Aneuploidy: Etiology, Disorders and Risk Factors : Etiology, Disorders and Risk Factors [1 ed.] 9781621002154, 9781621000709

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Aneuploidy: Etiology, Disorders and Risk Factors : Etiology, Disorders and Risk Factors, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Aneuploidy: Etiology, Disorders and Risk Factors : Etiology, Disorders and Risk Factors, Nova Science Publishers, Incorporated, 2012. ProQuest

GENETICS - RESEARCH AND ISSUES

ANEUPLOIDY

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

ETIOLOGY, DISORDERS AND RISK FACTORS

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GENETICS - RESEARCH AND ISSUES

ANEUPLOIDY ETIOLOGY, DISORDERS AND RISK FACTORS

SALVATORE DE ROSSI Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

AND

FILIPPO BIANCHI EDITORS

Nova Science Publishers, Inc. New York

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Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Aneuploidy : etiology, disorders, and risk factors / editors, Salvatore de Rossi and Filippo Bianchi. p. ; cm. Includes bibliographical references and index. ISBN:  (eBook) I. De Rossi, Salvatore. II. Bianchi, Filippo, 1964[DNLM: 1. Aneuploidy. 2. Genetic Predisposition to Disease. 3. Risk Factors. QU 500] LC classification not assigned 616'.042--dc23

Published by Nova Science Publishers, Inc.  New York Aneuploidy: Etiology, Disorders and Risk Factors : Etiology, Disorders and Risk Factors, Nova Science Publishers, Incorporated, 2012. ProQuest

Contents Preface Chapter I

Chapter II

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Chapter III

Chapter IV

vii Aneuploidy: Mechanisms, Cancer and the Role of Environmental Pollutants Amie L. Holmes and John Pierce Wise Ploidy in Mitosis and Meiosis: A Role of MAPK/Erk Network Franck B. Riquet, Pauline Vandame and Jean-François Bodart The Spindle Assembly Checkpoint and Aneuploidy Juliana Faria, Joana Barbosa, Inês M. B. Moura, Rui M. Reis and Hassan Bousbaa The Role of Centromere Cohesion and Associated Proteins in Alzheimer‘s Disease: A Relation to Aneuploidy? V. P. Bajic, D. J. Bonda, L. Zivkovic, Z Milicevic, B. Plecas-Solarovic, Xiongwei Zhu and B. Spremo-Potparevic

Chapter V

Cohesins, Genomic Stability, and Cancer José L. Barbero

Chapter VI

Adult Neurogenesis and Aneuploidy in Etiology, Pathogenesis and Pathology of Alzheimer‘s Disease Philippe Taupin

Chapter VII

Aneuploidy in Cultured Human Multipotent Mesenchymal Stromal Cells V. A. Nikitina, E. S. Voronina, L. D. Katosova and N. P. Bochkov

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vi Chapter VIII

Chapter IX

Contents Somatic And Germ Cell Spontaneous Aneuploidy Level In Healthy Fertile People N. Zotova, E. Markova, V. Artukhova and A. Svetlakov Sperm Aneuploidy and Male Fertility El-Sayed A. Mohamed and Myung-Geol Pang

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Index

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143 161

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Preface Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders. Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate properly between the two cells. Chromosome abnormalities occur in 1 of 160 live births. In this book, the authors present topical research in the study of the etiology, disorders and risk factors of aneuploidy, including the role of environmental pollutants as a mechanism of aneuploidy; ploidy in mitosis and meiosis; the spindle assembly checkpoint and aneuploidy; cohesions, genomic stability and cancer and aneuploidy in cultured human multipotent mesenchymal stromal cells. Chapter I - Chromosome instability, including changes in chromosome structure and/or number, is a common feature of many types of solid tumors. Specifically, aneuploidy is a frequent hallmark of cancer and is considered the most consistent marker of malignancy. However, the timing of aneuploidy in tumorigenesis remains unknown. It is still debated whether aneuploidy is an early or late event in tumorigenesis but recent data suggest that it serves more as a driving force rather than a consequence of tumorigenesis. Changes in chromosome number can alter the critical balance of proteins required to regulate the cell cycle, chromosome segregation, DNA synthesis and DNA repair. Deregulation of any number of these processes can further destabilize the genome and promote tumorigenesis. This chapter focuses on the biological mechanisms that have been identified for aneuploidy and puts these mechanisms into the context of a known environmental and occupational carcinogen, hexavalent chromium. Centrosome amplification, defects in the spindle assembly checkpoint, sister chromatid cohesion defects and kinetochore-microtubule attachment defects are the four primary mechanisms responsible for inducing aneuploidy. All of these mechanisms have been observed in human cancers and strongly correlate with aneuploidy. Centrosome amplification causes aneuploidy through multipolar spindle formation and unequal pulling of the chromosomes resulting in chromosome missegregation. Defects in the spindle assembly checkpoint also induce chromosome missegregation by allowing premature entry into anaphase. Defects in sister chromatid cohesion during mitosis lead to large scale chromosome segregation errors due to premature separation of sister chromatids. Lastly, kinetochore-microtubule attachment defects increase the rate of merotelic attachments and lagging chromosomes in anaphase leading to chromosome missegregation and aneuploidy.

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Salvatore de Rossi and Filippo Bianchi

Errors in chromosome segregation can arise spontaneously or be induced by exposure to chemical agents that alter normal mitotic progression. The mechanisms for many environmental and occupational carcinogens remain unknown but induction of aneuploidy may be a driving factor. The authors have been pioneering the study of metal-induced chromosome instability, specifically investigating the mechanisms involved in hexavalent chromium-induced aneuploidy. In this chapter, they focus on aneuploidy as an early and initiating event in tumorigenesis. The authors will review the four primary mechanisms of aneuploidy, discussing both the normal mechanisms of centrosome duplication, the spindle assembly checkpoint, sister chromatid cohesion and kinetochore-microtubule attachments and how dysregulation of these processes leads to aneuploidy and cancer, and will use hexavalent chromium as a representative aneugenic contaminant to apply these mechanisms to a known human carcinogen. Chapter II - Genetic unbalances resulting in gain or loss of chromosomes may affect cell evolution towards unregulated growth characteristics and diminished response to apoptosis, cellular damage and cell cycle checkpoints. Besides their dependency towards oncogenes, evidences have been raised towards the idea that cancers might also be dependent upon various abnormal assortments of chromosomes, aneuploidy or even polyploidy. In addition to their roles in transcription up-regulation at G1/S, which have been shown to be involved in tumorigenesis, Mitogen Activated Protein Kinases (MAPK) from the Extracellular Regulated Kinase (Erk) group may recruit transcription-independent transduction mechanisms involved in cell reorganization at division and particularly in genetic material segregation. While the role of Erk in M-phase cellular reorganization may be discussed in mammalian cells, meiotic models, including urochordates, molluscs, amphibian and mammals female gametes, have provided case studies where the deregulation of the Erk network lead to catastrophic events or uncontrolled division cycles. This chapter describes the functions explored for MAPK/Erk during M-phase cellular reorganization and the network of its regulators. The role of a particular MAPKKK, Mos, with regards to their involvement in genomic stability and ploidy, will be addressed and discussed. Chapter III - Abnormal chromosome number, or aneuploidy, is commonly observed in most solid tumors, and results from mis-segregation of whole chromosomes in a phenomenon referred to as chromosome instability (CIN). Dysregulation of the spindle assembly checkpoint (SAC) is thought as one of the mechanisms underlying CIN. The SAC is a signaling pathway that prevents precocious chromosome segregation until all chromosomes of a dividing cell are aligned at the metaphase plate. While complete loss of the SAC activity is lethal due to massive mis-segregation, partial loss of the SAC is a common feature of many aneuploid tumor cells allowing them to gain or lose a small number of chromosomes. The authors review the authors‘ current knowledge on the molecular mechanisms of SAC and discuss its contribution to CIN as well as its potential as a suitable target in cancer therapy. Chapter IV - Aside from its two characteristic pathologies, amyloid beta (Aβ) containing senile plaques and neurofibrillary tangles (NFT‘s), Alzheimer‘s disease (AD) exhibits a mitotically active phenotype. Despite being in a post-mitotic, quiescent state, affected cells show features of activation of a full cell cycle, such as activation of cyclins and cyclin dependent kinases and activation of cell cycle checkpoint control proteins. Replication of DNA in AD neurons (cyclin B and CDK 1 ) has been successfully correlated with various

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Preface

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cytogenetic alterations of neuronal chromosomes, one of which corresponds to the occurrence of tetraploidy in AD neurons. Other groups have found chromosome instability, i.e. premature centromere division (PCD) of the X chromosome in cortical neurons and peripheral blood lymphocytes and bi-nuclear neuronal cells in AD brains. These cytogenetic alterations not only show that replication has occurred in these cells, but that a number of proteins which constitute the centromere complex have become dys-regulated. Thus, this temporal instability of the centromere or premature centromere division (PCD) in neuronal and peripheral blood cells in AD display that cohesion is altered by an unknown mechanism which consequently may lead to aneuploidy. Until recently there has been no data on the role of proteins controlling the centromere region in postmitotic neurons. New data suggests that cohesin complexes and related proteins that mediate sister–chromatid cohesion in dividing cells may also contribute to gene regulation and other processes in postmitotic cells. Evidence demonstrating that instability of centromere-cohesion dynamics in the early phases of the cell cycle which coincides with re-entry alteration in cortical neurons enables the possibility to further elucidate initial processes leading to AD. The authors may thus become able to answer the question ―Is Alzheimer‘s disease a consequence of aneuploidy‘‘? In this review, they shall discuss the involvement of cohesion impairment and the related proteins in the involvement of chromosome instability leading to aneuploidy and how these processes are related to AD pathology. Chapter V - The multifunctional protein complex named cohesin remodels the chromatin structure in different essential cellular processes such as chromosome segregation, during cell division in mitosis and meiosis, heterochromatin formation, DNA-repair, DNA replication initiation, and regulation of gene expression. The control of the association/dissociation of cohesin complexes to chromatin is carried out by an increasing number of denominated cohesin-interacting proteins or cohesin-regulators. Malfunctions of cohesin complexes and/or its regulators lead to cell death, chromosomal instability, aneuploidy, or developmental syndromes, such as Cornelia de Lange and Roberts syndrome/SC phocomelia, which have been denominated cohesinopathies. In addition, the number of studies and results that implicate cohesin and cohesin-regulators in different human cancers and in mechanisms of resistance to anti-tumor therapy have grown. These findings show that the control of cohesin metabolism during cell life is essential to correct DNA dynamics and to preserve cellular euploidy and genome stability. Chapter VI - Alzheimer‘s disease (AD) is a neurodegenerative disease. The disease is characterized by widespread neurodegeneration, amyloid deposits, neurofibrillary tangles and aneuploidy. Patients with AD elicit learning and memory deficits and anosmia. Adult neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) of mammals. Neurogenesis is enhanced in the hippocampus and is reduced in the subventricular zone (SVZ) of patients with AD. On the one hand, enhanced neurogenesis in the brain patients with AD suggests a regenerative attempt to compensate for the neuronal loss. On the other hand, reduced neurogenesis would contribute to pathological processes associated with the disease. Aneuploidy is an underlying of the pathogenesis of AD, such as neurodegeneration. Neurogenesis has the potential to generate aneuploid neuronal cells in the adult brain. The generation of aneuploid neuronal cells in the brain of patients with AD would contribute to the etiology and pathogenesis of the disease. Hence,

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Salvatore de Rossi and Filippo Bianchi

adult neurogenesis would contribute to the pathology of AD, the understanding and contribution of which remain to be elucidated and determined. Chapter VII - The guarantee of safety is a main condition to clinical application of cell transplantation. During the cultivation procedures chromosome abnormalities can arise which can lead to long term consequences of cell therapy. The aneuploid cell frequency (nullisomy, monosomy and trisomy) in the interphase nuclei of mesenchymal stromal cells (MSC) had analyzed on autosomes 6, 8, 11 and sex chromosomes. The phenotype of MSCs on early and late passages had no difference. In spite of decrease proliferation possibilities by 11-12 passage, cells kept typical MSC immunophenotype. The aneuploid cell clone formation appeared in some cultures. Two cultures of MSC from bone marrow had clones of aneuploidy cells: with trisomy 8 and monosomy X. In two cultures of MSC from adipose tissue clones with monosomy 6 were revealed at the late passages. The results show potential of genetic transformation and selection of MSCs with abnormal karyotype during cultivation in vitro. The results substantiate the need for more profound study of stem cell genetics and development of quality control system for the cell therapy. Chapter VIII - In this chapter the authors are presenting an investigation of spontaneous aneuploidy level in somatic and germ cells of healthy fertile men and women analyzed by FISH technique. They performed molecular-cytogenetic investigation of blood cells on 28 fertile healthy persons, among them sperm cells from 10 men and polar bodies (PBs) from 6 women of reproductive age. Cultivated lymphocytes and fixed sperm were analyzed by FISH for five chromosomes (13, 18, 21, Х, and Y). The first and second PBs aneuploidy were analyzed during IVF (in vitro fertilization) plus PGD program with oocyte donation by FISH for chromosomes 13, 18, 21. In addition to standard statistics, limits and confidence intervals were scored. A total of 28,000 lymphocytes and 10,000 sperm were scored for five chromosomes, 54 first and 54 second PBs - for three chromosomes. The authors found differences in results, with direct and indirect labeling, and scored an absolute error of FISH technique. The average lymphocytes aneuploidy level for all chromosomes (13, 18, 21, Х, and Y) was 1.73±0.20% for women and 1.05±0.08% for men (P=0.014). The average sperm aneuploidy for five chromosomes was 1.17±0.14%, the limit was 2.82%. They determined limits of mutation detection for each chromosome separately, and for all five chromosomes in lymphocytes and sperm. The total lymphocyte limits were 3.40% for women (four chromosomes) and 1.44% for men (five chromosomes). The authors investigated correlations between aneuploidy in somatic and germ cells, finding a mild association between frequencies of monosomy in the 13th and 18th chromosomes in sperm and lymphocytes. Also, they revealed correlation between sperm aneuploidy and semen quality, r=0.644. Aneuploidy frequency in PBs of women was extremely variable (0-40%). The authors did not find any correlations between polar bodies aneuploidy and aneuploidy levels in lymphocytes. Chapter IX - Worldwide, infertility affects 20% of couples and male factor infertility is the primary problem in about half of these couples. Extensive studies have shown that infertile men have an increased frequency of aneuploid spermatozoa. Because aneuploidy is correlated with overall reproductive failure, the high frequency of cytogenetically abnormal sperm cells in male factor infertility raises concerns that there may be an increased incidence of abnormal babies, as well as a higher incidence of spontaneous abortions. Before a few decades, human spermatozoa/rodents egg (from mice and hamster) fusion techniques were

