Table of Contents

Volume 4, Issue 12

  December 2005

  Pages 1693 - 1869

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 Issue Catalogue (MARC XML)

REVIEW OF CONCEPT

Open Access Article

Why Therapeutic Response May Not Prolong the Life of a Cancer Patient: Selection for Oncogenic Resistance

Mikhail V. Blagosklonny
Pages 1693 - 1698
http://dx.doi.org/10.4161/cc.4.12.2259
Abstract | PDF

Most cancers do not respond to chemotherapy. Disappointedly, even objective clinical responses to anticancer therapy often do not translate into improvements in overall survival. To explain the response-survival paradox, it has been pointed out that effective therapy is ineffective against cancer stem cells, which replenish the tumor (causing relapse). In contrast, I discuss that, according to this scenario, patient survival will be prolonged at least by the duration of remission. Furthermore, stem-cell-based relapses will be sensitive to the initial therapy, and in theory cancer could be controlled indefinitely. To explain the paradox, I discuss that effective therapy selects for resistance among proliferating cancer cells. Importantly, mechanisms of resistance can be divided into non-oncogenic (e.g., drug transporters and mutation in drug-targets) and oncogenic (apoptosis and cell cycle dysregulation). The latter is associated with highly aggressive cancer phenotype and, therefore, there is no increase in overall survival. I further suggest that (a) therapeutic response is a prerequisite for successful therapy, (b) resistance can be exploited for therapeutic advantage, (c) each response can be translated into increased survival, and (e) in slow growing tumors, cancer can be stopped without tumor shrinkage. Therapy will control cancer if it can selectively suppress proliferating cancer cells and will improve survival as long as acquired resistance can be exploited.




EXTRA VIEWS

Open Access Article

SUMO Keeps a Check on Recombination during DNA Replication

Helle D. Ulrich, Sabina Vogel and Adelina A. Davies
Pages 1699 - 1702
http://dx.doi.org/10.4161/cc.4.12.2194
Abstract | PDF

The small ubiquitin-related modifier SUMO plays an important role in the maintenance of genome stability. Accordingly, DNA replication, repair and recombination factors as well as mediators of chromosome dynamics and cohesion are among its many targets. Attachment of SUMO can modulate the properties of the modified proteins by affecting localization, conformation, stability or enzymatic activity, but often its mechanism of action remains poorly defined. Recent findings demonstrate how SUMO modification of PCNA, the processivity clamp for replicative DNA polymerases, prevents unscheduled recombination during DNA replication by means of directly enhancing physical interactions with an anti-recombinogenic helicase, Srs2. This review highlights how the SUMO conjugation system exerts its effect on the replication fork and discusses the implications for ubiquitin-dependent DNA damage tolerance.

Open Access Article

PTPRV is a Key Mediator of p53-Induced Cell Cycle Exit

Gilles Doumont, Alain Martoriati and Jean-Christophe Marine
Pages 1703 - 1705
http://dx.doi.org/10.4161/cc.4.12.2207
Abstract | PDF

The p53 tumor suppressor functions as a sequence-specific DNA-binding transcription factor that promotes antiproliferative responses, including cell cycle checkpoints, cellular senescence and apoptosis. The precise nature of the p53 transcriptional programs and the complex mechanisms that govern whether or not a cell dies in response to p53 activation remain elusive. We have recently reported the identification of a new direct p53 target, Ptprv, encoding a transmembrane tyrosine phosphatase. Ptprv expression is dramatically and preferentially increased in cells undergoing p53-dependent cell cycle exit, but not in cells undergoing p53-mediated apoptosis. Importantly, while p53-induced apoptosis is intact in mice lacking Ptprv, Ptprv-null cells are defective in G1 checkpoint control. In addition, we report herein that Ptprv is induced at high cell density and mediates contact inhibition of cell growth. Together, the data suggest that Ptprv is a potent inhibitor of cell proliferation and a critical mediator of p53-induced cell cycle exit.

Open Access Article

New Talents for an Old Acquaintance: the SR Protein Splicing Factor ASF/SF2 Functions in the Maintenance of Genome Stability

Xialu Li and James L. Manley
Pages 1706 - 1708
http://dx.doi.org/10.4161/cc.4.12.2210
Abstract | PDF

ASF/SF2 is a well-studied SR protein that plays important roles in pre-mRNA splicing and other aspects of RNA metabolism. Genetic inactivation experiments have revealed the fundamental roles of ASF/SF2 and other SR proteins in cell viability and animal development. However, the nature of the events triggered by in vivo depletion of ASF/SF2 remained largely elusive. Recently, we have demonstrated a significant function of ASF/SF2 in the maintenance of genome stability by preventing the formation of R loops, which provided new insights into the essential roles of ASF/SF2 in cellular physiology.

