Table of Contents

Volume 6, Issue 21

  November 1, 2007

  Pages 2585 - 2718

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 Issue Catalogue (MARC XML)

EXTRA VIEWS

Open Access Article

Let-7 Prevents Early Cancer Progression by Suppressing Expression of the Embryonic Gene HMGA2

Sun-Mi Park, Scott Shell, Amir Reza Radjabi, Robert Schickel, Christine Feig, Ben Boyerinas, Daniela M. Dinulescu, Ernst Lengyel and Marcus E. Peter
Pages 2585 - 2590
http://dx.doi.org/10.4161/cc.6.21.4845
Abstract | PDF

The microRNA let-7 regulates late embryonic development by suppressing expression of a number of genes such as c-myc and RAS as well as the embryonic gene high mobility group, A2 (HMGA2). We now demonstrate that HMGA2 is more efficiently targeted by let-7 than RAS. Its expression inversely correlates with the expression of let-7 in the NCI60 cells lines, and the expression of RAS does not change when amounts of let-7 that efficiently silence expression of HMGA2 are introduced into tumor cells. We did not find a difference in the expression of HMGA2 between primary ovarian cancer samples and matching metastases, suggesting that the expression of HMGA2 represents an early event during cancer progression. The late repression of HMGA2 by let-7 during embryonic development, and the early reexpression of HMGA2 during cancer development, is in line with the hypothesis that cancer development represents a case of reverse embryogenesis.

Open Access Article

Interaction Between the Cdk2/Cyclin A Complex and a Small Molecule Derived from the pRb2/p130 Spacer Domain: A Theoretical Model

Antonio Giordano, Emanuele Bellacchio, Luigi Bagella and Marco G. Paggi
Pages 2591 - 2593
http://dx.doi.org/10.4161/cc.6.21.4878
Abstract | PDF

Retinoblastoma (RB) family proteins pRb, p107 and pRb2/p130 are important cellular factors which play a well-recognized role as tumor and growth suppressors. These proteins are actively involved in the negative control of the cell cycle and their function is modulated via complex homeostatic processes, most of them involving post-translational regulation of their phosphorylation status. Interestingly, the family members p107 and pRb2/p130 share the ability to physically interact and inhibit the kinase activity of the Cdk2/Cyclin A and Cdk2/Cyclin E complexes. Regarding pRb2/p130, its inhibitory effect on the Cdk2/Cyclin A activity has been attributed to the “spacer” region. Recently, a 39 aa-long pRb2/p130 spacer-derived peptide (Spa310, aa 641-679) has been selected as the sequence responsible for Cdk2/Cyclin A inhibition. Following the identification of this active sequence, here we propose a computer-generated three-dimensional model of the interaction between the Cdk2/Cyclin A complex and the N-terminal 9-amino acid sequence of the Spa310 peptide. We believe this model as useful for the rational development of peptide or peptidomimetic kinase inhibitors to be used for the negative modulation of cell cycle in cancer cells.

Open Access Article

Stimulus-Specific Transcriptional Regulation Within the p53 Network

Aaron Joseph Donner, Jennifer Michelle Hoover, Stephanie Aspen Szostek and Joaquín M. Espinosa
Pages 2594 - 2598
http://dx.doi.org/10.4161/cc.6.21.4893
Abstract | PDF

The p53 transcriptional network is composed of hundreds of effector genes involved in varied stress-response pathways, including cell cycle arrest and apoptosis. It is not clear how distinct p53 target genes are differentially activated to trigger stress-specific biological responses. We analyzed the p53 transcriptional program upon activation by two DNA-damaging agents, UVC and doxorubicin, versus the non-genotoxic molecule Nutlin-3. In colorectal cancer cells, UVC triggers apoptosis, doxorubicin induces transient cell cycle arrest followed by apoptosis, and Nutlin-3 leads to cell cycle arrest with no significant apoptosis. Quantitative gene expression analysis allowed us to group p53 target genes into three main classes according to their activation profiles in each scenario. The CDK-inhibitor p21 was classified as a Class I gene, being significantly activated under cell cycle arrest conditions (i.e. doxorubicin and Nutlin-3) but not during UVC-induced apoptosis. Chromatin immunoprecipitation analysis of the p21 locus indicates that the level of p53-dependent transcription is determined by the effects of stimulus-specific transcriptional coregulators acting downstream of p53 binding and histone acetylation. In particular, our analysis indicates that the subunits of the CDK-module of the human Mediator complex function as stimulus-specific positive coregulators of p21 transcription.