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Preface

xi

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used to study human sperm aneuploidy. While this method was effective to define chromosomes number and structure abnormality, it was very difficult to define the aneuploidy for specific chromosome; also it is time and effort consuming. An alternative technique for aneuploidy determination is fluorescence in situ hybridization (FISH) analysis with chromosome-specific DNA probe was developed in 1990s, which directly identifies both aneuploidy and polyploidy in spermatozoa. Using FISH protocol, it is become clear that some specific chromosomes show higher aneuploidy rate than other chromosomes. While the incidence of sperm aneuploidy was observed in men with normal and abnormal karyotype, its rate varies based on life style costume, age, diseases and other factors. However, the genetic factor remains the main controller in sperm aneuploidy. Many trials have been made in andrology laboratory to discriminate between sperm with normal or abnormal chromosome numbers, however none of them is reliable. Recent trends should be focused on the physiological differences between euploid and aneuploid spermatozoa.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter I

Aneuploidy: Mechanisms, Cancer and the Role of Environmental Pollutants Amie L. Holmes and John Pierce Wise* Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, University of Southern Maine; Department of Applied Medical Science, University of Southern Maine, Portland, ME, US

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Abstract Chromosome instability, including changes in chromosome structure and/or number, is a common feature of many types of solid tumors. Specifically, aneuploidy is a frequent hallmark of cancer and is considered the most consistent marker of malignancy. However, the timing of aneuploidy in tumorigenesis remains unknown. It is still debated whether aneuploidy is an early or late event in tumorigenesis but recent data suggest that it serves more as a driving force rather than a consequence of tumorigenesis. Changes in chromosome number can alter the critical balance of proteins required to regulate the cell cycle, chromosome segregation, DNA synthesis and DNA repair. Deregulation of any number of these processes can further destabilize the genome and promote tumorigenesis. This chapter focuses on the biological mechanisms that have been identified for aneuploidy and puts these mechanisms into the context of a known environmental and occupational carcinogen, hexavalent chromium. Centrosome amplification, defects in the spindle assembly checkpoint, sister chromatid cohesion defects and kinetochoremicrotubule attachment defects are the four primary mechanisms responsible for inducing aneuploidy. All of these mechanisms have been observed in human cancers and strongly correlate with aneuploidy. Centrosome amplification causes aneuploidy through multipolar spindle formation and unequal pulling of the chromosomes resulting in *

Correspondence: Dr. John Pierce Wise, Sr., PO Box 9300, 96 Falmouth St., Portland. ME 04104-9300. Tel: (207) 228-8050. Fax: (207) 228-8518. Email: [email protected].

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Amie L. Holmes and John Pierce Wise chromosome missegregation. Defects in the spindle assembly checkpoint also induce chromosome missegregation by allowing premature entry into anaphase. Defects in sister chromatid cohesion during mitosis lead to large scale chromosome segregation errors due to premature separation of sister chromatids. Lastly, kinetochore-microtubule attachment defects increase the rate of merotelic attachments and lagging chromosomes in anaphase leading to chromosome missegregation and aneuploidy. Errors in chromosome segregation can arise spontaneously or be induced by exposure to chemical agents that alter normal mitotic progression. The mechanisms for many environmental and occupational carcinogens remain unknown but induction of aneuploidy may be a driving factor. We have been pioneering the study of metal-induced chromosome instability, specifically investigating the mechanisms involved in hexavalent chromium-induced aneuploidy. In this chapter, we focus on aneuploidy as an early and initiating event in tumorigenesis. We will review the four primary mechanisms of aneuploidy, discussing both the normal mechanisms of centrosome duplication, the spindle assembly checkpoint, sister chromatid cohesion and kinetochore-microtubule attachments and how dysregulation of these processes leads to aneuploidy and cancer, and will use hexavalent chromium as a representative aneugenic contaminant to apply these mechanisms to a known human carcinogen.

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Introduction Cell cycle progression, the replication of DNA and the separation of chromosomes in mitosis are tightly regulated by the cell to maintain its genomic integrity. Numerous safeguards, such as DNA repair and cell cycle checkpoint pathways, are in place to protect cells at both the nucleotide and chromosome level. Loss of one or more of these safeguards can lead to genomic instability and ultimately tumorigenesis. The genomic instability model of carcinogenesis posits that alterations in genes that are responsible for controlling genomic integrity lead to a cascade of changes in the entire genome [1]. Genomic instability is classified into microsatellite instability and chromosome instability [1]. Microsatellite instability occurs at the nucleotide level when cells lose mismatch repair and are unable to repair normal insertion/deletion loops that arise from polymerase slippage during replication of microsatellites, repetitive non-coding DNA sequences abundant in the human genome [2]. Inability to repair these insertion/deletion loops leads to changes in microsatellite length which translates into frameshift mutations that are carried throughout the entire genome [2]. Loss of mismatch repair increases mutation frequency by more than 200-fold [2]. Chromosome instability occurs at the chromosome level when processes that maintain chromosome integrity and regulate chromosome segregation are disrupted [1]. Chromosome instability is further sub-classified into numerical and structural chromosome instability. Structural chromosome instability is characterized by chromosome translocations and breaks, while numerical chromosome instability involves loss and gain of whole chromosomes. Numerical chromosome instability is a hallmark of many solid tumors and is the most prevalent form of genomic instability [3]. Changes in chromosome number can alter the critical balance of proteins required to regulate the cell cycle, chromosome segregation, DNA

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synthesis and DNA repair. Deregulation of any number of these processes can further destabilize the genome and promote tumorigenesis. The four primary mechanisms responsible for inducing numerical chromosome instability include centrosome amplification, defects in the spindle assembly checkpoint, kinetochore dysfunction and sister chromatid cohesion defects [4]. In this chapter, we will review the four primary mechanisms of numerical chromosome instability, discussing both the normal mechanisms of centrosome duplication, the spindle assembly checkpoint, sister chromatid cohesion and kinetochore-microtubule attachments and how dysregulation of these processes leads to aneuploidy and cancer, and will use hexavalent chromium as a representative aneugenic contaminant to apply these mechanisms to a known human carcinogen.

Centrosomes

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Centrosome Duplication Cycle Like DNA replication, centrosome duplication occurs in a semi-conservative fashion as depicted in Figure 1. At the beginning of G1, there is one centrosome that consists of a mother and daughter centriole oriented orthogonally [5]. At the G1/S transition, the mother and daughter centriole lose their orthogonal orientation and during S phase new centrioles begin to form and elongate at each centriole. During late S and G2 phases, the centrosomes mature recruiting additional pericentriolar material (PCM) to the centrosomes which increase its microtubule nucleation ability. The two newly formed centrosomes then separate at the G2/M transition and move to opposite poles to form the mitotic spindles. Mitosis ensues and one centrosome is distributed to each daughter. Since the centrosome cycle is tightly coupled to the DNA replication cycle, the activation of cyclin-dependent kinase 2 (Cdk2)/cyclin E/A controls centrosome duplication; however, the exact mechanism governing centrosome duplication remains unclear. At the G1/S transition, active Cdk2/cyclin E phosphorylates nucleophosmin (NPM) releasing it from the centrosome, allowing centrosome duplication to begin. The release of NPM from the centrosome is thought to license the centrosome for duplication. Once released, NPM binds to and activates ROCK2 at the centrosome, driving centrosome duplication forward [6]. Two other targets for Cdk2 are CP110 which may be important for centriole disorientation [7] and multipolar spindle protein 1 (Mps1) which is required for centrosome duplication [8-9] but its exact role is unknown. Recently, Mps1 has been shown to bind to and phosphorylate mortalin at the centrosome which in turn creates a positive feedback loop further activating Mps1 [10]. This hyper-activation of Mps1 is responsible for the progression of centrosome duplication [10]. Centrins, tubulins, polo-like kinase 2 (Plk2), Plk4 and Aurora A are also required for centriole duplication [5, 11], but how these and other proteins assemble and organize into the newly formed centrioles remains unknown. Centrosome maturation in late S/G2 requires Plk1, Aurora-A and TPX2 [12-13]. These proteins recruit gamma-tubulin and other PCM

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components to the centrosome thereby increasing the centrosome‘s microtubule nucleation ability. Centrosome separation at the onset of mitosis is important for the generation of bipolar spindles and requires the kinase, Nek2A. Before the onset of mitosis, Nek2A is maintained in an inactive state by protein phosphatase 1 (PP1) [14-16]. Once the cell is ready to enter mitosis, Cdk1/cyclin B is activated which inactivates PP1 leading to Nek2A activation via autophosphorylation [17]. Activated Nek2A phosphorylates C-Nap1, a protein involved in centrosome cohesion, causing partial release of C-Nap1 from the centrosome and inducing centrosome separation [14-17].

Figure 1. The Centrosome Duplication Cycle. Late in G1, when the cell commits to entering into the cell cycle, the mother and daughter centrioles undergo centriole disorientation and lose their orthogonal orientation. During S phase, the centrosomes duplicate in a semi-conservative fashion with new centrioles forming from each existing centriole. In late S phase and G2, centrosome maturation occurs followed by centrosome separation at the onset of mitosis. Centrosome separation allows the centrosomes to move to opposite poles of the cell to form bipolar mitotic spindles. Following proper kinetochore-microtubule attachment to each chromosome, chromosome separation ensues and the cell divides into two with each newly formed cell containing one centrosome.

Activation of Cdk1/cyclin B also leads to the activation of Eg5 by Aurora A which is critical for centrosome migration to opposite poles [18-19]. The resulting bioriented centrosomes orchestrate chromosome segregation. When the chromosomes are aligned along the metaphase plate and ready to separate, separase is activated and induces sister chromatid separation. Recently, studies have found that separase also initiates centriole disengagement in anaphase which may function as a licensing factor preventing centrosome overduplication

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[20]. After disengagement, NPM re-associates with the centrosomes in mitosis possibly mediated by phosphorylation at T234 and T237 by Cdk1/cyclin B which may also serve as a licensing mechanism to prevent reduplication of the centrosome prior to the next cell cycle [11, 21].

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Centrosome Amplification and Cancer Coordination of the centrosome and DNA replication cycle is critical to regulate centrosome number and ensure that only two centrosomes are present in the cell upon mitotic entry [22]. Various stresses to the cell can disrupt this strict coordination and induce centrosome amplification [23]. Mitotic cells with greater than two centrosomes generate multipolar spindles leading to unequal pulling of the chromosomes into daughter cells and ultimately aneuploidy [24]. Aneuploid cells that survive and persist in the population can eventually result in tumor formation and cancer. Centrosome abnormalities are often observed in lung cancer and in numerous other types of cancers and a strong association between chromosome instability and centrosome amplification exists [25-27]. Commonly observed centrosome abnormalities in tumor cells include increases in centrosome size and number, supernumerary centriole formation, accumulation of excess pericentriolar material and inappropriate phosphorylation of centrosomal proteins [28-30]. In accordance with these observations, increased expression of proteins that comprise centrosomes, such as gamma-tubulin and pericentrin, are often observed in tumor cells [26]. Cyclin E, an important regulator of centrosome duplication, is also often overexpressed in human cancers [31] and experimental studies reveal that overexpression of cyclin E induces centrosome amplification in mammalian cells [32-33]. Another centrosome regulatory protein, Aurora A, exhibits gene amplification in primary breast tumors and in numerous cancer cell lines which correlates with poor prognosis [34-35]. These studies pinpoint numerous centrosome-associated proteins that are dysregulated in human cancers, suggesting that centrosome amplification may be a driving factor in tumorigenesis.

Mechanisms of Centrosome Amplification The major mechanisms for centrosome amplification include: 1) centrosome overduplication, 2) failure of cytokinesis, 3) centriole splitting and 4) acentriolar centrosome formation [as reviewed in 22]. Centrosome overduplication occurs when the cell cycle arrests in response to DNA damage while the centrosome duplication cycle continues generating supernumerary centrosomes. Unlike the DNA replication cycle, the centrosome cycle has no checkpoints to regulate the number of centrosomes, and overduplication of the centrosomes can occur in one cell cycle resulting in centrosome amplification [36]. This mechanism is the most well studied mechanism for centrosome amplification in response to DNA damage; however, multiple mechanisms exist and much remains unknown.

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Centrosome overduplication was first observed in Chinese hamster ovary cells arrested in S phase with hydroxyurea [37]. Later, it was shown that centrosome amplification only occurred in human cells arrested in S phase if p53 was absent and cyclin E was overexpressed [38]. Since those initial experiments, loss of numerous DNA damage repair proteins, including BRCA1, BRCA2, Rad51 and its paralogs, have been shown to induce centrosome overduplication [39-44], suggesting a link between DNA damage repair and centrosome amplification. Loss of critical DNA repair proteins would result in an accumulation of DNA damage leading to cell cycle arrest and time for centrosomes to reduplicate. In support of this link, prolonged arrest of cells in G2 mediated by ataxia telangectasia mutated protein (ATM) and checkpoint kinase 1 and 2 (Chk1 and Chk2) in response to DNA damage induces centrosome amplification [45-47]. The mechanism for G2 arrest mediated-centrosome amplification remains unknown, however, Cdk2, Rad51 and BRCA1 are implicated [45, 4749]. A second mechanism for centrosome amplification is failure of cytokinesis. In this mechanism, centrosome amplification is a byproduct of cytokinesis failure. When a cell fails to undergo cytokinesis, it enters into the next cell cycle with two nuclei and two centrosomes. During the next cell cycle, both the DNA and centrosomes are duplicated and the cell enters into mitosis with a tetraploid complement of chromosomes and four centrosomes. Checkpoint mechanisms exist to prevent the proliferation of these abnormal cells [50], but if the cell is able to escape arrest and undergoes another cell division cycle, it will enter into mitosis with four centrosomes instead of two. Defects in DNA damage and spindle assembly checkpoints can lead to failure of cytokinesis [51]. Over twenty-five proteins are involved in controlling and executing cytokinesis and disruption of any number of these proteins, including but not limited to Aurora B, Plk1, PRC1 and the central spindlin complex MKLP1, can result in cytokinesis failure [51]. The third mechanism, centriole splitting, occurs when the G2 centrosomes split to form at least two centrosomes each with one centriole [22, 48, 52]. Studies show that cells entering mitosis with DNA damage or incompletely replicated DNA undergo centriole splitting [48, 52]; however, the mechanism for DNA damage-induced centriole splitting remains unclear. Protein manipulation studies reveal that p16, shugoshin1 (Sgo1), importin β and exportin 1 may be involved in maintaining mother/daughter centriole association as depletion of these proteins results in centriole splitting [53-56], but whether these proteins are involved after DNA damage is unknown. The fourth mechanism is acentriolar centrosome formation, which is the generation of centrosomes that do not contain centrioles [5, 57-58]. Two potential mechanisms exist for formation of acentriolar centrosomes. The first mechanism involves centrosome fragmentation. If cells fail to undergo proper centrosome maturation in S and G2 phases, the centrosomes are unable to withstand the forces exerted on them by the spindle fibers in mitosis and they undergo fragmentation. The resulting acentriolar centrosomes are still able to function as centrosomes and nucleate microtubules in mitosis, creating multipolar spindles [5, 59]. The second mechanism, which is less common, is de novo formation of acentriolar centrosomes. De novo formation of acentriolar centrosomes is observed in cells where the centrosomes were experimentally removed [57]; however, it remains unknown whether de novo centrosome formation can occur in the presence of normal centrosomes.