Open Access Article

Cytokinesis and the Spindle Midzone

Henrik Bringmann
Pages 1709 - 1712
http://dx.doi.org/10.4161/cc.4.12.2215
Abstract | PDF

At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: 1. The position of the presumptive cytokinesis furrow is specified. 2. A contractile ring is formed. 3. The contractile ring contracts, resulting in furrow ingression. 4. Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements.

Open Access Article

ATP-Dependent Chromatin-Remodeling Complexes in DNA Double-Strand Break Repair: Remodeling, Pairing and (Re)pairing

Jian Huang, Bing Liang, Jiajing Qiu and Brehon C. Laurent
Pages 1713 - 1715
http://dx.doi.org/10.4161/cc.4.12.2222
Abstract | PDF

The genomic integrity of a eukaryotic cell is challenged by over 10,000 chromosomal lesions per day. Therefore the cell has evolved efficient mechanisms to recognize, signal, and repair DNA breaks. Defects in any of these steps can lead to chromosomal aberrations and cancers. As these lesions must be repaired in the context of chromatin, both chromatin-modifying and nucleosomeremodeling enzymes have been implicated in DNA damage repair. We reported recently that the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes are involved in DSB repair specifically by homologous recombination. Here we discuss how such enzymes might be recruited to DNA breaks, why so many remodelers are recruited to sites of DSBs, and a possible functional connection between RSC’s roles in sister chromatid cohesion and DSB repair.

Open Access Article

AML1-ETO Needs a Partner: New Insights into the Pathogenesis of t(8;21) Leukemia

F. Kuchenbauer, M. Feuring–Buske and C. Buske
Pages 1716 - 1718
http://dx.doi.org/10.4161/cc.4.12.2256
Abstract | PDF

The detailed characterization of genetic and molecular aberrations in acute myeloid leukemia (AML) has substantially improved our understanding of the pathogenesis of this disease. With an incidence of up to 12% in all AML cases, the translocation t(8;21), forming the AML1-ETO fusion gene, is one of the most common genetic aberrations in AML. Experimental data have shown that AML1-ETO is not sufficient to induce leukemia by itself, but has to collaborate with other genetic alterations for leukemic transformation. These data are supported by observations in AML patients, who recurrently show activating mutations of the receptor tyrosine kinase FLT3 or c-KIT together with the AML1-ETO fusion gene. These findings might have clinical implications and provide a rationale to test RTK inhibitors in the treatment of patients with core binding factor AML and concurrent activating RTK mutations.

Open Access Article

The Intersection of Inheritance and Metastasis: The Role and Implications of Germline Polymorphism in Tumor Dissemination

Kent Hunter
Pages 1719 - 1721
http://dx.doi.org/10.4161/cc.4.12.2258
Abstract | PDF

Metastasis is an enormously complex process that involves both spatial and temporal barriers. Metastatic cells must not only acquire all of the characteristics of a primary tumor, but additionally must be capable of invasion, survival during transit and in the secondary site, interact productively with a novel microenvironment and proliferate to form a clinically relevant lesion 1. Adding complexity to the process is the fact that it can be years or even decades after diagnosis of the primary tumor before the secondary tumors are apparent. A number of models have been proposed to explain the origins of metastasis. However, while all of the models can account for some aspects of the experimental observations, suggesting they may be at least in part true, none adequately explain all of the data. This implies that the existing models are likely to be too simplistic and additional factors must be considered to adequately account for existing and newly emerging data.

Open Access Article

The Senescent Side of Tumor Suppression

Manuel Collado and Manuel Serrano
Pages 1722 - 1724
http://dx.doi.org/10.4161/cc.4.12.2260
Abstract | PDF

Neoplastic transformation of human cells is a rare event that requires overcoming anti-tumoral cellular responses. Oncogene-induced senescence (OIS) is considered a crucial tumor suppressor mechanism controlling unchecked proliferation driven by oncogenic mutation. However, the analysis of OIS has been restricted so far to cultured cells. Recently, we have identified novel molecular markers of OIS and we have demonstrated the occurrence of senescence using mouse models of oncogenic activation. Importantly, we have found that senescent cells are abundant in premalignant lesions of the skin, the lung, and the pancreas. In contrast, senescent cells were rare in the malignant lesions developed by these same animals. These observations, together with similar ones by other investigators, strongly argue for the occurrence of OIS in vivo and for its active role in restricting tumor development. These results open the possibility of using senescence markers as diagnostic and prognostic tools, and prompt the investigation on the potential therapeutical use of senescence-inducing drugs.