Open Access Article

Control of Cell Cycle and Cell Growth by Molecular Chaperones

Martí Aldea, Eloi Garí and Neus Colomina
Pages 2599 - 2603
http://dx.doi.org/10.4161/cc.6.21.4920
Abstract | PDF

Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G1 and released by specific chaperones in late G1 to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.

Open Access Article

Modulation of the p53-MDM2 Interaction by Phosphorylation of Thr18: A Computational Study

Hui Jun Lee, Deepa Srinivasan, David Coomber, David P. Lane and Chandra S. Verma
Pages 2604 - 2611
http://dx.doi.org/10.4161/cc.6.21.4923
Abstract | PDF

The transcription factor p53 is under negative regulation by the ubiquitin ligase MDM2 and its close homologue MDM4. In the bound complex between MDM2 and p53, the transactivation domain of p53 adopts an amphipathic helical conformation which optimizes the spatial organization of 3 key hydrophobic residues (Phe19, Trp23, Leu26) for maximum interactions. The interaction with MDM2 is known to be abrogated by phosphorylation of Ser/Thr residues in the MDM2 N-terminal domain and in the p53 transactivation domain. In the latter, phosphorylation of Thr18 has been attributed to destabilize a key hbond between Thr18 and Asp21. This interaction has been thought to be critical for the formation of the helical conformation of the p53 transactivation domain. Molecular dynamics simulations of the p53 transactivation domain suggest that phosphorylation of either Thr18 or Ser20 does not disrupt its helical structure but does result in reduced affinities for MDM2. While interactions between the Thr18 and Asp21 are indeed broken due to charge-charge repulsions, the peptide has enough inherent flexibility to form alternate patterns of hbonds, resulting in the maintenance of helicity. Electrostatics of MDM2 reveal local anionic patches in the region where Thr18 docks. These suggest that repulsions will arise because the MDM2 surface will force the p53 to bind in a manner that will place the negatively charged phosphorylated Thr18 near this anionic region. A similar, albeit somewhat attenuated pattern of electrostatic modulations, is seen for a model of MDM4 that has been built. Mutants of MDM2 and MDM4 have been designed to attenuate this anionicity and have been computationally demonstrated to enhance the binding of the phosphorylated peptides.




PERSPECTIVES

Open Access Article

AIF-Mediated Programmed Necrosis: A Highly Orchestrated Way to Die

Hanan Boujrad, Olena Gubkina, Nadine Robert, Slavica Krantic and Santos A. Susin
Pages 2612 - 2619
http://dx.doi.org/10.4161/cc.6.21.4842
Abstract | PDF

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly regulated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.

Open Access Article

Effects of Hypoxia on Heterotypic Macrophage Interactions

Benjamin T. Spike and Kay F. Macleod
Pages 2620 - 2624
http://dx.doi.org/10.4161/cc.6.21.4879
Abstract | PDF

The role of macrophages in modulating the systemic response to hypoxia and oxidative stress is emerging from basic biological processes, such as the regulation of red blood cell production, and from analysis of tumor progression, as a key factor determining whether cells survive, proliferate or differentiate under micro-environmental pressures. Our recent work identified a novel role for macrophages in promoting expansion of erythroid progenitors in vitro while confirming previous data that macrophages are not required for red cell enucleation. This work emerged from analyses of hypoxia and cell death in the Rb null fetal liver where we demonstrated that defects in erythropoietic islands were due to deterioration in the fetal liver microenvironment that disrupted heterotypic interactions of macrophages with erythroblasts and not to intrinsic defects in Rb null macrophages. The significance of these findings for the effect of hypoxia on macrophage interactions and activity during tumor progression is also discussed.