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Spindle Assembly Checkpoint Mechanism of the Spindle Assembly Checkpoint The spindle assembly checkpoint functions at the transition between metaphase and anaphase to ensure the fidelity of chromosome segregation [60]. This checkpoint serves as a ‗stop/go‘ checkpoint. Unlike DNA damage checkpoints, the spindle assembly checkpoint is active as a cell enters mitosis and a ‗stop‘ signal is maintained until metaphase when spindle fibers are attached to all kinetochores and tension is felt at each kinetochore. Once all kinetochores are occupied by spindle fibers and an appropriate amount of tension is felt by the kinetochores, the spindle assembly checkpoint is turned off and a ‗go‘ signal is transduced allowing the cell to progress into anaphase. O

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Figure 2. Activation of the Spindle Assembly Checkpoint. In the absence of kinetochore-microtubule attachments, the activation of several SAC proteins results in the formation of the mitotic checkpoint complex (MCC) which inhibits anaphase progression by inactivating the APC/C. The Mad1/C-Mad2 complex is recruited to the Rod-ZW10-ZWILCH complex (RZZ) at the kinetochore and is regulated by Mps1. The Mad1-C-Mad2 complex facilitates the structural conversion of soluble O-Mad2 allowing it to bind to Cdc20 and be converted to C-Mad2. BubR1 and Bub3 also have fast turnover rates at the kinetochore and therefore cycle off the kinetochore to bind to C-Mad2-Cdc20 to form the MCC which potently inhibits Cdc20. The formation of the MCC is likely regulated by Aurora B, Cdk1 and Bub1 via phosphorylation of Cdc20, but the mechanism still remains unknown. The formation of MCC complex containing Cdc20, C-Mad2, BubR1 and Bub3 results in the inactivation of the anaphase promoting complex (APC/C). Therefore, securin remains bound to separase, holding it inactive and the sister chromatid cohesion remains intact.

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In order to prevent premature entry into anaphase, the spindle assembly checkpoint prevents sister chromatid separation by inhibiting the ability of Cdc20 to activate the anaphase promoting complex/cyclosome (APC/C) [60]. Maintaining the APC/C in an inactive state prevents the degradation of securin and cyclin B1 which are required for mitotic progression into anaphase. Once the spindle assembly checkpoint is satisfied, Cdc20 binds to and activates the APC/C, an ubiquitin ligase. The APC/C-Cdc20 complex poly-ubiquitinates cyclin B and securin, targeting them for degradation by the 26S proteosome. Degradation of securin releases separase, an enzyme which cleaves the cohesin complex responsible for holding the sister chromatids together, while degradation of cyclin B inactivates the mitotic kinase, Cdk1, allowing for mitotic exit. With the cleavage of the cohesin complex and the inactivation of Cdk1, the sister chromatids are pulled to opposite poles by the spindle fibers and mitotic exit ensues. Numerous spindle assembly checkpoint proteins are responsible for amplifying and executing the spindle assembly checkpoint ‗stop‘ signal, ultimately targeting Cdc20 activity (Figure 2). The upstream steps leading to the inhibition of Cdc20 are less understood. Inhibition of Cdc20 primarily occurs through the mitotic checkpoint complex (MCC) which is comprised of BubR1, Bub3, Mad2 and Cdc20 itself and to a lesser degree Mad2 alone can inhibit Cdc20 [60]. One of the first proteins recruited to the empty kinetochore is Bub1 in complex with Bub3 [61]. Bub1 is required for recruitment of numerous spindle assembly checkpoint proteins, including Mad1, Mad2, BubR1, and Bub3 and for MCC formation [6164]. BubR1 in complex with Bub3 is then recruited to the kinetochore followed by Mad1 and Mad2. Mad1 and Mad2 are recruited to the Rod-ZW10-ZWILCH complex (RZZ) at the kinetochore and their localization is regulated by Mps1. Mad1 binds stably to unattached kinetochores and recruits Mad2 to the empty kinetochore [65-66]. Mad2 tightly binds to Mad1 at the kinetochore and acquires a closed conformation (C-Mad2) [67-68]. This complex is rather stable and remains bound to the empty kinetochore and comprises one pool of Mad2 in the mitotic cell [66, 69]. The Mad1-C-Mad2 complex recruits Mad2 in its open conformation (O-Mad2), a second pool of Mad2, to the kinetochore where it transiently associates with C-Mad2 and is then released [66-68]. Association of O-Mad2 with the Mad1C-Mad2 complex facilitates its structural conversion allowing for soluble O-Mad2 to bind to Cdc20 and be converted to C-Mad2 [66-68, 70]. Similar to O-Mad2, BubR1 and Bub3 have fast turnover rates at the kinetochore and therefore cycle off the kinetochore to bind to CMad2-Cdc20 to form the MCC which potently inhibits Cdc20. The formation of the MCC is likely regulated by Aurora B, Cdk1 and Bub1 via phosphorylation of Cdc20, but the mechanism still remains unknown. Attachment defects that result in a lack of tension at the kinetochore, such as syntelic attachments, are detected by Aurora B [60]. Aurora B is part of the chromosomal passenger complex located in the centromere chromatin [71]. Aurora B along with other kinetochore complexes destabilize faulty microtubule-kinetochore interactions creating an empty kinetochore which is detected by the spindle assembly checkpoint [60, 71-72]. Once the kinetochore is occupied by microtubules and an appropriate amount of tension is sensed, spindle assembly checkpoint proteins are stripped from the kinetochore and the spindle assembly checkpoint is turned ‗off‘ (Figure 3). Binding of spindle fibers to CENP-E facilitates the release of BubR1 from the kinetochore while dynein, a motor protein, actively

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strips other spindle assembly checkpoint proteins, such as Mad1, Mad2, RZZ complex and Mps1, from the kinetochore [60, 73-74]. The release of spindle assembly checkpoint proteins from kinetochores diminishes the spindle assembly checkpoint signal resulting in Cdc20 activation and anaphase progression.

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Figure 3. Inactivation of the Spindle Assembly Checkpoint. In response to microtubule-kinetochore attachment, SAC proteins are stripped off the kinetochore. Mps1, the RZZ complex and Mad1-C-Mad2 are removed by the dynein/dynactin complex. The interaction of CENP-E with the microtubules induces the inactivation and removal of BubR1 from the kinetochore. Formation of C-Mad1 and the MCC cease and Cdc20 binds to and activates APC/C. The APC/C polyubiquitinates securin, targeting it for degradation by the 26S proteosome. Degradation of securin releases separase which cleaves the Scc1/Rad21 subunit of cohesin resulting in sister chromatid separation and anaphase.

Spindle Assembly Checkpoint Defects and Cancer The spindle assembly checkpoint protects cells from acquiring an aneuploid state. Defects in the spindle assembly checkpoint can lead to chromosome instability and cancer [75]. Alterations in the expression of spindle assembly checkpoint genes are observed in lung cancer [25]. A study investigating spindle assembly checkpoint function in human lung

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cancer cell lines showed that 40% of the cell lines exhibited spindle assembly checkpoint defects [76]. Even though mutations in key spindle assembly checkpoint genes are observed in lung cancer including Mad1 and Bub1 mutations, these mutations are infrequent [77-79]. Therefore, inactivating mutations in spindle assembly checkpoint genes may not be responsible for the high incidence of spindle assembly checkpoint defects in lung cancer. Haploinsufficiency of Mad2 results in a defective spindle assembly checkpoint and an increased incidence of lung tumors in mice [80] and numerous studies report the loss of heterozygosity in 4q, a region where Mad2 is located [81-85]. These data suggest that loss of heterozygosity instead of mutations may be a frequent event in lung cancer and contribute to spindle assembly checkpoint defects.

Kinetochores

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Kinetochore Structure and Function The kinetochore serves to initiate, control and monitor chromosome movements during mitosis. Kinetochores assemble at the centromere and primarily serve to link microtubules from the mitotic spindle to the chromosomes. The kinetochore is comprised of two regions, the inner kinetochore which persists throughout the cell cycle and the outer kinetochore, which assembles and functions only in mitosis. Figure 4 illustrates the organization and location of numerous proteins within the kinetochore. The inner plate of the kinetochore contains nucleosomes with specialized histones, DNA, numerous CENP family proteins, and other proteins [86]. The components and organization of the inner kinetochore are not well understood. The outer kinetochore contains an outer plate that is made up primarily, if not solely, of proteins which form on the inner kinetochore during nuclear envelope breakdown. About 20 microtubule attachment sites exist on the outer plate [86]. The fibrous corona forms on the outermost regions of the kinetochore and is very dynamic. It is comprised mostly of proteins responsible for eliciting the spindle assembly checkpoint, and therefore, is usually only present when microtubules are absent from the kinetochore. CENP-A, a centromere specific variant of histone H3, is the first protein to bind to the kinetochore during kinetochore assembly and is required for the recruitment of other inner kinetochore proteins including CENP-C, CENP-H and CENP-I/MIS6 [86-87]. CENP-A also controls the recruitment and assembly of outer kinetochore proteins in a CENP-A dependent pathway, including Plk, Rod, ZW10, ZWINT-1, CENP-E and spindle assembly checkpoint proteins, Mps1, BubR1, Mad1 and Mad2 [86, 88-90]. BubR1, Bub3 and Bub1 also play an important role in spindle assembly checkpoint protein recruitment to the outer kinetochore but their position in the assembly pathway remains controversial [64, 91]. The chromosomal passenger complex comprised of Aurora B, INCENP, survivin and borealin/dasra B associates with the centromeric heterochromatin in the beginning of mitosis but in a CENP-A independent manner [72, 92]. Kinetochores serve as the anchoring sites for spindle microtubule attachment to chromosomes. Microtubules generated from the centrosomes search the cytoplasm to capture chromosomes at the kinetochore. Once microtubules bind to the kinetochores, their

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interaction is stabilized and chromosome alignment proceeds. CENP-E, a component of the fibrous corona, is involved in the initial binding and anchoring of microtubules to the kinetochore [93-94]. After the initial binding mediated by CENP-E, kinetochore-microtubule stabilization and subsequent chromosome alignment is dependent on the Ndc80/Hec1 complex and the RanGAP1-RanBP2/Nup358 complex [86, 95]. kMTs - - -

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CENP-A nucleosomes

Figure 4. Kinetochore Structure. This figure shows the organization of the centromere-kinetochore region and the locations of some of its protein constituents. kMTs, kinetochore microtubules; Bor, Borealin; Sur, Survivin; MCAK, mitotic centromere-associated kinesin; RZZ, Rod-ZW10-ZWILCH complex; Plk1, polo-like kinase 1; Mps1, multipolar spindle 1. [Adapted from ref. 60]

Another important function of the kinetochore is the correction of erroneous kinetochoremicrotubule attachments. Microtubules bind to kinetochores by chance and therefore, attachment errors are common. Merotelic attachments occur if one or both of the sister chromatids are attached to microtubules originating from both poles. These attachments are not detected by the spindle assembly checkpoint because both attachment and tension

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requirements are satisfied and if left uncorrected will result in chromosome missegregation. The chromosomal passenger complex is responsible for correcting attachment errors before anaphase ensues. This complex and specifically aurora B kinase destabilizes improper microtubule-kinetochore interactions to create an amphitelic orientation [72].

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Kinetochore Dysfunction and Cancer Kinetochore dysfunction can lead to chromosome instability and ultimately cancer. Kinetochore defects that result in enhanced kinetochore-microtubule stability or defects in attachment error correction result in increased merotelic attachments and lagging chromosomes in anaphase. Merotelic attachments and lagging chromosomes are commonly observed in cancer cells. Kinetochore-microtubule attachments in cancer cells are more stable than in normal cells suggesting that attachment defects are persistent and potentially underlie the chromosome instability observed in cancer cells [96-97]. Cellular studies reveal numerous kinetochore proteins that are linked to chromosome stability. Dysregulation of CENP-E, NDC80/Hec1 complex, Aurora B, Kif2b, MCAK, APC and numerous other kinetochore proteins increase merotely and chromosome missegregation in anaphase [4]. In addition, studies show that restoration of normal gene expression levels in cancer cells can restore faithful chromosome segregation. For example, over expression of downstream targets of Aurora B, Kif2b and MCAK, in cancer cells that exhibited chromosome instability decreased the kinetochore-microtubule stability and restored proper chromosome segregation [98]. Even though the link between kinetochore defects and chromosome instability is strong in cell-based studies, only one kinetochore protein, APC, is commonly mutated in tumors [99-100]. Cells with mutations in APC exhibit a high rate of lagging chromosomes in anaphase [101]. Even though mutations in kinetochore proteins appear to be rare in human tumors, kinetochore dysfunction is still likely to be a driving factor in tumorigenesis. Protein imbalances, rather than loss of function mutations, are more likely to result in stabilization of kinetochore-microtubule attachments leading to chromosome missegregation [4]. In support of this hypothesis, aberrant expression of kinetochore proteins is observed in numerous cancers. Overexpression of CENP-A, CENP-H, Aurora B and INCENP are observed in cancer tissues or in cancer cell lines [102-106].