PERSPECTIVES

Open Access Article

CDK-Dependent Stabilization of Cdc6: Linking Growth and Stress Signals to Activation of DNA Replication

Anja M. Duursma and Reuven Agami
Pages 1725 - 1728
http://dx.doi.org/10.4161/cc.4.12.2193
Abstract | PDF

Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G1 phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.

Open Access Article

Thrombopoietin Stimulates Vascular Endothelial Cell Growth factor (VEGF) Production in Hematopoietic Stem Cells

Keita Kirito and Kenneth Kaushansky
Pages 1729 - 1731
http://dx.doi.org/10.4161/cc.4.12.2197
Abstract | PDF

Thrombopoietin (TPO) is a pivotal and non-redundant hematopoietic cytokine, supporting the survival, self-renewal activity and proliferation of hematopoietic stem and progenitor cells, the growth and differentiation of megakaryocytes, and the functional activation of their progeny, blood platelets. . TPO exerts these effects through regulating the abundance or subcellular localization of several transcription factors, including the homeodomain proteins HOXB4 and HOX A9. In addition to these effects, TPO helps orchestrate a cytokine-network in the bone marrow microenvironment that supports hematopoietic stem cell (HSC) function. In recent studies we have shown that TPO stimulates production of vascular endothelial cell growth factor (VEGF), another cytokine vital for HSC physiology, promoting their survival and expansion into committed hematopoietic progenitors. . Like several other effects of the cytokine, the effect of TPO on VEGF expression is mediated by stabilization and activation of the primary transcription factor responsible for VEGF expression, the oxygen tension responsive hypoxia inducible factor-1 (HIF-1). Together with the observation that bone marrow microenvironment is hypoxic and hypoxia simulates the repopulating activity of HSCs, our observations suggest that TPO mimics hypoxia and controls important genes required for HSC cycling, including VEGF, adding to our understanding of how the hormone contributes to HSC function.

Open Access Article

From Immature Lamin to Premature Aging: Molecular Pathways and Therapeutic Opportunities

Juan Cadiñanos, Ignacio Varela, Carlos López-Otín and José M.P. Freije
Pages 1732 - 1735
http://dx.doi.org/10.4161/cc.4.12.2202
Abstract | PDF

Accelerated aging or progeria has been a puzzling disease for many years. The recent findings involving the lamin A/FACE-1 (substrate/protease) system in the etiology of Hutchinson-Gilford progeria syndrome and related pathologies have shed some light on the mechanisms underlying the development of these devastating conditions. Thus, genetic defects in the nuclear envelope protein prelamin A or in the FACE-1 metalloprotease (also called Zmspte24) involved in prelamin A proteolytic maturation, causes the accumulation of an abnormal form of this protein and the subsequent disruption of nuclear envelope integrity. Recently, we and others have observed how this disruption leads to alterations in chromatin organization, genomic instability, transcriptional changes, and activation of a p53-linked signaling pathway. By using genetic manipulation approaches in mouse, we have shown that lowering prelamin A levels results in a total recovery of Zmpste24-deficient mice from the accelerated aging process. Moreover, p53 nullizygosity allows a modest but significant improvement in the premature aging phenotype, and contributes to delay the onset of the progeroid condition. On the basis of these results, we propose different potential therapeutic approaches that could be tested in Zmpste24-deficient mice. These strategies, some of which are based on existing drugs, might contribute to the development of effective treatments for these dramatic pathologies.