Open Access Article

Turning the Tables: Myc Activates Wnt in Breast Cancer

Victoria H. Cowling and Michael D. Cole
Pages 2625 - 2627
http://dx.doi.org/10.4161/cc.6.21.4880
Abstract | PDF

Previous molecular and genetic data implicate the c-myc gene as a critical downstream effector of the Wnt/TCF pathway in colon cancer. However, the involvement of c-myc in mammary epithelial cell transformation had not been explored. We recently showed that c-Myc induces a profound morphological transformation in human mammary epithelial cells accompanied by anchorage-independent growth. The mechanism of c-Myc transformation was revealed in part through the finding that, in contrast to colon cancer, c-Myc activates the Wnt pathway and endogenous TCF activity by suppressing the Wnt inhibitors DKK1 and SFRP1. Notably, DKK1 and SFRP1 were found to be strongly suppressed in human breast cancer cell lines and their re-expression inhibited the transformed phenotype. We demonstrated that breast cancer cells become dependent on repression of the Wnt inhibitors for cell proliferation, i.e. they have acquired an “oncogene addiction”, suggesting that the Myc-Wnt pathway is an attractive therapeutic target. We propose that a positive feedback loop of c-myc and Wnt signaling operates in breast cancer.

Open Access Article

Canonical and Alternative MAPK Signaling

Genaro Pimienta and Jaime Pascual
Pages 2628 - 2632
http://dx.doi.org/10.4161/cc.6.21.4930
Abstract | PDF

The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38α/β. Also, a form of dosage compensation between homologues has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.




REVIEWS

Open Access Article

Deciphering AP-1 Function in Tumorigenesis: Fra-ternizing on Target Promoters

Pasquale Verde, Laura Casalino, Francesco Talotta, Moshe Yaniv and Jonathan B. Weitzman
Pages 2633 - 2639
http://dx.doi.org/10.4161/cc.6.21.4850
Abstract | PDF

Multi-gene families of transcription factors pose a formidable challenge to molecular and functional analysis. Dissecting distinct functions for individual family members requires a combination of approaches in different cellular and animal models. The AP-1 transcription factor complex serves as a paradigm for understanding the dynamics of transcriptional regulation. Knockout, knockdown and transgenic strategies, inducible alleles, mutational analysis, chemical genetics, etc.; researchers have applied all the tricks of the trade to understand how AP-1 works. AP-1 refers to a mixture of dimers formed between members of the Jun, Fos and ATF families. The complexity of the AP-1 biological functions reflects the wide combinatorial diversity of its components.¹ AP-1 has been linked to cancer and neoplastic transformation ever since the first jun and fos genes were cloned as cellular homologues of viral oncogenes twenty years ago. Because of the oncogenic or tumor suppressive activity exhibited by distinct Jun and Fos nuclear proteins depending on the cell context and the genetic background of the tumor, the AP-1 complex has been called a “double-edged sword” in tumorigenesis.² The cumulating results over the last decade are finally leading to the identification of specific functions for individual AP-1 components and their contribution to neoplastic disease. Here, we focus on the Fra-1 protein in tumorigenesis, which offers an illustrative example of this helter-skelter voyage.

Open Access Article

Morphogens and the Control of Cell Proliferation and Patterning in the Spinal Cord

Fausto Ulloa and James Briscoe
Pages 2640 - 2649
http://dx.doi.org/10.4161/cc.6.21.4822
Abstract | PDF

The development of animal embryos depends on accurate coordination of the growth and specification of precursor cells. Morphogens, extracellular signals that act at a distance to control cell fate, are crucial in the patterning of embryonic tissues. One of the most extensively studied examples of a morphogen patterned tissue is the developing vertebrate spinal cord. The distribution of distinct neuronal subtypes along the dorsoventral (DV) axis of the spinal cord is determined by counteracting gradients of long-range signals. Wnt and BMP signals promote dorsal identities, while Shh signaling induces ventral identities. Simultaneous to their specification, neural progenitors proliferate, facilitating the growth of the neural tube. In this review we discuss evidence indicating that the signals governing progenitor specification also control proliferation and survival of progenitor cells. Moreover, evidence of reciprocal transcriptional interactions and cross-talk between the signaling pathways has emerged from recent studies. Together these studies suggest ways in which patterning and growth may be coordinated in the spinal cord. One level of interaction is an inhibitory regulation of repressor forms of the transcription factor Gli3—generated in the absence of Shh—on β-catenin activity, the transcription factor activated by Wnt signaling. This interaction may also be relevant in other tissues and situations in which the two signaling pathways are known to participate.