Sister Chromatid Cohesion Mechanism of Sister Chromatid Cohesion Sister chromatid cohesion is essential for proper segregation of chromosomes in metaphase, because the sister chromatids must remain together until the chromosomes are ready to segregate. Sister chromatid cohesion is maintained by a multi-subunit complex called cohesin. Cohesin is comprised of Smc1A, Smc3, Rad21/Scc1 and SA1/2/Scc3 [107]. The core of the cohesin complex is the formation of a heterodimer between Smc1A and Smc3. Each subunit contains a hinge domain in the center of the protein while the C and N terminal

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domains contain ATP-nucleotide-binding domains (NBD) (Figure 5A) [108-110]. The Smc1A and Smc3 subunits interact via the hinge domain creating a V-shaped structure [111]. The role of ATP at the NBD in the formation or function of cohesin remains unknown, but it has been suggested that ATP binding to the subunits forces the NBDs together, while hydrolysis of ATP allows for the dissociation of the domains and the opening of the subunits into a prominent V-shaped structure [111-112]. The third subunit in the cohesin complex is Rad21/Scc1 which binds to the NBDs of Smc1A and Smc3 to form a tripartite ring [108]. The last subunit in the core cohesin complex is SA1/2/Scc3 which binds to Rad21/Scc1. Other accessory proteins involved in loading of cohesin onto chromosomes or in cohesion establishment or release include NIPBL/Scc2, Scc4, ESCO1/2, Pds4A/B, and Wapl/Wapal. The most popular model for cohesin-mediated sister chromatid cohesion is the ring model; however, the exact mechanism remains unknown (Figure 5). The ring model suggests that the interaction between DNA and cohesin is topological. One strong version of the ring model suggests that sister chromatids are enclosed with one tripartite cohesin ring [108] (Figure 5A). One weak version of the ring model, often referred to as the handcuff model, postulates that sister chromatids are held together by two tripartite rings surrounding each chromatid and the tripartite rings are bound together by a single SA1/2/Scc3 subunit (Figure 5B). Another version of the weak ring model suggests that two cohesin rings enclosed around the sister chromatids are topologically interconnected [111] (Figure 5C). In vertebrates, cohesin is loaded onto the DNA during telophase and G1 along the length of the chromosomes. As the cell progresses into S phase and sister chromatids are produced, cohesin establishes cohesion between the sister chromatids with the highest density at the centromeres. As the cell enters into prophase, 90% or more of the cohesin is removed from the chromosome, but the cohesin localized at the centromere remains intact [113]. The release of cohesin from the arms of the chromosomes at the beginning of mitosis creates a soluble pool of cohesin complexes that are not affected by separase activity. This allows a large pool of cohesin complexes to be available for re-association with the chromosomes after anaphase is complete [111, 113]. Release of cohesin from the chromosome arms is mediated by Plk1dependent phosphorylation of Rad21/Scc1 and SA1/2/Scc3 [114]. Cohesin complexes at the centromere are protected from dissociation by shugoshin1 (Sgo1) and protein phosphatase 2A (PP2A) [113]. It is hypothesized that Sgo1 recruits PP2A to the centromere and reverts Plk1mediated phosphorylation of cohesin subunits protecting cohesin from dissociation [113, 115116]. Once the spindle assembly checkpoint is satisfied in mitosis, separase is activated, cleaving the Rad21/Scc1 subunit of cohesin at two sites. Cleavage of Rad21/Scc1 subunit destroys the cohesin ring, releasing the cohesin complex from the centromeres and allowing for sister chromatid separation [113].

Sister Chromatid Cohesion Defects and Cancer Sister chromatid cohesion is important for the maintenance of genomic stability and defects in sister chromatid cohesion during mitosis can lead to large scale chromosome segregation errors. The importance of sister chromatid cohesion is exemplified in the recent findings that numerous human cancers carry mutations in cohesin genes [117]. Smc1A,

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Smc3, and NIPBL are commonly mutated in colorectal cancers resulting in non-functional proteins [118]. These missense and nonsense mutations could disrupt sister chromatid cohesion by preventing proper association of cohesin subunits, by altering Smc ATPase activity or by changing the displacement of cohesin from chromosomes [117]. The dysregulation of the cohesin pathway could then cause chromosome segregation errors in mitosis leading to chromosome instability. Consistent with this hypothesis, colorectal cancer cells exhibit a 100-fold increase in chromosome missegregation compared to normal cells [117, 119].

A)

ATP-Nucleotide Binding Domain (NBD)

Smc1A Smc hinge dimerization domain

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B)

C)

Figure 5. Cohesin Ring Models. The mechanism by which cohesin holds sister chromatids together remains unknown, but three models have been proposed. A) The strong version of the ring model predicts that the sister chromatids are enclosed together within one cohesin ring. B) One weak version of the ring model, also known as the handcuff model, hypothesizes that one cohesin ring encircles each sister chromatid and the two cohesin rings are bound together by one Scc1/Rad21 subunit. C) Another version of the weak ring model proposes the interconnection of two cohesin rings, each encircling one sister chromatid. [Adapted from ref. 111]

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Changes in expression of cohesin and cohesin-associated proteins have also been observed in human cancers. Overexpression of Wapal and Esco2 correlate with cervical cancer and melanoma malignancy, respectively [120-121]. Overexpression of separase is observed in breast cancer, prostate cancer and osteosarcomas [122-123] and leads to premature chromatid separation and lagging chromosomes [122]. The cohesin subunit, Rad21/Scc1, is upregulated in breast and prostate cancer [124-126]. Therefore, alterations in the control of sister chromatid cohesion have the potential to lead to chromosome instability and cancer.

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Hexavalent Chromium: An Aneugenic Environmental and Occupational Carcinogen Hexavalent chromium (Cr(VI)) compounds are potent human lung carcinogens, but their mechanism of action remains unknown. One probable mechanism for carcinogenicity based on current chromium data and on lung tumor data, in general, is the genomic instability model [127]. Molecular studies in chromate-induced lung tumors reveal that mutations in key tumor suppressor genes and oncogenes rarely occur, but genomic instability and epigenetic modifications are prevalent in these tumors. Chromate-induced tumors exhibit both chromosome instability and microsatellite instability. The frequency of microsatellite instability was significantly higher in chromateexposed workers compared to non-exposed individuals and it was strongly correlated with longer exposure time and decreased expression of hMLH1, a mismatch repair protein [128129]. In addition to microsatellite instability, chromosome instability was also observed in chromate-induced lung tumors. Loss of heterozygosity at 6 different loci was observed in 5075% of chromate-induced lung tumors [128]. Although, this increase was not Cr(VI) specific, the high frequency indicates that it is likely an important event in chromate-induced tumors and lung cancer, in general, as most lung cancers exhibit chromosome instability [25]. Epidemiology, whole animal and cell culture studies indicate that solubility plays a key factor in the carcinogenicity of Cr(VI), with particulate or insoluble Cr(VI) compounds being the most carcinogenic [130-133]. Cr(VI) deposits and persists at the lung bifurcation sites where Cr-induced tumors form which is consistent with a particulate exposure [134-135]. The reason for the potency differences between particulate and soluble Cr(VI) compounds remains unknown, but one proposed mechanism is exposure duration differences. Inhalation of soluble Cr(VI) compounds will result in rapid clearance from the lung, but particulate Cr(VI) impacts in the lung bifurcation sites leading to more prolonged exposures to Cr(VI). Aneuploidy has not been assessed in human lung tumors, but cell culture studies indicate that Cr(VI) is a potent aneugenic metal [136-141]. Prolonged exposure to particulate Cr(VI) induces concentration- and time-dependent increases in aneuploidy in both human lung fibroblast and epithelial cells [136-139]. Aneuploidy is not detected after a 24 h exposure but increases in hypodiploid, hyperdiploid and tetraploid metaphases are observed in response to chronic particulate chromate exposure, suggesting exposure duration plays a key role in

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Cr(VI)-induced aneuploidy [136-138]. More importantly, particulate Cr(VI)-induced aneuploidy is a permanent phenotypic change as particulate chromate-induced foci cells exhibit aneuploidy even after 10 passages in chromium-free media [139]. Of the four mechanisms of chromosome instability, centrosome amplification and defects in the spindle assembly checkpoint have been investigated after particulate Cr(VI) exposure. Disruption of the spindle assembly checkpoint is understudied for environmental contaminants, in general. Lung cancers, in general, exhibit expression changes in spindle assembly checkpoint genes [25], but these alterations have yet to be studied in Cr-induced tumors. Cell culture studies show that chronic exposure to particulate chromate induces concentration- and time-dependent increases in spindle assembly checkpoint bypass in the form of centromere spreading, premature centromere division and premature anaphase [136,138]. These data are consistent with the aneuploidy data, showing no increase after only a 24 h exposure but concentration and time-dependent increases after longer exposures [136,138]. Mitotic stage analysis data support the spindle assembly checkpoint bypass data showing the percentage of cells in anaphase increases while the percentage of cells in metaphase decreases after chronic particulate Cr(VI) exposure [136]. Particulate Cr(VI)-induced spindle assembly checkpoint bypass decreases in the presence of mitotic spindle poisons [136] suggesting that the spindle assembly checkpoint is weakened instead of abolished in particulate chromate-treated cells. This outcome is consistent with previous reports that show chromosomally unstable cancer cells are still able to arrest in metaphase in response to spindle poisons [142] and that complete loss of the spindle assembly checkpoint through homozygous mutations in key spindle assembly checkpoint genes results in embryonic lethality [143-145]. These data suggest that complete inactivation of the spindle assembly checkpoint may lead to such a high degree of chromosome instability that it does not offer a selective advantage. The mechanism for particulate Cr(VI)-induced spindle assembly checkpoint bypass remains unknown. It is possible that the improper localization and/or decreased expression of key spindle assembly checkpoint proteins after particulate chromate exposure leads to a weakened spindle assembly checkpoint and eventual spindle assembly checkpoint bypass. Consistent with this hypothesis, chronic exposure to particulate chromate decreases expression of Mad2 [136], but further work is needed to investigate potential localization changes and the role of other proteins in particulate Cr(VI)-induced spindle assembly checkpoint bypass. Centrosome amplification is also strongly associated with numerical chromosome instability and is a common phenotype in numerous cancers including lung cancer [25, 27]. Centrosome amplification has not been investigated in Cr-induced tumors or in animal models, but studies in human cells show that Cr(VI) induces centrosome amplification [137139]. Consistent with the aneuploidy data, no centrosome amplification is observed after only a 24 h exposure, but more chronic exposures to particulate Cr(VI) induce a concentrationand time-dependent increase in centrosome amplification in both mitotic and interphase cells [137-138]. Mitotic stage analysis reveals an increase in cells in mitotic catastrophe and disorganized anaphase after particulate Cr(VI) exposure and these phenotypes are most likely due to centrosome amplification [137]. Mitotic catastrophe is a programmed cell death pathway that

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takes place in mitosis and occurs when cells with DNA damage or abnormal microtubule formation bypass the G2 or spindle assembly checkpoint [146]. One study shows that centrosome overduplication can result in mitotic catastrophe, but cells able to escape mitotic catastrophe can undergo multipolar division or slip out of mitosis without undergoing cytokinesis [146-147]. Bypass of mitotic catastrophe can lead to the generation aneuploid cells and may be an important step in tumorigenesis. It has been argued that centrosome amplification is an epiphenomenon rather than a driving force in tumorigenesis [148]. However, exposure to particulate chromate for only two to five days induces a dramatic increase in centrosome amplification [137-138]. In addition, the increase in centrosome amplification correlates with aneuploidy. More importantly, centrosome amplification continues to be present in cells transformed by particulate Cr(VI) [139]. Mitotic cells with multiple centrosomes are observed in lead chromate-induced foci, most likely resulting in abnormal chromosome segregation during mitosis. Together, these data suggest that centrosome amplification may be an early and persistent event in particulate chromate-induced carcinogenesis and a driving factor for aneuploidy. Centrosome amplification occurs through multiple mechanisms involving centrioles. Centrioles are barrel-shaped structures comprised of nine triplet microtubules located within the centrosome. The most studied mechanisms include centrosome overduplication, failure of cytokinesis, centriole splitting and acentriolar centrosome formation [22]. These mechanisms can be distinguished from each other by studying the centrioles in the centrosome. Failure of cytokinesis is characterized by a binucleated cell containing four centrosomes, with all four centrosomes having two centrioles each. Centriole splitting is distinguished by centrosomes that contain only one centriole. Acentriolar centrosome formation is identified by centrosomes that contain no centrioles. Finally, centrosome overduplication is characterized by a mononucleated cell with greater than two centrosomes, each centrosome containing two centrioles. Failure of cytokinesis is a common mechanism for centrosome amplification and leads to the induction of a binucleated tetraploid cell with four centrosomes each containing two centrioles [149]. Chronic exposure to particulate Cr(VI) induces an increase in tetraploid cells and an increase in cells with amplified centrosomes and normal centrin [138], which are consistent with this mechanism. However, no increases in binucleated cells are observed [137-138]. Thus, failure of cytokinesis does not appear to be involved in particulate Cr(VI)induced centrosome amplification. Particulate chromate also induces centriole splitting [138]. A general mechanism for centriole splitting is not yet defined. Centriole splitting generates multiple centrosomes with only one centriole and these cells can form multipolar spindles and undergo abnormal division. Studies show that centriole splitting can occur when cells enter mitosis with either DNA damage or incompletely replication DNA. Ionizing radiation exposure induces a concentration-dependent increase in centriole splitting [48, 150]. Therefore, these data suggest that Cr(VI)-induced DNA damage is most likely responsible for centriole splitting. Alterations in p16, Sgo1, importin β and exportin 1 expression all lead to centriole splitting [53-56], so it is possible that one or more of these proteins are involved in particulate chromate-induced centriole splitting.

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Acentriolar centrosome formation also occurs after particulate Cr(VI) exposure [138]. The general mechanism for acentriolar centrosome formation is also not well understood. Data suggest that acentriolar centrosome formation may be due to excess centrosomal components [22] or centrosome fragmentation [5]. Centrosomes that fail to undergo proper maturation in late S and G2 are unable to withstand forces exerted on them during mitosis resulting in centrosome fragmentation. Interestingly, cells depleted of DNA repair proteins, XRCC2, XRCC3, Rad51D and Rad51B, all exhibit centrosome fragmentation suggesting that DNA repair proteins may also be involved in maintaining centrosome integrity [41-43]. The centrosome fragmentation observed after depletion of XRCC2 and XRCC3 is not dependent on ATM or ataxia telangectasia related protein (ATR) as treatment with caffeine does not affect centrosome fragmentation suggesting that the G2 checkpoint may not be involved in centrosome fragmentation [151]. XRCC2, XRCC3, Rad51D and Rad51B are all involved in homologous recombination repair of DNA double strand breaks, the primary repair pathway used for Cr(VI)-induced DNA double strand breaks in late S and G2 [152-154]. Therefore, Cr(VI)-induced DNA damage and repair may also be involved in centrosome fragmentation. In addition to DNA repair proteins, chromosome cohesion also plays an important role in maintenance of centrosome integrity. Studies show that depletion of proteins involved in sister chromatid cohesion, such as haspin, Sgo2 and Scc1, induce cells that contain both centriolar and acentriolar centrosomes [155]. Interestingly, particulate Cr(VI)-treated cells with acentriolar centrosomes exhibit a similar mitotic phenotype to those depleted of haspin [137, 155]. More studies are needed to determine the mechanism of Cr(VI)-induced acentriolar centrosome formation, but DNA repair or chromosome cohesion defects are promising mechanisms. Lastly, particulate chromate induces centrosome overduplication [138]. Of the four mechanisms for centrosome amplification, this mechanism is the most studied. Studies show that cells can undergo centrosome overduplication during S and G2 phase arrests [37-38, 45, 156]. Consistent with this mechanism, Cr(VI) exposure induces a persistent G2 arrest [138, 157-161], suggesting that the cells with amplified centrosomes and normal centrin may undergo centrosome overduplication during a particulate chromate-induced G2 arrest. Studies indicate that chromate exposure may induce a small G1 arrest [138, 162]; however, centrosome overduplication most likely occurs during prolonged G2 arrest. Centrosome amplification is linked to DNA damage and a recent study shows that centrosome amplification occurs at a much higher rate after irradiation in a G2-enriched cell population compared to G1-enriched cells suggesting that the DNA damage signal allows for centrosome overduplication in G2-arrested cells [45-47, 163]. Specifically, induction of DNA double strand breaks cause the DNA replication cycle to arrest in G2 while the centrosome duplication cycle continues. DNA damage-induced centrosome amplification requires ATM and Chk1/2, but studies suggest that just a delay in G2 is not sufficient to induce centrosome amplification; rather a specific signal is required [45-47]. The specific signal and mechanism is unknown, but Cdk2, Rad51 and BRCA1 have been suggested to be involved [45, 47-49]. One proposed mechanism hypothesizes that in response to DNA damage, activated Chk1 phosphorylates and activates Cdk2 driving centrosome amplification [47].