Open Access Article

Blimp1 and the Emergence of the Germ Line during Development in the Mouse

Mitinori Saitou, Bernhard Payer, Dónal O’Carroll, Yasuhide Ohinata and M. Azim Surani
Pages 1736 - 1740
http://dx.doi.org/10.4161/cc.4.12.2209
Abstract | PDF

To elucidate the mechanism for the specification of primordial germ cells (PGCs) in mice, we have developed and exploited the methods of single cell analysis. Based on these studies, we proposed a molecular programme associated with this process, a key event of which is the repression of homeobox genes that are, without exception, up regulated in somatic neighbors. We have now identified Blimp1, a potent transcriptional repressor of a histone methyltransferase subfamily, as a key regulator of PGC specification. Indeed, the unexpected early onset of Blimp1 expression in a few cells at the most proximal-posterior epiblast cells marks the origin of the germ cell lineage. Disruption of Blimp1 function resulted in aberrant PGC-like cells with a deregulated intrinsic gene expression programme at a very early stage, which demonstrates that Blimp1 is a critical determinant of the germ line in mice.

Open Access Article

Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma

Chitra Subramanian, Anthony W. Opipari, Valerie P. Castle and Roland P.S. Kwok
Pages 1741 - 1743
http://dx.doi.org/10.4161/cc.4.12.2212
Abstract | PDF

Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specially cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.

Open Access Article

Cancer Gene Discovery Using the Sleeping Beauty Transposon

Timothy K. Starr and David A. Largaespada
Pages 1744 - 1748
http://dx.doi.org/10.4161/cc.4.12.2223
Abstract | PDF

Epidemiological and molecular data support the hypothesis that cancer results from a series of acquired somatic mutations. Discovering the initial mutations required for oncogenesis has long been a goal of cancer research. To date, the majority of causative mutations have been identified based on their ability to act in a dominant fashion and/or because they are activated by chromosomal translocations. Forward genetic screens are necessary for unbiased discovery of the remaining unknown oncogenic mutations. Two recent projects have demonstrated the feasibility of using the Sleeping Beauty transposon as an insertional mutagen for cancer gene discovery. In this article we discuss the history of cancer gene discovery and propose novel forward genetic screens using Sleeping Beauty transposon aimed at specific tissues and accelerating the discovery of recessive tumor suppressor genes.

Open Access Article

Switching On-Off Snail: LOXL2 Versus GSK3?

Héctor Peinado, Francisco Portillo and Amparo Cano
Pages 1749 - 1752
http://dx.doi.org/10.4161/cc.4.12.2224
Abstract | PDF

Epithelial-mesenchymal transition (EMT) is considered as an essential determinant of carcinoma progression. The transcription factor Snail controls EMT by repressing Ecadherin gene expression and other epithelial genes. Snail protein stability and cellular localization is finely controlled by GSK3?-dependent phosphorylation and subsequent ubiquitination. GSK3? phosphorylates Snail at two different motifs which induce its nuclear export and association with ?-Trcp thus leading to Snail degradation. Recently, Snail was found to interact physical and functionally with LOXL2, a member of the lysyl oxidase gene family. Interestingly, LOXL2 seems to attenuate the GSK3?- dependent Snail degradation. Here, we discuss the relevance of this new potential mechanism of regulation and the role of LOXL2 during carcinoma progression.




REVIEW

Open Access Article

G-CSF (Granulocyte-Colony Stimulating Factor) in the Central Nervous System


Pages 1753 - 1757
http://dx.doi.org/10.4161/cc.4.12.2213
Abstract | PDF

G-CSF (Granulocyte-colony stimulating factor) is a hematopoietic growth factor that has been known for 20 years, and has been named for its role in the proliferation and differentiation of cells of the myeloic lineage. We have uncovered a novel spectrum of activities of G-CSF in the central nervous system. G-CSF and its receptor are expressed by neurons in many brain regions, and are upregulated upon experimental stroke. In neurons, G-CSF acts anti-apoptotically by activating several protective pathways. In vivo, G-CSF decreases infarct volumes in acute stroke models in rodents. Moreover, G-CSF stimulates neuronal differentiation of adult neural stem cells in the brain, and improves long-term recovery in more chronic stroke models. Thus, G-CSF is a novel neurotrophic factor, and a highly attractive candidate for the treatment of neurodegenerative conditions. Here we discuss this new property of G-CSF in contrast to its known functions in the hematopoietic system, summarize data from other groups on G-CSF’s actions in cerebral ischemia, compare G-CSF to Erythropoietin (EPO) in the CNS and highlight clinical implications.




