation initiation factor eIF4E is a potent oncogene.1 In fact, its overexpression in human cancer often correlates with poor prognosis.1, 2 Traditionally, eIF4E plays a role in translation initiation where it binds the 5’ m7G cap found on mRNAs.3 More recent studies indicate that a significant fraction of eIF4E (up to 68%) resides in the nucleus where it regulates the nuclear export of specific mRNAs. Additionally, eIF4E may play a role in mRNA sequestration and stability in cytoplasmic processing bodies (P-bodies).4, 5 Our recent studies suggest that eIF4E governs cell cycle progression and cellular proliferation by coordinately orchestrating the expression of several genes at the post-transcriptional level. Hence, eIF4E functions as a central node of an RNA regulon (described below), which plays an essential role in normal differentiation and development and is frequently dysregulated in cancer. Several cellular factors, such as the promyelocytic leukemia protein PML, modulate the function of this regulon by altering the activity of eIF4E. Here, the physiological implications of these observations are described and the clinical implications of directly targeting eIF4E, and the related regulon, are discussed.




ANALYTICAL REVIEW

Open Access Article

Mitotic Arrest and Cell Fate: Why and How Mitotic Inhibition of Transcription Drives Mutually Exclusive Events

Mikhail V. Blagosklonny
Pages 70 - 74
http://dx.doi.org/10.4161/cc.6.1.3682
Abstract | PDF

Here I discuss how the same mechanism - namely, inhibition of transcription during mitosis - can explain (a) apoptosis during mitotic arrest, (b) mitotic slippage, (c) G1 arrest after mitotic slippage and (d) secondary apoptosis after mitotic slippage. In fact, during mitotic arrest transcription is absent, thus depleting those short-lived proteins that have short-lived RNAs. Depletion of anti-apoptotic proteins (IAP, Mcl-1) can trigger apoptosis during prolonged mitotic arrest (apoptosis I). On the other hand, simultaneous depletion of cyclin B allows a cell to exit mitosis without division (mitotic slippage), thus preventing apoptosis I. Depletion of Mdm-2 causes accumulation of p53 during mitotic arrest. If a cell exits mitosis, transcription resumes. In turn the accumulated p53 induces p21 and Bax (and other pro-apoptotic proteins). In turn p21 can cause G1 arrest, whereas Bax can cause apoptosis II. Also, I discuss that mitotic depletion of short-lived proteins collaborates with mitotic phosphorylation of p53, Bcl-2 and BclxL. Finally this article clarifies notions of apoptosis-prone and -reluctant cells and mitotic catastrophe.




BRIEF REPORTS

Open Access Article

The Nuclear Envelope and Spindle Pole Body-Associated Mps3 Protein Bind Telomere Regulators and Function in Telomere Clustering

Lisa M. Antoniacci, Margaret Kenna and Robert V. Skibbens
Pages 75 - 79
http://dx.doi.org/10.4161/cc.6.1.3647
Abstract | PDF

It has long been posited that the nuclear envelope is a key regulator of both the spatial organization of chromatin and gene transcription. Mps3p is an integral nuclear envelope membrane protein with a single trans-membrane domain that is essential for spindle pole body duplication. More recently, Mps3p was shown to associate with the cohesion establishment factor Ctf7p and found to be critical for cohesion establishment. Here, we provide new evidence that the nuclear envelope, via Mps3p, plays a pivotal role in telomere foci formation. Results from in vitro pull-downs and in vivo co-precipitations also show that Mps3p associates with the telomerase-assembly component Est1p. Moreover, pair-wise combinations of mps3, est1 or ctf7 alleles all produce conditional lethality. Findings that Mps3p and the nuclear envelope recruit/sequester soluble chromatin metabolism factors such as Ctf7p and Est1p describe, at the molecular level, a new mechanism of nuclear envelope-dependent chromatin regulation.

Open Access Article

CBF-1 (RBP-Jk) Binds to the PTEN Promoter and Regulates PTEN Gene Expression

Jarrett T. Whelan, Siiri L. Forbes and Fred E. Bertrand
Pages 80 - 84
http://dx.doi.org/10.4161/cc.6.1.3648
Abstract | PDF

The PTEN gene regulates multiple signaling pathways that influence cell proliferation, survival and differentiation. Loss of PTEN expression is closely linked with oncogenesis. Little is known regarding regulation of PTEN gene expression. The PTEN promoter region has been reported and is regulated in part by p53. In a previous study, we found that Notch-1 signaling resulted in increased PTEN protein expression. Herein, we tested the hypothesis that the PTEN gene is a direct target of Notch-1 signal transduction, through binding of the Notch-activated transcription factor CBF-1 to the PTEN minimal promoter. 293 cells expressing constitutively active Notch-1 exhibited increased PTEN gene expression and promoter transactivation. Overexpression of CBF-1 in 293 cells resulted in decreased PTEN gene expression. Mobility shift assays and supershift assays demonstrated that CBF-1 binds to the PTEN minimal promoter. These data indicate that the Notch-1 receptor pathway is a key regulator of PTEN gene transcription.