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The mechanism for how Cr(VI) overduplicates centrosomes during a G2 arrest is uncertain. However, consistent with the proposed mechanism for DNA damage-induced centrosome overduplication, exposure to particulate Cr(VI) induces DNA damage in the form of DNA adducts and crosslinks, DNA double strand breaks, and chromosome aberrations [157, 164-174]. In addition, studies indicate that particulate Cr(VI)-induced DNA double strand breaks are produced only in G2 [152, 157, 175-177] and activate ATM, ATR, Chk2, Chk1 and Mre11 [152, 157, 162, 175, 178]. In turn, these proteins elicit the G2 arrest observed after particulate Cr(VI) exposure. Cr(VI)-induced DNA double strand breaks are caused indirectly and likely involve repair of ternary Cr-DNA adducts manifested as DNA cross-links [157, 179-180]. Repair of these crosslinks likely involves proficient excision and mismatch repair as these repair systems are essential for Cr(VI)-induced mutagenesis, clastogenesis and double strand break formation [159, 180-181]. To allow time for Cr(VI)-induced DNA double strand breaks to be repaired, cells arrest the DNA replication cycle in G2, in an ATR and ATM-dependent manner [157, 175, 178]. Studies using ionizing radiation reveal that ATR responds to stalled replication forks while ATM is activated in response to DNA double strand breaks, and both of these proteins transduce cell cycle arrest and DNA repair signals [182-183]. Expression of Chk1 and Chk2, two downstream targets of ATR and ATM, also increase in response to Cr(VI) [179]. Altogether, these data suggest that a DNA damage-induced G2 arrest induced by ATM, ATR and Chk1/2 may be involved in Cr(VI)-induced centrosome amplification. Interestingly, a significant amount of cross-talk exists between the spindle assembly checkpoint and centrosome duplication cycle suggesting that perturbations of one or more proteins by particulate chromate may affect both outcomes. For example, Nek2 is involved in both centrosome duplication and the spindle assembly checkpoint. In the centrosome duplication cycle, Nek2 is responsible for inducing centrosome separation in G2 [184], and in the spindle assembly checkpoint, Nek2 is required for Mad2 localization to the kinetochore and proper kinetochore-microtubule attachment [185-187]. Under normal conditions, DNA damage-induced G2 arrest inhibits Nek2 activity and centrosome separation. However, Nek2 is often overexpressed in numerous human cancers and overexpression is correlated with centrosome amplification [188-191]. Dysregulation of Nek2 also impairs the spindle assembly checkpoint signaling and induces premature chromosome segregation in mitosis [185]. Nek2 is just one example of cross-talk between the centrosome duplication cycle and the spindle assembly checkpoint. Other cross-talk proteins include Mps1 and Plk1, among others. Another interesting possibility for cross-talk is that there are numerous overlaps between sister chromatid cohesion and centrosome integrity and duplication. Even though centromere spreading, premature centromere division and premature anaphase are typically associated with spindle assembly checkpoint bypass, defects in kinetochore structure or sister chromatid cohesion may also contribute to these phenotypes. Kinetochore dysfunction can cause improper recruitment or localization of spindle assembly checkpoint proteins, resulting in spindle assembly checkpoint bypass. Defects in sister chromatid cohesion can cause premature dissociation of sister chromatids and phenotypes similar to spindle assembly checkpoint bypass.

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In addition, cohesin, Sgo1 and separase all play a role at the centrosome and are involved in either maintaining or dissolving chromatid cohesion. Cohesin complexes have recently been found at the centrosomes and depletion of cohesin causes centrosome defects, but it remains unknown if these effects are direct or secondary to mitotic defects [192]. Sgo1 is responsible for protecting cohesin complexes at the centromere during mitosis, but is also implicated in maintaining centriole cohesion [54]. Depletion of Sgo1 induces centriole splitting, resulting in multipolar spindle formation in mitosis [54]. Lastly, separase, which is responsible for cleaving the cohesin complex at the onset of anaphase, is also responsible for centriole disengagement in anaphase, proposed to be a licensing event for subsequent centrosome duplication [20]. Therefore, dysregulation of any of these proteins could lead to both sister chromatid cohesion defects and centrosome amplification. In summary, longer exposures to particulate chromate amplify centrosomes and interfere with the spindle assembly checkpoint. Multiple mechanisms are involved in particulate chromate-induced centrosome amplification, which is not uncommon as evidenced by studies in human breast cancer cells that show centrosomes contained one, two, supernumerary or no centrioles at all [27]. Specific mechanisms involved in particulate Cr(VI)-induced centrosome amplification include centrosome overduplication, uncontrolled centriole splitting and acentriolar centrosome formation induced, in part, by DNA damage-induced G2 arrest.

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Conclusion Loss of genomic stability is an important step in carcinogenesis. Centrosome amplification, defects in the spindle assembly checkpoint, kinetochore dysfunction and sister chromatid cohesion defects can all give rise to numerical chromosome instability, a hallmark of cancer. The induction of aneuploidy is one potential mechanism by which environmental and occupational carcinogens can cause cancer. Hexavalent chromium is one example of a human lung carcinogen that induces aneuploidy via centrosome amplification and bypass of the spindle assembly checkpoint. More work is needed to assess the role of aneuploidy and genomic instability in the mechanism of other environmental and occupational carcinogens.

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[154] Camyre, E; Wise, SS; Milligan, P; Gordon, N; Goodale, B; Stackpole, M; Patzlaff, N; Aboueissa, AM; Wise, JP Sr. Ku80 deficiency does not affect particulate chromateinduced chromosome damage and cytotoxicity in Chinese hamster ovary cells. Toxicol Sci, 2007, 97, 348-354. [155] Dai, J; Kateneva, AV; Higgins, JM. Studies of haspin-depleted cells reveal that spindle-pole integrity in mitosis requires chromosome cohesion. J Cell Sci, 2009, 122, 4168-4176. [156] D'Assoro, AB; Busby, R; Suino, K; Delva, E; Almodovar-Mercado, GJ; Johnson, H; Folk, C; Farrugia, DJ; Vasile, V; Stivala, F; Salisbury, JL. Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint. Oncogene, 2004, 23, 4068-4075. [157] Xie, H; Holmes, AL; Young, JL; Qin, Q; Joyce, K; Pelsue, SC; Peng, C; Wise, SS; Jeevarajan, AS; Wallace, WT; Hammond, D; Wise, JP Sr. Zinc chromate induces chromosome instability and DNA double strand breaks in human lung cells. Toxicol Appl Pharmacol, 2009, 234, 293-299. [158] O'Brien, TJ; Fornsaglio, JL; Ceryak, S; Patierno, SR. Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae. DNA Repair (Amst), 2002, 1, 617-627. [159] Peterson-Roth, E; Reynolds, M; Quievryn, G; Zhitkovich, A. Mismatch repair proteins are activators of toxic responses to chromium-DNA damage. Mol Cell Biol, 2005, 25, 3596-3607. [160] Zhang, Z; Leonard, SS; Wang, S; Vallyathan, V; Castranova, V; Shi, X. Cr (VI) induces cell growth arrest through hydrogen peroxide-mediated reactions. Mol Cell Biochem, 2001, 222, 77-83. [161] Wakeman, TP; Wyczechowska, D; Xu, B. Involvement of the p38 MAP kinase in Cr(VI)-induced growth arrest and apoptosis. Mol Cell Biochem, 2005, 279, 69-73. [162] Ha, L; Ceryak, S; Patierno, SR. Chromium (VI) activates ataxia telangiectasia mutated (ATM) protein. Requirement of ATM for both apoptosis and recovery from terminal growth arrest. J Biol Chem, 2003, 278, 17885-17894. [163] Inanç, B; Dodson, H; Morrison, CG. A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage. Mol Biol Cell, 2010, 21, 3866-3877. [164] Wise, SS; Holmes, AL; Ketterer, ME; Hartsock, WJ; Fomchenko, E; Katsifis, S; Thompson, WD; Wise, JP Sr. Chromium is the proximate clastogenic species for lead chromate-induced clastogenicity in human bronchial cells. Mutat Res, 2004, 560, 7989. [165] Wise, JP; Leonard, JC; Patierno, SR. Clastogenicity of lead chromate particles in hamster and human cells. Mutat Res, 1992, 278, 69-79. [166] Wise, JP Sr; Wise, SS; Little, JE. The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells. Mutat Res, 2002, 517, 221-229. [167] Wise, SS; Elmore, LW; Holt, SE; Little, JE; Antonucci, PG; Bryant, BH; Wise, JP Sr. Telomerase-mediated lifespan extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or genotoxicity. Mol Cell Biochem, 2004, 255, 103-111.

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[168] Wise, SS; Schuler, JH; Holmes, AL; Katsifis, SP; Ketterer, ME; Hartsock, WJ; Zheng, T; Wise, JP Sr. Comparison of two particulate hexavalent chromium compounds: Barium chromate is more genotoxic than lead chromate in human lung cells. Environ Mol Mutagen, 2004, 44, 156-162. [169] Xie, H; Holmes, AL; Wise, SS; Gordon, N; Wise, JP Sr. Lead chromate-induced chromosome damage requires extracellular dissolution to liberate chromium ions but does not require particle internalization or intracellular dissolution. Chem Res Toxicol, 2004, 17, 1362-1367. [170] Wise, JP; Orenstein, JM; Patierno, SR. Inhibition of lead chromate clastogenesis by ascorbate: relationship to particle dissolution and uptake. Carcinogenesis, 1993, 14, 429-434. [171] Wise, JP Sr; Stearns, DM; Wetterhahn, KE; Patierno, SR. Cell-enhanced dissolution of carcinogenic lead chromate particles: the role of individual dissolution products in clastogenesis. Carcinogenesis, 1994, 15, 2249-2254. [172] Wise, SS; Schuler, JH; Katsifis, SP; Wise, JP Sr. Barium chromate is cytotoxic and genotoxic to human lung cells. Environ Mol Mutagen, 2003, 42, 274-278. [173] Wise, SS; Holmes, AL; Wise, JP Sr. Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to human lung epithelial cells. Mutat Res, 2006, 610, 2-7. [174] Holmes, AL; Wise, SS; Sandwick, SJ; Wise, JP Sr. The clastogenic effects of chronic exposure to particulate and soluble Cr(VI) in human lung cells. Mutat Res, 2006, 610, 8-13. [175] Xie, H; Wise, SS; Holmes, AL; Xu, B; Wakeman, TP; Pelsue, SC; Singh, NP; Wise, JP Sr. Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells. Mutat Res, 2005, 586, 160-172. [176] Reynolds, M; Stoddard, L; Bespalov, I; Zhitkovich, A. Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair. Nucleic Acids Res, 2007, 35, 465-476. [177] Reynolds, MF; Peterson-Roth, EC; Bespalov, IA; Johnston, T; Gurel, VM; Menard, HL; Zhitkovich, A. Rapid DNA double-strand breaks resulting from processing of CrDNA cross-links by both MutS dimers. Cancer Res, 2009, 69, 1071-1079. [178] Hill, R; Leidal, AM; Madureira, PA; Gillis, LD; Waisman, DM; Chiu, A; Lee, PW. Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNAPK) and differential induction of p53 target genes. DNA Repair (Amst), 2008, 7, 14841499. [179] Ha, L; Ceryak, S; Patierno, SR. Generation of S phase-dependent DNA double-strand breaks by Cr(VI) exposure: involvement of ATM in Cr(VI) induction of gammaH2AX. Carcinogenesis, 2004, 25, 2265-2274. [180] Brooks, B; O'Brien, TJ; Ceryak, S; Wise, JP Sr; Wise, SS; Wise, JP Jr; Defabo, E; Patierno, SR. Excision repair is required for genotoxin-induced mutagenesis in mammalian cells. Carcinogenesis, 2008, 29, 1064-1069. [181] Dronkert, ML; Kanaar, R. Repair of DNA interstrand cross-links. Mutat Res, 2001, 486, 217-247. [182] Falck, J; Coates, J; Jackson, SP. Conserved modes of recruitment of ATM; ATR and DNA-PKcs to sites of DNA damage. Nature, 2005, 434, 605-611.

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[183] Lee, JH; Paull, TT. ATM activation by DNA double-strand breaks through the Mre11Rad50-Nbs1 complex. Science, 2005, 308, 551-554. [184] Faragher, AJ; Fry, AM. Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindles. Mol Biol Cell, 2003, 14, 2876-2889 [185] Lou, Y; Yao, J; Zereshki, A; Dou, Z; Ahmed, K; Wang, H; Hu, J; Wang, Y; Yao, X. NEK2A interacts with MAD1 and possibly functions as a novel integrator of the spindle checkpoint signaling. J Biol Chem, 2004, 279, 20049-20057. [186] Liu, Q; Hirohashi, Y; Du, X; Greene, MI; Wang, Q. Nek2 targets the mitotic checkpoint proteins Mad2 and Cdc20: a mechanism for aneuploidy in cancer. Exp Mol Pathol, 2010, 88, 225-233. [187] Du, J; Cai, X; Yao, J; Ding, X; Wu, Q; Pei, S; Jiang, K; Zhang, Y; Wang, W; Shi, Y; Lai, Y; Shen, J; Teng, M; Huang, H; Fei, Q; Reddy, ES; Zhu, J; Jin, C; Yao, X. The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability. Oncogene, 2008, 27, 4107-4114. [188] Barbagallo, F; Paronetto, MP; Franco, R; Chieffi, P; Dolci, S; Fry, AM; Geremia, R; Sette, C. Increased expression and nuclear localization of the centrosomal kinase Nek2 in human testicular seminomas. J Pathol, 2009, 217, 431-441. [189] Tsunoda, N; Kokuryo, T; Oda, K; Senga, T; Yokoyama, Y; Nagino, M; Nimura, Y; Hamai, M. Nek2 as a novel molecular target for the treatment of breast carcinoma. Cancer Sci, 2009, 100, 111-116. [190] Hayward, DG; Clarke, RB; Faragher, AJ; Pillai, MR; Hagan, IM; Fry, AM. The centrosomal kinase Nek2 displays elevated levels of protein expression in human breast cancer. Cancer Res, 2004, 64, 7370-7376. [191] Hayward, DG; Fry, AM. Nek2 kinase in chromosome instability and cancer. Cancer Lett, 2006, 237, 155-166. [192] Kong, X; Ball, AR Jr; Sonoda, E; Feng, J; Takeda, S; Fukagawa, T; Yen, TJ; Yokomori, K. Cohesin associates with spindle poles in a mitosis-specific manner and functions in spindle assembly in vertebrate cells. Mol Biol Cell, 2009, 20, 1289-1301.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter II

Ploidy in Mitosis and Meiosis: A Role of MAPK/Erk Network Franck B. Riquet, Pauline Vandame and Jean-François Bodart* EA4479 Laboratoire de Régulation des Signaux de Division University of Lille 1, Cell Signalomics Group, Parc de la Haute Borne, France

Abstract

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Genetic unbalances resulting in gain or loss of chromosomes may affect cell evolution towards unregulated growth characteristics and diminished response to apoptosis, cellular damage and cell cycle checkpoints. Besides their dependency towards oncogenes, evidences have been raised towards the idea that cancers might also be dependent upon various abnormal assortments of chromosomes, aneuploidy or even polyploidy. In addition to their roles in transcription up-regulation at G1/S, which have been shown to be involved in tumorigenesis, Mitogen Activated Protein Kinases (MAPK) from the Extracellular Regulated Kinase (Erk) group may recruit transcription-independent transduction mechanisms involved in cell reorganization at division and particularly in genetic material segregation. While the role of Erk in M-phase cellular reorganization may be discussed in mammalian cells, meiotic models, including urochordates, molluscs, amphibian and mammals female gametes, have provided case studies where the deregulation of the Erk network lead to catastrophic events or uncontrolled division cycles. This chapter describes the functions explored for MAPK/Erk during M-phase cellular reorganization and the network of its regulators. The role of a particular MAPKKK, Mos, with regards to their involvement in genomic stability and ploidy, will be addressed and discussed. *

Corresponding author : telephone (33)320436867 ;Fax (33)320434038 ; e-mail : jean-francois.bodart @univlille1.fr, EA4479 Laboratoire de Régulation des Signaux de Division University of Lille 1, USR 3078 CNRS. Cell Signalomics Group, Parc de la Haute Borne. F-59655 Villeneuve d‘Ascq cedex, France.