BRIEF REPORTS

Open Access Article

Individual Adult Human Neurons Display Aneuploidy: Detection by Fluorescence In Situ Hybridization and Single Neuron PCR

Svetlana D. Pack, Robert J. Weil, Alexander O. Vortmeyer, Weifen Zeng, Jie Li, Hiroaki Okamoto, Makoto Furuta, Evgenia Pak, Irina A. Lubensky, Edward H. Oldfield and Zhengping Zhuang
Pages 1758 - 1760
http://dx.doi.org/10.4161/cc.4.12.2153
Abstract | PDF

Neurons, once committed, exit the cell cycle and undergo maturation that promote specialized activity and are believed to operate upon a stable genome. We used fluorescence in situ hybridization, selective cell microdissection, and loss of heterozygosity analysis to assess degree of aneuploidy in patients with a neurodegenerative disease and in normal controls. We found that aneuploidy occurs in approximately 40% of mature, adult human neurons in health or disease and may be a physiological mechanism that maintains neuronal fate and function; it does not appear to be an unstable state. The fact that neuronal stem cells can be identified in adult humans and that somatic mosaicism may be found in neuronal precursor cells deserves further investigation before using adult neural stem cells to treat human disease.

Open Access Article

BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia: Not Quite Enough to Cause Resistance to Imatinib Therapy?

Thoralf Lange, Byung Park, Stephanie G. Willis and Michael W. Deininger
Pages 1761 - 1766
http://dx.doi.org/10.4161/cc.4.12.2261
Abstract | PDF

Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of 2 copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.




METHODOLOGICAL REPORT

Open Access Article

In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification

Jia Li, C.S.H. Young, Paul M. Lizardia and David F. Stern
Pages 1767 - 1773
http://dx.doi.org/10.4161/cc.4.12.2211
Abstract | PDF

We have developed a method to localize DNA double strand breaks (DSBs) in situ in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HO endonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circle amplification (RCA), a sensitive method that allows the detection of single molecular event by rapid isothermal amplification, was used to localize the broken ends in situ. Punctate RCA signals were only seen in the cells that had been infected with both adenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mockinfected or infected with the site or endonuclease virus only. With use of a chemical crosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneously on the same sample. This methodology provides a novel approach for investigation of DNA recombination, DNA repair, and checkpoint controls in mammalian cells.




REPORTS

Open Access Article

Stathmin Prevents the Transition from a Normal to an Endomitotic Cell Cycle during Megakaryocytic Differentiation

Camelia Iancu-Rubin, Chris A. Nasrallah and George F. Atweh
Pages 1774 - 1782
http://dx.doi.org/10.4161/cc.4.12.2171
Abstract | PDF

Physiological polyploidy is a characteristic of several cell types including the megakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy by switching from a normal to an endomitotic cell cycle characterized by the absence of late mitotic stages. During an endomitotic cycle, the cells enter into mitosis and proceed normally through metaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. This abortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosome segregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation of the mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leads to aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathmin is switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle. Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitotic spindle and the exit from mitosis. Previous studies had shown that stathmin expression is downregulated as MK become polyploid and inhibition of its expression in K562 cells increases their propensity to become polyploid. In this report, we describe our studies of the mechanism by which stathmin plays its role in MK polyploidization. We show that stathmin overexpression prevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease in the number of multipolar mitotic spindles. These observations support a model in which downregulation of stathmin expression in megakaryocytes and other polyploid cells may be a critically important factor in endomitosis and polyploidy.

Open Access Article

The Absence of p53 Aggravates Polyploidy and Centrosome Number Abnormality Induced by Aurora-C Overexpression

Stéphanie Dutertre, Elise Hamard-Péron, Jean-Yves Cremet, Yann Thomas and Claude Prigent
Pages 1783 - 1787
http://dx.doi.org/10.4161/cc.4.12.2172
Abstract | PDF

Aurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimics Aurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to the centrosome, like Aurora-A. We then examined the phenotype generated by Aurora-C overexpression. Basically it looked similar to the phenotypes observed after overexpression of the other Aurora kinases. We observed an augmentation of polyploid cells containing more than two centrosomes. More interestingly this phenotype was aggravated in the absence of a functional p53. Although the physiological function of Aurora-C in somatic cells remains to be clarified, our results, just like for the two other Aurora kinases, raised the question of a role of Aurora-C in the development and progression of cancer especially in the presence of mutated p53.