Open Access Article

The Role of Gas1 in Embryonic Development and its Implications for Human Disease

David C. Martinelli and Chen-Ming Fan
Pages 2650 - 2655
http://dx.doi.org/10.4161/cc.6.21.4877
Abstract | PDF

Growth Arrest Specific Gene 1 (Gas1) has long been regarded as a cell cycle inhibitor of the G0 to S phase transition. How GAS1, a GPI-anchored plasma membrane protein, directs intracellular changes without an extracellular ligand or a transmembrane protein partner has been puzzling. A recent series of biochemical and molecular genetic studies assigned the mammalian Hedgehog (HH) growth factor to be a ligand for GAS1 in vitro and in vivo. HH has enjoyed considerable attention for its profound role in embryonic patterning as a classic morphogen, i.e. inducing various cell types in a concentration-dependent manner. GAS1 appears to help transform the HH concentration gradient into its morphogenic activity gradient by acting cooperatively with the HH receptor, the 12-transmembrane protein Patched 1 (PTC1). These findings provoke intriguing thoughts on how HH and GAS1 may coordinate cell proliferation and differentiation to create biological patterns. The role of HH extends to human genetic diseases, stem cell renewal, and cancer growth, and we consider the possibility of GAS1’s involvement in these processes as well.

Open Access Article

Tiny Actors, Great Roles: microRNAs in p53's Service


Nina Raver-Shapira and Moshe Oren
Pages 2656 - 2661
http://dx.doi.org/10.4161/cc.6.21.4915
Abstract | PDF

In the course of the last several years, microRNAs (miRNAs) have become a focus of great interest, owing to their unsuspected important roles in the regulation of many critical biological processes. Not surprisingly, miRNAs are also turning out to be intimately involved in cancer, through either excessive or decreased activity. A series of recent studies reveal a close link between miRNAs and the p53 tumor suppressor: as a transcription factor, p53 controls the expression of specific miRs, and this additional capacity of p53 contributes to its biological activities. This Perspective will discuss the recent studies and their implications.




BRIEF REPORT

Open Access Article

Inhibition of Paclitaxel-Induced Proteasome Activation Influences Paclitaxel Cytotoxicity in Breast Cancer Cells in a Sequence-Dependent Manner

Héctor Hernández-Vargas, Cayetano von Kobbe, Carolina Sánchez-Estévez, Mercedes Julián-Tendero, José Palacios and Gema Moreno-Bueno
Pages 2662 - 2668
http://dx.doi.org/10.4161/cc.6.21.4821
Abstract | PDF

Although the anti-tumour effects of paclitaxel result mainly from mitotic arrest, recent evidences suggest alternative mechanisms of cytotoxicity. Cell cycle, cell death, and gene expression assays were used to understand the molecular mechanisms of paclitaxel cytotoxicity in breast cancer cells. G₂/M cell cycle arrest and cell death coincided with the regulation of genes involved in cell death, cell cycle control, microtubule-based processes, oxidative stress, and ubiquitin-proteasome system. Induction of proteasome genes was also correlated with an accumulation of protein for proteasome subunits. Furthermore, a schedule-dependent regulation of paclitaxel-induced cytotoxicity was observed after combining paclitaxel and the proteasome inhibitor MG132. Proteasome inhibition after paclitaxel exposure induced the highest rate of growth inhibition and apoptosis, with no effect on mitotic arrest. These findings give support to clinical combinations of taxanes with proteasome inhibitors, outlining the importance of considering the sequence when designing such regimens.