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Keywords: Aneuploidy, Mitosis, Meiosis, Erk, Mos, p53, polyploidy, genomic instability

1. Crucial to Cell Integrity, Genomic Stability Is Controlled Throughout Cell Cycle

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1.1. Genomic Stability The ability to dissect the cancer genome has revealed extensive genetic changes both at the chromosomal and the nucleotide levels. These changes have been referred to genetic or genomic instability. Genetic instability may be observed under different forms and debates have arisen whether this genetic instability represents the cause or the consequence of tumorigenesis. Aneuploidy, designating an abnormal number of chromosomes, is opposed to euploidy, which refers to a normal set of chromosomes that is either diploid or an exact multiple of the diploid number. Several evidences have underlined the role of aneuploidy in precocious steps in carcinogenesis [1]: (i) Aneuploidy is ubiquitous in solid cancer, (ii) carcinogens cause aneuploidy, (iii) aneuploidy leads to unbalance dosage or expression of thousands of normal genes, and (iv) aneuploidy immortalizes cells. According to this model, random, but minor and non-cancerous, aneuploidy first accumulates in an emerging population of cells. Then, aneuploid cells ‗autocatalytically‘ generate new karyotypes, including lethal, pre-neoplastic or neoplastic ones [2]. The latter are characterized by high genomic instability and malignancy. The degree of malignancy appears to be also proportional to the degree of aneuploidy in cancer cells and attention has now been focused on mechanisms driving aneuploidy or more widely, on genomic instability, since genomic integrity is a crucial issue to any therapeutically approaches dealing with tumorigenesis and long term survival of treated-patients.

1.2. Failures in the Mitotic Machinery The cell cycle is controlled in a way to avoid ―madness‖ at the helm. Nevertheless the intrinsic robustness of the cell cycle and redundancies within the network, which are both in charge of the control checkpoints, enable transgression and progression in ―wrong‖ ways, which could result, for instance, in aneuploidy. Then, unbalance in the number of chromosomes is thought to result in failure of the organization or disorganization of the mitotic spindle, or in failure in the tight control of cell cycle progression. Four main phases, in addition to G0 or quiescence, may be distinguished: the phase Gap 1 (G1) precedes the replication and duplication of chromosomes (S-Phase), while the Gap 2 (G2) phase prepares the division of M-Phase (mitosis), which can be controlled either at its entry, at the G2/M border by mitogen signals, or at the metaphase-anaphase transition, prior to chromosome segregation (figure 1A). The events driving cell division itself are timely and spatially strictly controlled. Cell division aims at taking the duplicated genome and ensuring its equal distribution to each daughter cell. Cell division even culminates in the partition of the condensed chromosomes, which is achieved by the mitotic spindle. This complex machinery

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exhibits a bipolar geometry between centromeres or MicroTubules-Organizing Centers (MTOC) that enable correct segregation of the genetic material.

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(A)

(B) Figure 1. Cell cycle and regulatory machinery to M-Phase entry. A, basic scheme for cell cycle, with regulatory checkpoints at G1/S, G2/M, metaphase-anaphase and to DNA damage at G2. B, MPF regulation and DNA damage response. Main regulators are mentioned, which are discussed in the different sections of this chapter. G1/S transition is driven by the association of Cdks (2,4,6) with G1Cyclins D and E. At G2/M transition, MPF is activated by dual-phosphatase Cdc25 and CAK (Cdk Activating Kinase), which promotes Thr161 phosphorylation and association of the two subunit of the MPF dimmer. An autoamplification loop exists between MPF and Cdc25, which is also regulated by phosphorylation driven by Plk1. MPF is negatively regulated by kinases Myt1 and Wee1. DNA damage response is linked to M-phase entry machinery, and upon ATR/ATM activation, can delay or inhibit MPF activation, through Cdc25 inhibition or Wee1/Myt1 activation.

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Franck B. Riquet, Pauline Vandame and Jean-François Bodart

Spindle morphogenesis and cell reorganization at G2/M, which includes nuclear envelope breakdown, chromosome condensation, disappearance of Golgi apparatus and reorganization of endomembrane system, are complex events requiring the coordination of a regulatory network of protein through post-traductionnal modifications (phosphorylation, ubiquitynation, farnesylation,...) or through stimulation of protein synthesis. Protein kinases play key roles in these phenomena. Protein kinases covalently transfer the c-phosphate of ATP to recipient amino acid side chains on target proteins. These processes can be mediated by Serine / Threonine kinases (on Serine or Threonine residues) or by Tyrosine kinases (on Tyrosine residues). Mitotic decision is tightly linked to the activation of MPF (M-phase Promoting Factor). MPF is a universal heterodimer composed of at least one regulatory subunit, the cyclin B, and a catalytic subunit, cdk1. Its existence was first reported in the Northern Leopard Frog, Rana pipiens, through the pioneer experiments of Yoshio Masui [3], and the conundrum of its nature was solved at the biochemical level more than a decade and a half later, in Xenopus oocytes [4]. Cyclin B levels are periodically regulated by transcriptiontranslation / degradation cycles while Cdk1 has to be (1) phosphorylated on Thr 161 on its Tloop to produce an active kinase, and (2) to be dephosphorylated on Thr14 and Tyr15 for activation. The latter residues are targeted by antagonistic inhibitory kinases (Wee1, Myt1) and activatory dual-phosphatase Cdc25 (figure 1B) [5,6]. Similar mechanisms are involved in progression through G1 and S since the latter is closely associated to the activation and mobilization of so called Cyclin G1 (Cyclins D and E) and Cdks 2,4 and 6. Phosphorylation of protein kinases target proteins results in changes in proteins activities, interactions, localizations, or stability, thereby propagating a signal, which influences cellular decisions and functions. Signaling cascade propagation through mobilization of protein kinases is tightly controlled by combinations of regulation motifs, including feedforward and feedback mechanisms, which ensure irreversible, sustained or transitory decisions [7-9]. These mechanisms are closely controlled to prevent aberrant protein kinases activation or inactivation, and cellular decisions are themselves subjected to regulatory checkpoint like those controlling cell cycle progression throughout replication and division [5,6]. Deregulation of these pathways lead to severe pathologies such as carcinogenesis. Cells with more than two centrosomes during mitosis may assemble multipolar spindles that unequally distribute chromosomes to the daughter cells. Cells with persistent chromosomal abnormalities may undergo cell death, but some of them would survive and become malignant clones bearing defective genomic informations. These cells can be characterized by gains or losses of whole chromosomes along with karyotypic alteration such as nuclear pleomorphism and multi/giant-nucleated cells [10,11].

2. MAPK Network Members Are Involved in Genomic Stability 2.1. MAPK, a Node in a Network for Signal Integration The Mitogen Activated Protein Kinases (MAPK) / Extracellular regulated kinases (Erk) network is considered as one of the most important node in proliferation and thus, in cell

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cycle control and cancer models. Typically, the MAPK/Erk cascade is a highly conserved signaling pathway throughout eukaryotic cells, bridging cell surface receptors to diverse executor proteins, integrating signals and modulating many aspects of cell life such as cell cycle, survival, differentiation and cell migration.

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Figure 2. MAPK/Erk network. Erk cascade is built from three layers activated upon the activation of a signaling complex (upstream levels of regulation like GTPases have not been added not to increase the complexity of this interacting network), and serves as a node to integrate cell response. More than 160 substrates have been identified for Erk and may be grouped. See text for details about isoforms of MEK and Erk.

Being crucial element in the network of signal integration, the MAPK cascade (figure 2) consists of three layers, each one being composed of a kinase (MAPKKK or MAP3K, MAP2K or MEK and MAPK/Erk). The MAP3K tier of the cascade contains a few components, which include Raf-kinases, Mos, Tpl2 and MEKK1, and operates under distinct and different conditions [12]. The two other tiers of the cascade contain two groups of closely related proteins. First, the 45 kDa MEK1, 46 kDa MEK2, and 43 kDa MEK1b, which lacks 26 amino-acids in its kinase domain and has been suggested to be an inactive MEK1 isoform [13,14], is an evolutionarily conserved group of proteins sharing high degrees of homology [15,16]. MEK1 and MEK2 are mostly cytoplasmic where they interact with the downstream layer of Erk cascade through a N-terminal domain, providing cytoplasmic anchoring and rapid Erk1/2 activation upon mobilization of the cascade [17,18]. Second, the MAPK tier includes 44 kDa Erk1 and 42 kDa Erk2, which share 70 % of homology [19]. It is to note that several alternatively spliced variants of Erk have been described: rodent Erk1b [20], primate Erk1c [21,22] and primate Erk2b [23]. Differences in the role of MEK and Erk have been described elsewhere (i.e. [24]) and will not be further discussed here. This network acts as a transducer activated by several key growth factors or oncogenes. There have been compelling evidences for the involvement of MAPK/Erk in pathogenesis progression and oncogenic behavior in many human tumors including breast carcinoma,

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glioblastoma, as well as primary tumor cells derived from kidney, colon and lung tissues [2529]. Deregulation of the MAPK/Erk pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous pathologies including cancers, resulting in the constitutive activation of ERK and continual cell proliferation [30,31]. Nevertheless, while involvement of Raf (MAPK kinase kinase/MAP3K/MEKK)-MEK (MAPK kinase/MAP2K)-Erk cascade has been considered for control of growth signals, cell survival, tumor progression and invasion (angiogenesis and loss of cell adhesion), aneuploidy and its consequences has not received much attention.

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2.2. Requirement for MAPK/Erk in G2/M Progression in Mitotic Models Contribution of MAPK/Erk to cell cycle G2/M transition has been explored in somatic cells. Treatment of synchronized mouse NIH 3T3 cells with U0126 has been reported to lead to spindle defects: chromosome misalignment, abnormal chromosome segregation and multipolar spindles [32]. In contrast, spindle assembly in HeLa and RPE1 cells was not affected by MEK 1/2 inhibition with U0126 or PD184352 [33]. These results were confirmed by observations made by Shinohara and co-workers [34] in NIH3T3, HeLa, Ptk or RPE1 cells, where inhibition of Erk activity seemed to have no deleterious effects on spindle assembly. These contradictory observations have shaped the hypothesis that negative deregulation of MAPK activity drives catastrophic events during M phase progression. Nevertheless, the involvement of the MAPK/Erk network in mitosis regulation has been more clearly established since then [35]. There is a body of evidences demonstrating that upon pharmacologic inhibition, siRNA mediated or dominant negative MEK1 strategies, there is a noticeable delay in G2/M kinetics [36-40]. Consistent with the lack of Erk1/2 activation, down regulation of mitotic Ser/Thr kinases, such as Cdk1, Plk1, Aurora A and B, can be observed [39]. Similarly, when treated by Erk1/2 inhibitors, bladder cancer cells lines exhibit G2 arrest, presumably linked to the loss of Cyclin B1 expression [41]. In addition to above-mentioned mitotic regulators, Erk network has been involved in the regulation of Cdc25 [42,43] CENP-E [44], SWI/SNF [45] and Plk3 [46]. Noteworthy, this network has also been involved in the Golgi apparatus fragmentation during mitosis [47,48]. Finally, formation of centromere-containing micronuclei induced in Chinese hamsters fibroblasts by alkylating agents (Methylnitrosurea, MNU) was suggested to be linked together with the inhibition of Rsk [49].