Open Access Article

Deciphering Transcriptional Regulatory Elements That Encode Specific Cell-Cycle Phasing by Comparative Genomics Analysis

Chaim Linhart, Ran Elkon, Yosef Shiloh and Ron Shamir
Pages 1788 - 1797
http://dx.doi.org/10.4161/cc.4.12.2173
Abstract | PDF

Transcriptional regulation is a major tier in the periodic engine that mobilizes cell cycle progression. The availability of complete genome sequences of multiple organisms holds promise for significantly improving the specificity of computational identification of functional elements. Here, we applied a comparative genomics analysis to decipher transcriptional regulatory elements that control cell-cycle phasing. We analyzed genome-wide promoter sequences from 12 organisms, including worm, fly, fish, rodents and human, and identified conserved transcriptional modules that determine the expression of genes in specific cell cycle phases. We demonstrate that a canonical E2F signal encodes for expression highly specific to the G1/S phase, and that a cis-regulatory module comprising CHR-NF-Y elements dictates expression that is restricted to the G2 and G2/M phases. B-Myb binding site signatures occur in many of the CHR-NF-Y target genes, suggesting a specific role for this triplet in the regulation of the cell cycle transcriptional program. Remarkably, E2F signals are conserved in promoters of G1/S genes in all organisms from worm to human. The CHR-NF-Y module is conserved in promoters of G2/M regulated genes in all analyzed vertebrates. Our results reveal novel modules that determine specific cell-cycle phasing, and identify their respective putative target genes with remarkably high specificity.

Open Access Article

Subcellular Localization of the Cyclin Dependent Kinase Inhibitor Sic1 is Modulated by the Carbon Source in Budding Yeast

Riccardo L. Rossi, Vittoria Zinzalla, Andrea Mastriani, Marco Vanoni and Lilia Alberghina
Pages 1798 - 1807
http://dx.doi.org/10.4161/cc.4.12.2189
Abstract | PDF

The cyclin dependent kinase inhibitor Sic1 and the cyclin Clb5 are essential regulators of the cyclin dependent kinase Cdc28 during the G1 to S transition in budding yeast. Yeast enters S phase after ubiquitin-mediated degradation of Sic1, an event triggered by Cln1,2-Cdc28 mediated phosphorylation. We recently showed that Sic1 is involved in carbon source modulation of the critical cell size required to enter S phase. Here we show that the amount and sub-cellular localization of Sic1 are also carbon source-modulated. We identify a bipartite nuclear localization sequence responsible for nuclear localization of Sic1 and for correct cell cycle progression in a carbon-source dependent manner. Similarly to Cip/Kip proteins ? Sic1 mammalian counterparts ? Sic1 facilitates nuclear accumulation of its cognate cyclin, since cytoplasmic building-up of Clb5 is observed upon switching off expression of the SIC1 gene. Our data indicate a previously unrecognized inhibitor/activator dual role for Sic1 and put it among key molecules whose activity is regulated by their nuclear-cytoplasmic localization.

Open Access Article

Caveolin-1-Deficient Mice Have An Increased Mammary Stem Cell Population with Upregulation of Wnt/?-Catenin Signaling

Federica Sotgia, Terence M. Williams, Alex W. Cohen, Carlo Minetti, Richard G. Pestell and Michael P. Lisanti
Pages 1808 - 1816
http://dx.doi.org/10.4161/cc.4.12.2198
Abstract | PDF

Here, we show that a caveolin-1 (Cav-1) deficiency leads to an amplification of the adult mammary stem cell population, both in vivo and in vitro. First, the expression of two stem cell markers, Sca-1 and Keratin 6, is dramatically increased in the hyperplastic mammary ducts of Cav-1 deficient mice, suggesting that loss of Cav-1 induces the accumulation of a progenitor cell population in the mammary gland. To independently validate these results, we reconstituted mammary acini formation in vitro via a 3D Matrigel assay system--using primary cultures of mammary epithelial cells derived from WT and Cav-1 deficient mice. We show that Cav-1 null 3D epithelial structures display an intense increase in the expression of three stem cell markers, i.e., Sca-1, keratin 6 and keratin 5. Overall, we observed a 2-to-3 fold increase in the number of Cav-1 KO acini that are positive for a given stem cell marker. Also, we show that such amplification of progenitor cells has functional consequences, as demonstrated by the abnormal presence of myoepithelial cells in the hyperplastic lesions of Cav-1 deficient mammary glands. Finally, we provide evidence that hyper-activation of Wnt/?-catenin signaling may constitute one of the down-stream mechanisms leading to mammary stem cell accumulation. The longevity and slow-dividing properties of mammary stem cells facilitates the accumulation of genetic alterations, and renders these progenitor cells the likely precursors of malignant derivatives. As such, we propose that loss of Cav-1 induces the accumulation of mammary stem cells, and that this event may be an initiating factor during mammary tumorigenesis.