PRIORITY REPORT

Open Access Article

SIRT3 is Pro-Apoptotic and Participates in Distinct Basal Apoptotic Pathways

Simon J. Allison and J Milner
Pages 2669 - 2677
http://dx.doi.org/10.4161/cc.6.21.4866
Abstract | PDF

SIRT3, one of seven mammalian sirtuins, is a NAD-dependent deacetylase. SIRT3 localises to mitochondria where it deacetylates and thus activates acetyl-CoA synthetase 2 (AceCS2), indicating a role for SIRT3 in metabolism. Here we provide evidence that SIRT3 also impacts upon apoptosis and cell growth control. Using RNAi under basal (non-stress) conditions we show that SIRT3 is required for apoptosis induced by selective silencing of Bcl-2 in HCT116 human epithelial cancer cells. Identical treatment of ARPE19 epithelial non-cancer cells induces G1 growth arrest which also proved to be SIRT3-dependent. Previously we have identified SIRT1 and JNK2 as constitutive suppressors of apoptosis in HCT116 cells. We now demonstrate that SIRT3 functions in JNK2-regulated apoptosis but is dispensable for SIRT1-regulated apoptosis. SIRT3 is also dispensable for stress-induced apoptosis. Thus the pro-apoptotic functioning of SIRT3 is selectively coupled with defined pathways regulating cell survival under basal conditions.




REPORTS

Open Access Article

Loss of CABLES1, a Cyclin-dependent Kinase-interacting Protein that Inhibits Cell Cycle Progression, Results in Germline Expansion at the Expense of Oocyte Quality in Adult Female Mice

Ho-Joon Lee, Hideo Sakamoto, Hongwei Luo, Malgorzata E. Skaznik-Wikiel, Anne M. Friel, Teruko Niikura, Jacqueline C. Tilly, Yuichi Niikura, Rachael Klein, Aaron K. Styer, Lawrence R. Zukerberg, Jonathan L. Tilly and Bo. R. Rueda
Pages 2678 - 2684
http://dx.doi.org/10.4161/cc.6.21.4820
Abstract | PDF

Recent studies have shown that cell cycle inhibitors encoded by the Ink4a gene locus constrain the self-renewing activity of adult stem cells of the hematopoietic and nervous systems. Here we report that knockout (KO) of the Cables1 [cyclin-dependent kinase (CDK)-5 and ABL enzyme substrate 1] cell cycle-regulatory gene in mice has minimal to no effect on hematopoietic stem cell (HSC) dynamics. However, female Cables1-null mice exhibit a significant expansion of germ cell (oocyte) numbers throughout adulthood. This is accompanied by a dramatic elevation in the number of atretic immature oocytes within the ovaries and an increase in the incidence of degenerating oocytes retrieved following superovulation of CABLES1-deficient females. These outcomes are not observed in mice lacking p16^INK4a alone or both p16^INK4a and p19^ARF. These data support recent reports that adult female mice can generate new oocytes and follicles but the enhancement of postnatal oogenesis by Cables1 KO appears offset by a reduction in oocyte quality, as reflected by increased elimination of these additional germ cells via apoptosis. This work also reveals cell lineage specificity with respect to the role that specific CDK-interacting proteins play in restraining the activity of adult germline versus somatic stem cells.

Open Access Article

p53 is Nuclear and Functional in Both Undifferentiated and Differentiated Neuroblastoma

Lindi Chen, Archie J. Malcolm, Katrina M. Wood, Michael Cole, Sadick Variend, Catherine Cullinane, Andrew D.J. Pearson, John Lunec and Deborah A. Tweddle
Pages 2685 - 2696
http://dx.doi.org/10.4161/cc.6.21.4853
Abstract | PDF