2.3. Antagonistic Effects of Erk Pathway: Towards Genomic Integrity MAPK is activated in a variety of transformed cells and the MEK-MAPK pathway also mediates, if not totally, transformation induced by Ras, Raf and other oncoproteins [50]. Induction of ectopic H-Ras expression in p53-/- NIH3T3 cells drives premature entry into S phase, increase permissivity for gene amplification and generation of aberrant chromosomes within one cell cycle [51,52]. Thus, oncogenic H-Ras has been shown to induce karyotypic

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instability: when overexpressed, oncogenic Ras also generates chromosomes aberrations in rat mammary carcinoma cells, rat prostatic tumors cells [53] and in human colon carcinoma cell lines [54]. These chromosomes aberrations lead to improper chromosome segregation and subsequent exclusion of chromosomes from daughter nuclei [55,56]. Predominent defects induced by v-Ras are (i) formation of mitotic bridges, resulting from the acquisition of one or more centromeres, and (ii) centrosome amplification, which results in the formation of multiple mitotic spindles and chromosomes missegregation [57,58]. On one hand, Saavedra and coworkers stressed that transformed phenotypes associated with high genomic instability induced by Ras [micronuclei formation, abnormal centrosome amplification and DNA bridges] were strongly dependent upon MAPK activity [58]. On the other hand, NIH3T3 cells transformed by H-Ras V12 produce large amount of reactive oxygen species (ROS) superoxide [59], and induction of chromosomal instability by H-Ras V12 might also be dependent upon ROS production, since it can be rescued by N-acetyl-cysteine, a ROS scavenger [60,61]. In addition to studies on the role of the cascade Ras-Raf-MEK-Erk on promoting genomic instability, there have been evidences that Erk sustained activity was also involved in genomic instability induced by other carcinogens such as hepatitis B virus X oncoprotein (HBx) and arsenite. Integration of Hepatitis B virus has been suggested to facilitate genetic alterations of the host genome at stages of chronic viral hepatitis and cirrhosis, thereby increasing the risk for hepatocarcinoma development [62]. Ectopic expression of HBx in hepatoma cells leads to multinucleated cell population increase, resulting from aberrant mitosis progression, and to chromosomal aberrations like chromosomes rearrangement and micronuclei formation [63-66]. Similarly, ectopic expression of HBx in Chang liver cell line induced multipolar spindle formation and chromosomal missegregation during mitosis but also increased cell population with multinuclei or micronuclei. These effects, related to centrosome amplication, were mediated by the Ras-MEK-Erk pathway [67]. Arsenite induces injuries like chromosomal aberrations [68], micronuclei [69] and aneuploidy [70]. Arsenite activates Ras, Raf and Erk, through EGF receptor activation, in Src-dependent and ligand independent manner [71]. Under exposure to arsenite, genomic action of Erk involves the transcription of c-fos and c-jun and the DNA binding activity of AP-1 in the promotion of anchorage-independent growth [72] whereas transcriptionindependent and multi-functions of Erk contribute to genomic instability [73]. Though MAPK/Erk signals is clearly involved in genomic stability, it may also exert a protective role in maintaining genomic stability under a variety of carcinogens such as cadmium [74] or ionizing radiations [75], and protecting cell from aneuploidy through delaying cell cycle progression. Indeed, activation of the MAPK/Erk network either by growth factors or phorbol esters has been involved in G2-phase delay [76]. Several mechanisms have been proposed for this G2-delay, implicating (1) cdk inhibitor p21WAF1/CIP1 [76] or (2) response to DNA damage machinery with ATM and related ATR-dependent pathways, which are upstream regulators of signal negatively controlling the activation of mitotic cyclin/Cdks through the action of Chk1 and Chk2 [77,78]; or (3) MEKdependent pathway destabilizing Cdc25B by increasing the phosphorylation of residue Ser249 [43]. In response to stresses that trigger G2 delay through ATR/ATM signaling (figure 1) or extracellular stresses driving MAPK/Erk network activation, entry into mitosis

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would be delayed until intercellular / extracellular stresses have been solved. In this context, such delay in G2-phase is thought to provide enough time i.e. for reparation [79]. Such delay induced by MAPK/Erk has been reported to be required to maintain genomic integrity, or at least, not to increase the rate of chromosomal aberrations. Indeed, Rsk knock-down lead to an increase in the HGF-induced chromosomal defects [80]. Then, in contrast to pathological contexts induced by Ras and MAPK/Erk network, increased MAPK activity may also be linked to maintenance of genomic integrity and decreased activity may be associated to increase in chromosomal defects. Nevertheless, the relationship between growth factorinduced delay in G2/M, mitotic regulator and MAPK/Erk, as well as the physiological significance of such delay, remain to be fully clarified.

3. MAPK Pathway in G2/M, Lessons from Vertebrates Gametes In addition to its role in transcription up-regulation at G1/S, which is largely involved in tumorigenesis, the notion that MAPK/Erk pathway exerted functions in cell reorganization at M-phase and genetic material segregation, came primarily from studies in vertebrates oocytes. Oocytes have offered for many decades biochemical and physiological playgrounds, where kinases network could be explored in absence of p53-dependent checkpoints that exist in somatic cells.

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3.1. MAPK Activation in the South African Clawed Xenopus Oocytes, a Case of Study for Physical Properties of MAPK Activation Oocytes from many species have provided numerous insights in spindle morphogenesis and provide physiological model for chromosome segregation, which does not rely on transcription. In many species, MAPK/Erk network has appeared as a crucial component in the network regulating meiosis (figure 3, 4). In vertebrates‘ oocytes, the oncoprotein Mos acts as the upstream activator of MEK and MAPK/Erk levels. This oocyte-expressed kinase appeared early during animal evolution and functioned ancestrally in regulating specializations of female meiosis [81]. Once accumulated, Mos phosphorylates MEK, which in turn activates MAPK/Erk by dual phosphorylation of a TEY motif [82]. In amphibian, Mos-activated cascade prevents DNA synthesis during meiosis and promotes spindle morphogenesis as well as the cytostatic activity present in metaphase-II arrested oocytes [8386]. In this model, physiological role of Raf has been minored and Mos is thought to literally hijack the control of the MAPK cascade. A specific all-or-none response for MAPK/Erk activation is observed in these oocytes, in contrast to the gradual response of MAPK/Erk to external stimuli in mammalian somatic cells. The cascade arrangement of the signaling network generates the steepness of MAPK/Erk response in Xenopus oocytes. The physical properties of the cascade, which includes ultrasensitivity, bistability and irreversibility [8791], are thought to mainly arise for bistability and ultrasensitivity from the existence of a feed-back loop motif. Indeed, the Mos-MEK-MAPK/Erk network has been found to be

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embedded in a positive feed-back loop, driven by MAPK/Erk itself [88,92-94] and / or through MPF action [95-97], which promotes both Mos stability and synthesis.

Figure 3. MAPK inhibition prevents M-phase entry in mollusk Patella vulgata. Patella is a rare model where MAPK is required for meiotic resumption and breakdown of oocyte nucleus, or germinal vesicle. Indeed, when MAPK activation is impaired by U0126, the germinal vesicle remains intact and no spindle formation is observed in these conditions (ASW, Artificial Sea Water, U0126, Chemical inhibitor of MEK; GVBD: Germinal Vesicle BreakDown).

3.2. Role of MAPK/Erk Network in Spindle Organization MAPK/Erk activity has been suggested to be required for functional spindle assembly checkpoint in oocyte extracts [98-100]. Though presence of MAPK/Erk on kinetochores has been suspected in somatic cells [101], proteomic studies failed to find MAPK/Erk associated to isolated human metaphase chromosomes [102]. Thus, whether MAPK/Erk in its active form is a component of kinetochore, and whether it is required for spindle assembly checkpoint function, remains somehow controversial.

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Figure 4. Multiple roles of MAPK/ERk network in meiosis and interplay with MPF pathways. A, Interplay between MAPK/Erk and MPF pathways in Xenopus oocytes. Oocyte M-Phase initiation machinery activates these pathways through Mos and Cyclin B synthesis (*). The MAPK/Erk cascade is embedded in a positive feedback loop from MAPK to Mos. Since MPF and MAPK/Erk pathways are intimately linked, the latter exhibits a typical dynamics, which depends partially on MPF dynamics. B, Multiple and conserved role for MAPK/Erk in Urochordates and Amphibian.

From observations made in many species like starfish, jellyfish, urochordates, amphibian and mice, it is thought that control in meiotic spindle morphogenesis and positioning and chromatin organization are conserved functions for Mos and MAP/Erk network. First observations were made in nullizygous mice for Mos, where MEK activation is impaired and interphase-like structure for microtubules and chromosomes may be found between meiotic division, as well as formation of monopolar half-spindle [103-106]. Similar observations were made in other biological systems. MAPK/Erk cascade regulates spindle bipolarity through its direct or indirect effects on microtubule dynamics in amphibian oocytes [86,107]. No bipolar spindle anchored at the plasma membrane is observed when MAPK/Erk activity is inhibited by chemical inhibitors of MEK such as U0126 [32,108,109]. Rana japonica oocytes treated with U0126 also fail to organize a Microtubule Organizing Center (MTOC) at the bottom of

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the germinal vesicle and chromosomes are only partially condensed [110]. MAPK inhibitors, both in vitro [32] and in vivo [86], lead to the formation of monopolar spindle or aster-like structures, attesting the failure to establish a bipolar organization (figure 5). Similarly, when Mos accumulation is prevented in vivo, Xenopus oocytes exhibit aster-like structures. Such structures remain to be fully characterized but do not enable oocytes to properly segregate their genomic content [86]. Finally, the latter observation stressed by inhibiting the network at different levels that Mos and MAPK/Erk were playing distinct but complementary roles in spindle morphogenesis [86], suggesting that MAPK/Erk was made up with functional modules [111], which may exert distinct actions at different levels of spindle organization and potentially result, when deregulated, in different type of aneuploidy. Such particular role for Mos may contribute to the chromosome instability of tumor cells exhibiting an up-regulation of Mos.

Figure 5. Aster formation in absence of MAPK activity in Xenopus oocytes.

4. Mos, Particularities of a MAPKKK in Tumor Cells and Gametes 4.1. Transformation and Aneuploidy Driven By Mos Recombinant v-Mos was among the first oncogenes shown to transform cells efficiently in DNA transfection assays [112,113]. v-Mos and c-Mos have been demonstrated to bear essentially similar biological activities [112]. Moreover, their inappropriate expressions have been shown to be sufficient for morphological transformation [112-115]. When constitutively expressed, Mos could induce either cell death or oncogenic transformation, depending on its expression level [116]. Infection of Swiss 3T3 with Mo-MuSV (Moloney Murine Sarcoma Virus) causes a large percentage of the cells to round-up and detach from the monolayer to form floating cells [117,118]; only the remaining attached cells grow as morphologically

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transformed cells. When transformed by v-Mos, cells exhibit genetic instability [119], and binucleated tumor cells were one of the earliest unexplained histopathological observations found in Mo-MuSV-induced sarcomas in mice [120]. Mos associates with microtubules [121,122] and kinetochores [123]. When a bacterially expressed maltose-binding protein (MBP)-Mos Xenopus fusion protein is microinjected into potoroo epithelial cells (PtK1), it blocks mitosis by preventing normal metaphase spindle organization. Localization of Mos fusion protein at kinetochores was proposed to result in congression failure and in increased chromosomal instability [123]. Among the growth arrested floatting cells induced by v-Mos infection in Swiss 3T3, one third, which DNA content was 4C, were binucleated and underwent nuclear division [karyokinesis] but not cytokinesis. In the floatting cells precursors, mitotic spindle lacked astral microtubules and the spindle apparatus was asymetrically positioned with one spindle pole juxtaposed to the cell membrane, reminding the spindle topology during oocyte meiosis [116]. Apoptosis and growth arrest induced by Mos-MAPK in these conditions appeared to be p53 dependent [124]. p53-/- MEF (Mouse Embryo Fibroblasts) display abnormal mitotic spindles with three or more spindle poles [125] and chromosome instability is often observed in tumor cells lacking p53 [127-129]. In the absence of p53, Mos-induced apoptosis during S phase was abrogated and cells continued to cycle: nonpartitioning chromosomes and multinucleation cells rate were dramatically enhanced by Mos-MAPK [130]. Thus, surimposing a meiotic program, which orchestrates sister chromatid exchange and chromosome segregation in an unique way, on a mitotic cell cycle could possibly influence and compromise somatic cell checkpoint functions [130]. Nevertheless it is quite unlikely that Mos-driven transformation depends upon Mos distinct functions and that chromosomal changes are causatively related to oncogenic transformation. Mos-driven transformation depends mainly upon its ability to activate the MAPK cascade. Indeed, MEK overexpression is sufficient to induce transformation [131] and greatly enhances Mos transformation activity [132,133] whereas co-expression of dominant negative MEK1 or CL100 MAPK-phosphatase both inhibit Mos-driven transformation [132,133].

4.2. Number of M Phases Cycles: Does Mos Rule the Limits? Mitotic exit is irreversible and irremediably followed by interphase, based on the degradation mechanisms of Cyclin B [5,6,134]. This is not the case during meiosis, where exit from the first meiotic division is not followed by interphase and replication but immediately followed by the onset of a second division. This lack of irreversibility has raised the question how oocytes limit the number of M phases to just two during maternal meiosis. Switching off the activity Mos – MAPK/Erk network appeared as an attractive hypothesis for ruling the number of M phases, though it was clear from studies in jellyfish, starfish and amphibian that this mechanisms could not be a universal one. First evidences supporting this hypothesis was recorded in mouse oocytes where maintaining MAPK activity inhibits pronucleus formation [135,136] and where entry in meiosis III is observed in presence of high level of MAPK/Erk activity [137]. The hypothesis that Mos-MAPK/Erk network could be the determining factor limiting the number of meiosis to two was recently more formally tested in

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urochordates, which are at the crossroad between invertebrates and vertebrates [138]. In ascidian eggs, prolonging MAPK activity by expressing murine Mos leads to entry into supernumerary rounds of M phases, as attested by the increased number of polar bodies [138]. Then, urochordates offer an attractive model to gather new observations on conserved role of MAPK/Erk on spindle morphogenesis and to decipher the mechanisms leading to uncontrolled division and polyploidy since the successive rounds of M phases observed in this cases occur without intervening replication. Tetraploidization has been associated with the overexpression of meiotic-specific proteins such as Mos [120,134]. As mentioned earlier, p53-/- cells tolerate high levels of Mos in contrast to control one [126,139], suggesting that the stress generated by ectopic expression of Mos may kill cells or arrest their cell cycle if the p53 system is running intact. Inactivation of p53 could favor the presence of supernumerary centrosomes [126,140] and increase cell survival to tetraploidization [141,142]. Absence of p53 exerts also a permissive role on the up-regulation at the post-transcriptional level of Mos, which can be observed [125,139,143]. Survival of p53-/- cells overexpressing Mos is clearly associated to an unstable phenotype, with gradual increase the number of sub-tetraploid cells. Knockdown strategies in these conditions prevented multipolar mitoses and brought genome-stabilizing effects [144]. Though exact mechanisms remain to be deciphered, Mos has been then propose to act as an inhibitor of centrosome clustering, inhibiting then the coalescence of supernumerary centrosomes that allows occurrence of normal bipolar mitoses of tetraploid cells [144].

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Concluding Remarks The role of MAPK/Erk network in genomic integrity has been underlined in different models, though the effects of MAPK/Erk may be either protective towards genomic integrity by slowing down cell division or promoting genomic instability. The controversy that arise on the role of this network of kinases on aneuploidy and cell cycle control may be linked to cell dependent lineage or due to the dynamic of the network, which may be modulated according to external and internal stresses. Further work on the interplay between MAPK and MPF pathways will provide new insight on how MAPK may exert an influence on G2/M transition in somatic cells. Interestingly, meiotic MAPKKK Mos seems to play distinct and original role in spindle morphogenesis and aneuploidy through the regulation of ploidy cycles. Substrates targeted by MAPK/Erk and Mos remains to be identified to further understand their respective roles in aneuploidy and tumorigenesis.

Acknowledgments We thank Drs R. Beaujois, C. Russo, M. Jeseta, J.P. Vilain and R. Blossey for fruitfull discussions. Our work was supported by the ‗Ligue Régionale contre le Cancer‘ and the University of Lille 1.