Open Access Article

Loss of Caveolin-1 Causes the Hyper-Proliferation of Intestinal Crypt Stem Cells, with Increased Sensitivity to Whole Body ?-Radiation

Jiangwei Li, Ghada. S. Hassan, Terence M. Williams, Carlo Minetti, Richard G. Pestell, Herbert B. Tanowitz, Philippe G. Frank, Federica Sotgia and Michael P. Lisanti
Pages 1817 - 1825
http://dx.doi.org/10.4161/cc.4.12.2199
Abstract | PDF

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.

Open Access Article

Mechanistic Insight into the Cdc28-related Protein Kinase Ime2 through Analysis of Replication Protein A Phosphorylation

Dawn M. Clifford, Kara E. Stark, Kathryn E. Gardner, Susanne Hoffmann-Benning and George S. Brush
Pages 1826 - 1833
http://dx.doi.org/10.4161/cc.4.12.2214
Abstract | PDF

In budding yeast, the meiosis-specific protein kinase Ime2 is required for normal meiotic progression. Current evidence suggests that Ime2 is functionally related to Cdc28, the major cyclin-dependent kinase in yeast that is essential for both cell cycle and meiosis. We have previously reported that a natural target of Ime2 activity is replication protein A (RPA), the cellular single-stranded DNA-binding protein that performs critical functions during DNA replication, repair, and recombination. Ime2-dependent RPA phosphorylation first occurs early in meiosis and targets the middle subunit of the RPA heterotrimeric complex (Rfa2). We now demonstrate that Rfa2 serine 27 (S27) is required for Ime2-dependent Rfa2 phosphorylation in vivo. S27 is also required for Rfa2 phosphorylation in vitro catalyzed by immunoprecipitated Ime2. In addition, Ime2 mediates in vitro phosphorylation of a short peptide containing Rfa2 amino acids 23 through 29, thereby providing evidence that S27 itself is the phosphoacceptor. Phosphorylation site mapping supports this conclusion, as mass spectrometry analysis has revealed that at least three residues within Rfa2 amino acids 2 through 35 become phosphorylated specifically during meiosis. Although S27 is embedded in a motif that is recognized by several protein kinases, this sequence is not a typical target of cyclin-dependent kinases. Therefore, the mechanism underlying Ime2 substrate recognition could differ from that of Cdc28.

Open Access Article

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

François-Michel Boisvert, Alexandre Rhie, Stéphane Richard and Aidan J. Doherty
Pages 1834 - 1841
http://dx.doi.org/10.4161/cc.4.12.2250
Abstract | PDF

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.

Open Access Article

Preferential Occurrence of Chromosome Breakpoints within Early Replicating Regions in Neuroblastoma

Isabelle Janoueix-Lerosey, Philippe Hupé, Zofia Maciorowski, Philippe La Rosa, Gudrun Schleiermacher, Gaëlle Pierron, Stéphane Liva, Emmanuel Barillot and Olivier Delattre
Pages 1842 - 1846
http://dx.doi.org/10.4161/cc.4.12.2257
Abstract | PDF

Neuroblastoma (NB) is a frequent paediatric extra cranial solid tumour characterized by the occurrence of unbalanced chromosome translocations, frequently, but not exclusively, involving chromosomes 1 and 17. We have used a 1 Mb resolution BAC array to further refine the mapping of breakpoints in NB cell lines. Replication timing profiles were evaluated in 7 NB cell lines, using DNAs from G1 and S phases flow sorted nuclei hybridised on the same array. Strikingly, these replication timing profiles were highly similar between the different NB cell lines. Furthermore, a significant level of similarity was also observed between NB cell lines and lymphoblastoid cells. A segmentation analysis using the Adaptative Weights Smoothing procedure was performed to determine regions of coordinate replication. More than 50% of the breakpoints mapped to early replicating regions, which account for 23.7% of the total genome. The breakpoints frequency per 108 bases was therefore 10.84 for early replicating regions, whereas it was only 2.94 for late replicating regions, these difference being highly significant (p<10-4). This strong association was also observed when chromosomes 1 and 17, the two most frequent translocation partners in NB were excluded from the statistical analysis. These results unambiguously establish a link between unbalanced translocations, whose most likely mechanism of occurrence relies on break-induced replication, and early replication of the genome.