ANALYTICAL REPORT

Open Access Article

Exploring Mechanisms of the DNA-Damage Response: p53 Pulses and their Possible Relevance to Apoptosis

Tongli Zhang, Paul Brazhnik and John J. Tyson
Pages 85 - 94
http://dx.doi.org/10.4161/cc.6.1.3705
Abstract | PDF

The transcription factor p53 plays a central role in maintaining genomic integrity. Recent experiments in MCF7 cells have shown that p53 protein level rises and falls in distinct pulses in response to DNA damage. The amplitudes of and intervals between pulses seem to be independent of the extent of damage, and some cells generate regular pulses of p53 over many days. Identifying the molecular mechanisms responsible for such interesting behavior is an important and challenging problem. This paper describes four dual-feedback mechanisms that combine both positive and negative feedback loops, which have been identified in the signaling network responsible for p53 regulation. Mathematical models of all four mechanisms are analyzed to determine if they are consistent with experimental observations and to characterize subtle differences among the possible mechanisms. In addition, a novel molecular mechanism is proposed whereby p53 pulses may induce, at first, cell cycle arrest and, if sustained, cell death. The proposal accounts for basic features of p53-mediated responses to DNA damage and suggests new experiments to probe the dynamics of p53 signaling.




REPORTS

Open Access Article

CYFIP2, a Direct p53 Target, is Leptomycin-B Sensitive

Roger S. Jackson II, Yong-Jig Cho, Susanne Stein and Peng Liang
Pages 95 - 103
http://dx.doi.org/10.4161/cc.6.1.3665
Abstract | PDF

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes, we have identified Cytoplasmic FMR Interacting Protein 2 (CYFIP2) as a p53-inducible gene. Here we show that the CYFIP2 promoter contains a p53-responsive element that confers p53 binding as well as transcriptional activation of a heterologous reporter. Inducible expression of CYFIP2 is sufficient for caspase activation and cellular apoptosis, reminiscent of p53 activation. Together, these results suggest that CYFIP2 is a direct p53 target gene that may be part of a redundant network of genes responsible for p53-dependent apoptosis. In addition, the sensitivity of CYFIP2 protein subcellular localization to Leptomycin-B, a Crm-1/Exportin inhibitor, suggests that the biological functions of CYFIP2 may extend from the cytoplasmic compartment into the nucleus of the cell.

Open Access Article

Pharmacological Abrogation of S-Phase Checkpoint Enhances the Anti-Tumor Activity of Gemcitabine In Vivo

David J. Matthews, Michael Yakes, Jason Chen, Michele Tadano, Lester Bornheim, Douglas O. Clary, Albert Tai, Jill M. Wagner, Nicole Miller, Yong D. Kim, Scott Robertson, Louis Murray and Larry M. Karnitz
Pages 104 - 110
http://dx.doi.org/10.4161/cc.6.1.3699
Abstract | PDF

Chk1 and Chk2 kinases are critically involved in modulating DNA damage checkpoints. In particular, Chk1, a key activator of the S-phase DNA damage response, may be involved in resistance to genotoxic therapies that target DNA synthesis. We studied the in vitro and in vivo effects of EXEL-9844 (XL844), a potent, orally available, and specific inhibitor of Chk1 and Chk2, in combination with gemcitabine. In clonogenic assays using multiple cell lines in vitro, EXEL-9844 had only minor effects as a single agent but substantially enhanced gemcitabine-induced cell killing. Correspondingly, in PANC-1 cells, EXEL-9844 increased gemcitabine-induced H2AX phosphorylation, blocked Cdc25A phosphorylation, and induced premature mitotic entry. In a PANC-1 xenograft model, EXEL-9844 significantly enhanced gemcitabine antitumor activity but had limited effect as a single agent. Together, these data show that cell cycle checkpoint inhibitors may have significant clinical utility in potentiating the activity of gemcitabine.