Aberrant cytoplasmic sequestration has been reported as an alternative mechanism of p53 inactivation to mutation in neuroblastoma. We hypothesized that p53 localization and function in neuroblastoma is related to differentiation status. Eighty-two untreated and 24 paired pre and post-chemotherapy neuroblastomas were studied by immunocytochemistry for p53, p21^WAF1, BAX, Bcl2 and Ki67. Predominantly nuclear p53 was detected in undifferentiated neuroblastoma, and both nuclear and cytoplasmic p53 in differentiating neuroblastoma. The nuclear p53 labeling index (LI) correlated with the Ki67 LI (r = 0.51, p <0.001), and weakly with p21^WAF1 (r = 0.37), but not with BAX or Bcl2. There was a significant reduction in p53, p21^WAF1 and Ki67 LI after chemotherapy (p < 0.01), an increase in BAX (p <0.05), but no change in Bcl2. p53 localization and function were examined in two p53 wild-type undifferentiated and 9-cis retinoic acid differentiated neuroblastoma cell lines. Using immunocytochemistry, immunofluorescence and cell fractionation, p53 was found to be predominantly nuclear in both undifferentiated and differentiated cells. Following irradiation, there was upregulation of p53, p21^WAF1 and MDM2, but less induced PARP and caspase 3 cleavage in differentiated cells, suggesting intact p53 transcriptional function, but resistance to apoptosis. p53 function in undifferentiated and differentiated cells was confirmed by upregulation of p21^WAF1 and MDM2 following Nutlin-3 treatment. In conclusion, p53 is predominantly nuclear and functional in neuroblastoma regardless of differentiation status.

Open Access Article

E2F-Dependent Induction of p14ARF During Cell Cycle Re-entry in Human T Cells

Ana Gutierrez del Arroyo, Selma El Messaoudi, Paula A. Clark, Marion James, Francesca Stott, Adrian Bracken, Kristian Helin and Gordon Peters
Pages 2697 - 2705
http://dx.doi.org/10.4161/cc.6.21.4857
Abstract | PDF

The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have recently been attributed to a variant E2F binding site but attempts to map specific response elements within the ARF promoter have generally yielded confusing answers. Here we show that in IL2-dependent T-lymphocytes, ARF expression is induced as cells progress from G0 into S phase, in parallel with other bona fide E2F target genes. This is accompanied by increased association of E2F1 with the endogenous ARF promoter. Our findings suggest that the ability of ARF to register normal proliferative cues depends on the levels of E2F generated in different settings and argue against the idea that it reacts exclusively to oncogenic signals.

Open Access Article

Imaging of Nucleolar Dynamics During the Cell Cycle of Cancer Cells in Live Mice

Kensuke Yamauchi, Meng Yang, Katsuhiro Hayashi, Ping Jiang, Norio Yamamoto, Hiroyuki Tsuchiya, Katsuro Tomita, A.R. Moossa, Michael Bouvet and Robert M. Hoffman
Pages 2706 - 2708
http://dx.doi.org/10.4161/cc.6.21.4861
Abstract | PDF

The synthesis and assembly of ribosomal subunits take place in the nucleolus. The nucleolus forms in the nucleus around the repeated ribosomal gene clusters and undergoes cyclic changes during the cell cycle. Although the nucleolus is easily visualized by light microscopy of cells in vitro, the nucleolus has not been imaged in cells in vivo. We report here development of a mouse model to visualize the nucleolus cycle of cancer cells in live mice. HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. The nucleolus was visualized by contrast to the fluorescence of GFP expressed in the nucleus. HT-1080 dual-color cells were seeded on the surface of a skin-flap of nude mice. The inside surface of the skin-flap was directly imaged with a laser scanning microscope 24 hours after seeding. The nucleoli of the cancer cells were clearly imaged in real-time. The appearance of the nucleoli changed dramatically during the cell cycle. During mitosis, the nucleolus disappeared. After mitosis, the nucleoli decreased in number and increased in size. The nucleolus appears to have a major role in cell cycle regulation effected at least in part by sequestering proteins which affect cell cycle progression. Nucleolar imaging could be used for more precise determination of cancer-cell position in the cell cycle in vivo.

Open Access Article

Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells

José E. Capó-Aponte, Zheng Wang, M.A. Murat Akinci, J. Mario Wolosin, Kathryn S. Pokorny, Pavel Iserovish and Peter S. Reinach
Pages 2709 - 2718
http://dx.doi.org/10.4161/cc.6.21.4881
Abstract | PDF

Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium–chloride cotransporter (KCC) in mediating (i) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (ii) increases in cell cycle progression; and (iii) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G0/G1-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G2/M-phase and accelerated the transition from G0/G1-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (i) activation of the p44/42MAPK signaling pathway, (ii) cell cycle progression, and (iii) increases in cell proliferation.