Open Access Article

Resistance to Microtubule-Stabilizing Drugs Involves Two Eventse: β-Tubulin Mutation in One Allele Followed by Loss of the Second Allele

Yuefang Wang, Aurora O’Brate, Wei Zhou and Paraskevi Giannakakou
Pages 1847 - 1853
http://dx.doi.org/10.4161/cc.4.12.2264
Abstract | PDF

Resistance to paclitaxel (PTX) or the epothilones (Epo) occurs via the acquisition of point mutations in β-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with PTX or Epo A which harbor distinct β-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant β-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant β-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for β-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for β-tubulin and mitotic index by immunofluorescence were performed to correlate the β-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt b-tubulin in addition to a β-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant b-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.

Open Access Article

53BP1 Oligomerization is Independent of its Methylation by PRMT1

Melissa M. Adams, Bin Wang, Zhenfang Xia, Julio C. Morales, Xiongbin Lu, Lawrence A. Donehower, Daniel A. Bochar, Stephen J. Elledge and Phillip B. Carpenter
Pages 1854 - 1861
http://dx.doi.org/10.4161/cc.4.12.2282
Abstract | PDF

p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs) where it is recruited to or near sites of DNA damage. Although little is known about the biochemical functions of 53BP1, the protein possesses several motifs that are likely important for its role as a DNA damage response element. This includes two BRCA1 C-terminal repeats, tandem Tudor domains, and a variety of phosphorylation sites. Here we show that a glycine-arginine rich (GAR) stretch of 53BP1 lying upstream of the Tudor motifs is methylated. We demonstrate that arginine residues within this region are important for asymmetric methylation by the PRMT1 methyltransferase. We further show that sequences upstream of the Tudor domains that do not include the GAR stretch are sufficient for 53BP1 oligomerization in vivo. Thus, although Tudor domains bind methylated proteins, 53BP1 homo-oligomerization occurs independently of Tudor function. Lastly, we find that deficiencies in 53BP1 generate a “hyper-rec” phenotype. Collectively, these data provide new insight into 53BP1, an important component in maintaining genomic stability.

Open Access Article

Similar Tumor Suppressor Gene Alteration Profiles in Asbestos-Induced Murine and Human Mesothelioma

Céline Lecomte, Pascal Andujar, Annie Renier, Laurence Kheuang, Vincent Abramowski, Lucile Mellotte, Jocelyne Fleury-Feith, Jessica Zucman-Rossi, Marco Giovanni and Marie-Claude Jaurand
Pages 1862 - 1869
http://dx.doi.org/10.4161/cc.4.12.2300
Abstract | PDF

The status of tumor suppressor genes (TSGs) relevant to human malignant mesothelioma (HMM) pathogenesis was examined in cultures of mesothelioma cells from tumoral ascites developed in mice exposed to asbestos (asb) fibers. The status of the respective hortologous human genes was also investigated in 12 HMM cell cultures. Eleven primary cultures from mice hemizygous for Nƒ2 (asb-Nf2KO3/+) and 4 wild type counterparts (asb-Nf2+/+) were analyzed for mutations in Nf2, p16/Cdkn2a, p19/Arf and Trp53 genes and protein expression of p15/Cdkn2b and Cdk4. TSG alterations in both mouse and human mesothelioma cells consisted in frequent inactivation of p16/Cdkn2a, p19/Arf (or P14/ARF) and p15/Cdkn2b, co-inactivation of p16/Cdkn2a and p15/Cdkn2b and low rate of Trp53 mutations in both asb-Nf2KO3/+ and asb-Nf2+/+ mesothelioma cells. In both mouse and human mesothelioma cells, inactivation of the hortologous genes p16/Cdkn2a or P16/CDKN2A was due to deletions at the Ink4/Arf locus encompassing p19/Arf or P14/ARF, respectively. Loss of heterozygosity at the Nf2 locus was detected in 10 of 11 asb-Nf2KO3/+ cultures and Nf2 gene rearrangement in one asb-Nf2+/+ culture. These data show that the profile of TSG alterations in asbestos-induced mesothelioma is similar in mice and humans. Thus, the mouse mesothelioma model could be useful for human risk assessment, taking into account interindividual variations in genetic sensitivity to carcinogens.