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[123] Wang XM, Yew N, Peloquin JG, Vande Woude GF, Borisy GG. Mos oncogene product associates with kinetochores in mammalian somatic cells and disrupts mitotic progression. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8329-33. [124] Fukasawa K, Vande Woude GF. Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability. Mol Cell Biol. 1997 Jan;17(1):506-18. [125] Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Abnormal centrosome amplification in the absence of p53. Science. 1996 Mar 22;271(5256):1744-7. [126] Carder P, Wyllie AH, Purdie CA, Morris RG, White S, Piris J, Bird CC. Stabilised p53 facilitates aneuploid clonal divergence in colorectal cancer. Oncogene. 1993 May;8(5):1397-401. [127] Donehower LA. The p53-deficient mouse: a model for basic and applied cancer studies. Semin Cancer Biol. 1996 Oct;7(5):269-78. [128] Livingstone LR, White A, Sprouse J, Livanos E, Jacks T, Tlsty TD. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell. 1992 Sep 18;70(6):923-35. [129] Martínez-Cruz AB, Santos M, García-Escudero R, Moral M, Segrelles C, Lorz C, Saiz C, Buitrago-Pérez A, Costa C, Paramio JM. Spontaneous tumor formation in Trp53deficient epidermis mediated by chromosomal instability and inflammation. Anticancer Res. 2009 Aug;29(8):3035-42. [130] Fukasawa K, Murakami MS, Blair DG, Kuriyama R, Hunt T, Fischinger P, Vande Woude GF. Similarities between somatic cells overexpressing the mos oncogene and oocytes during meiotic interphase. Cell Growth Differ. 1994 Oct;5(10):1093-103. [131] Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994 Aug 12;265(5174):966-70. [132] Okazaki K, Sagata N. The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells. EMBO J. 1995 Oct 16;14(20):5048-59. [133] Okazaki K, Sagata N. MAP kinase activation is essential for oncogenic transformation of NIH3T3 cells by Mos. Oncogene. 1995 Mar 16;10(6):1149-57. [134] Potapova TA, Daum JR, Pittman BD, Hudson JR, Jones TN, Satinover DL, Stukenberg PT, Gorbsky GJ. The reversibility of mitotic exit in vertebrate cells. Nature. 2006 Apr 13;440(7086):954-8. [135] Moos J, Visconti PE, Moore GD, Schultz RM, Kopf GS. Potential role of mitogenactivated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs. Biol Reprod. 1995 Sep;53(3):692-9. [136] Moos J, Xu Z, Schultz RM, Kopf GS. Regulation of nuclear envelope assembly/disassembly by MAP kinase. Dev Biol. 1996 May 1;175(2):358-61. [137] Verlhac MH, Kubiak JZ, Weber M, Géraud G, Colledge WH, Evans MJ, Maro B. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development. 1996 Mar;122(3):815-22.

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[138] Dumollard R, Levasseur M, Hebras C, Huitorel P, Carroll M, Chambon JP, McDougall A. Mos limits the number of meiotic divisions in urochordate eggs. Development. 2011 Mar;138(5):885-95. [139] Kalejs M, Ivanov A, Plakhins G, Cragg MS, Emzinsh D, Illidge TM, Erenpreisa J. Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe. BMC Cancer. 2006 Jan 9;6:6. [140] Carroll PE, Okuda M, Horn HF, Biddinger P, Stambrook PJ, Gleich LL, Li YQ, Tarapore P, Fukasawa K. Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene. 1999 Mar 18;18(11):1935-44. [141] Castedo M, Ferri K, Roumier T, Métivier D, Zamzami N, Kroemer G. Quantitation of mitochondrial alterations associated with apoptosis. J Immunol Methods. 2002 Jul 1;265(1-2):39-47. [142] Senovilla L, Vitale I, Galluzzi L, Vivet S, Joza N, Younes AB, Rello-Varona S, Castedo M, Kroemer G. p53 represses the polyploidization of primary mammary epithelial cells by activating apoptosis. Cell Cycle. 2009 May 1;8(9):1380-5. [143] Gorgoulis VG, Zacharatos P, Mariatos G, Liloglou T, Kokotas S, Kastrinakis N, Kotsinas A, Athanasiou A, Foukas P, Zoumpourlis V, Kletsas D, Ikonomopoulos J, Asimacopoulos PJ, Kittas C, Field JK. Deregulated expression of c-mos in non-small cell lung carcinomas: relationship with p53 status, genomic instability, and tumor kinetics. Cancer Res. 2001 Jan 15;61(2):538-49. [144] Vitale I, Senovilla L, Jemaà M, Michaud M, Galluzzi L, Kepp O, Nanty L, Criollo A, Rello-Varona S, Manic G, Métivier D, Vivet S, Tajeddine N, Joza N, Valent A, Castedo M, Kroemer G. Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos. EMBO J. 2010 Apr 7;29(7):1272-84.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter III

The Spindle Assembly Checkpoint and Aneuploidy Juliana Faria1, Joana Barbosa1, Inês M. B. Moura1, Rui M. Reis2,3 and Hassan Bousbaa*,1,4 1

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Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde – Norte, CESPU, Gandra PRD, Portugal 2 Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal 3 Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil 4 Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Porto, Portugal

Abstract Abnormal chromosome number, or aneuploidy, is commonly observed in most solid tumors, and results from mis-segregation of whole chromosomes in a phenomenon referred to as chromosome instability (CIN). Dysregulation of the spindle assembly checkpoint (SAC) is thought as one of the mechanisms underlying CIN. The SAC is a signaling pathway that prevents precocious chromosome segregation until all chromosomes of a dividing cell are aligned at the metaphase plate. While complete loss of the SAC activity is lethal due to massive mis-segregation, partial loss of the SAC is a common feature of many aneuploid tumor cells allowing them to gain or lose a small number of chromosomes. We review our current knowledge on the molecular mechanisms of SAC and discuss its contribution to CIN as well as its potential as a suitable target in cancer therapy.

*

Corresponding author: Prof. Hassan Bousbaa, Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde - Norte, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal. Phone: +351 – 224157186. Fax: +351 – 224157102, [email protected].

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Juliana Faria, Joana Barbosa, Inês M. B. Moura et al.

Introduction

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The cell cycle is a ubiquitous and complex process that is required for cell growth, proliferation, genetic material transmission and tissue regeneration (Schafer, 1998). It consists of temporally coordinated events that ensure proper embryogenesis and subsequent cell differentiation. Four checkpoint mechanisms are responsible for its tight control: DNA damage checkpoints at G1/S, S and G2/M, and the spindle assembly checkpoint (SAC) during mitosis (Tyson and Novak, 2008). These checkpoint mechanisms consist of complex signaling cascades that only allow cells to progress through the cell cycle if their requirements are met (Rieder, 2011; Tyson and Novak, 2008). The spindle assembly checkpoint (SAC) is a surveillance mechanism that is constitutively expressed during the transition from prometaphase to metaphase in eukaryotic dividing cells (Kops et al., 2005). It detects improper kinetochore-microtubule attachments, imposing a mitotic delay by preventing anaphase onset, in order to allow cells to correct them. This ‗wait anaphase‘ mechanism is sustained until all chromosomes are correctly connected to the microtubule network, bi-oriented and aligned at the metaphase plate (Logarinho and Bousbaa, 2008; May and Hardwick, 2006; Rieder et al., 1994; Zich and Hardwick, 2010). Therefore, the SAC activity accounts for equal chromosome segregation to cell progeny and, hence, for an effective reduction in mitotic error rates. Not surprisingly, weakened SAC activity has been reported in many aneuploid tumors (Bannon and Mc Gee, 2009; Chi and Jeang, 2007; Dalton and Yang, 2009; Suijkerbuijk and Kops, 2008). Given its importance in genomic stability and cancer prevention, we will focus on the molecular mechanism of SAC activity, its relation to cancer, and its use in current anti-cancer strategies.

The Molecular Mechanism of SAC In order to be accurately segregated at the onset of anaphase, chromosomes must attach, through their sister kinetochores, to the microtubules emanating from the opposite poles of the mitotic spindle. This bi-orientation ensures their alignment at the metaphase equator so that each chromatid is transported toward the corresponding pole to be delivered to the future daughter cell. However, attachment of chromosomes to microtubules is a stochastic and asynchronous event and, upon nuclear envelope breakdown at prometaphase, many chromosomes experience improper attachments before successful bi-orientation. Such misattachments include monotelic attachment (with one kinetochore of a chromosome attached to microtubules from one pole and its sister unattached), syntelic attachment (with two sister kinetochores attached to microtubules from the same pole), and merotelic attachment (with a sister kinetochore attached to microtubules from both poles). These erroneous attachments, if left undetected and uncorrected, can lead to chromosome mis-segregation and genomic instability. Fortunately, they are detected by the SAC, which delays anaphase onset until all mis-attachments are corrected. Next, we will address the mechanism by which the SAC halts mitosis to prevent precocious sister chromatid separation. The components involved in the

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SAC molecular pathway have been reviewed elsewhere (Cheeseman and Desai, 2008; Musacchio and Salmon, 2007). Sister chromatids are held together at the centromere by a ring-like structure consisting of a complex of cohesin proteins synthesized in S phase (Marangos and Carroll, 2008; Suijkerbuijk and Kops, 2008; Zhou et al., 2002). Their separation at the onset of anaphase requires degradation of one of cohesin subunits, Scc1, which is promoted by the proteolytic activity of separase (Bannon and Mc Gee, 2009; Bolanos-Garcia and Blundell, 2010; Nasmyth, 2005; Przewloka and Glover, 2009). This caspase-like protein is normally kept inactive by Securin. When all chromosomes are aligned at the metaphase plate with correct bipolar attachment to spindle microtubules, Securin becomes ubiquitinated by the anaphase promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase (Logarinho and Bousbaa, 2008; Morgan, 1999; Reddy et al., 2007; Stegmeier et al., 2007). Ubiquitination targets securin for degradation by the 26S proteasome (Decordier et al., 2008; Suijkerbuijk and Kops, 2008). Separase is then activated and cleaves Scc1, no longer holding sister chromatids together and, thus, anaphase begins (Bannon and Mc Gee, 2009; Bharadwaj and Yu, 2004; Bolanos-Garcia and Blundell, 2010; Morgan, 1999; Nasmyth, 2005). Degradation of cyclin B is also accomplished through APC/C-mediated ubiquitination, leading to the inactivation of cyclin-dependent kinase 1 (Cdk1) and subsequent mitotic exit (Logarinho and Bousbaa, 2008; May and Hardwick, 2006; Schmidt and Medema, 2006; Suijkerbuijk and Kops, 2008). The main downstream target of the spindle assembly checkpoint is Cdc20, a protein required for APC/C activation. Once the nuclear envelope is broken down, SAC proteins, namely Mad2, Bub3, and BubR1, are recruited to the outer kinetochore surface of all unattached chromosomes. These proteins use the kinetochore as a platform to generate, in near equal stoichiometry, the so called mitotic checkpoint complex (MCC) that diffuses through the cytosol to prevent Cdc20 from activating the APC/C (Figure 1) (Sudakin et al., 2001). This diffusible inhibitory signal is generated as long as unattached or mis-attached kinetochores are present, hence preventing anaphase onset until all chromosomes achieve correct attachment to bipolar spindle and align at the metaphase equator (Bharadwaj and Yu, 2004; Kops et al., 2005; May and Hardwick, 2006; Zich and Hardwick, 2010). Additionally, according to the Mad2-template model, a closed conformation of Mad2 (C-Mad2) in complex with Mad1 resides at unattached kinetochores and serves as receptor to convert cytosolic open conformation of Mad2 (O-Mad2) into C-Mad2 bound to Cdc20. This latter leaves the kinetochore and promotes inhibitory signal amplification by converting more O-Mad2 into CMad2 in the cytosol (De Antoni et al., 2005). The C-Mad2 form is a more potent inhibitor of APC/C in vitro given its higher affinity for Cdc20 (Chan et al., 2005; Musacchio and Salmon, 2007; Suijkerbuijk and Kops, 2008). The nature of the signal that triggers SAC response is still controversial. It is more likely to be the result of a redundant combination of both absence of kinetochore-microtubule attachment and of the lack of physical tension between sister kinetochores (Bharadwaj and Yu, 2004; May and Hardwick, 2006; Pinsky and Biggins, 2005). When all chromosomes undergo bipolar attachments to spindle microtubules, tension between sister kinetochores promotes SAC silencing and anaphase onset (Schmidt and Medema, 2006; Zhou et al., 2002). Anaphase inhibitory complexes are then disassembled and

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SAC proteins withdrawn from the kinetochores, both through free diffusion into the cytosol and through motor protein-mediated transport along microtubules to the spindle poles (Lu et al., 2009). Mad1 and Mad2 become undetectable at the kinetochores, while Bub1 and BubR1 levels are diminished three to four-fold (Chan and Yen, 2003; Zhou et al., 2002). When cells are not capable of satisfying the SAC after a long mitotic arrest, they may have different fates: some undergo apoptotic death during mitosis, others exit mitosis but die via apoptosis in G1 phase, and others exit mitosis but are tetraploid and reproductively dead (Niikura et al., 2007; Suijkerbuijk and Kops, 2008). In this context, Bub1 and BubR1 have been shown to play an important role in eliminating cells that adapt to prolonged mitosis and undergo defective mitotic events (Suijkerbuijk and Kops, 2008).

Figure 1. Molecular basis of spindle assembly checkpoint. Unattached kinetochore activates the SAC (Checkpoint On) by recruiting the Mad2, BubR1, and Bub3. These proteins form the Mitotic Checkpoint Complex (MCC), the diffusible inhibitory signal that sequesters Cdc20, keeping the APC/C inactive thereby preventing it from targeting Securin and Cyclin B for degradation. As a consequence, sister-chromatid cohesion is maintained and the cell cycle is arrested. The MCC disassembles (Checkpoint Off) once all kinetochores become properly attached and aligned. Cdc20 is the free to activate the APC/C which results in Securin and Cyclin B ubiquitination (U) and degradation. Securin degradation leads to the activation of the protease Separase, which cleaves cohesin, leading to sisterchromatid separation. Cyclin B degradation decreases the cyclin-dependent kinase (Cdk) 1 activity, which results in mitotic exit.

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SAC Relevance to Aneuploidy and Cancer Aneuploidy is a common feature in human cancers. It has been more than one century since Hansemann reported aberrant mitotic figures in cancer cells (Ando et al., 2010; Chi and Jeang, 2007; Foijer, 2010) and Boveri first hypothesized an association between chromosomal abnormalities and carcinogenesis (Fang and Zhang, 2011; Foijer, 2010; Holland and Cleveland, 2009; Thompson et al., 2010). Indeed, genomic instability, frequently manifested in the form of chromosomal instability (CIN) – a term that may refer to a loss or gain of complete or partial chromosomes –, is a hallmark of many solid tumours (Fang and Zhang, 2011; Lopez-Saavedra and Herrera, 2010; Thompson et al., 2010). Cells that have undergone chromosome gain or loss are said to be aneuploid (Foijer, 2010). It is estimated that 70-80% of cancers display some degree of aneuploidy, most of them showing both numerical and structural chromosomal abnormalities (Foijer, 2010). Aneuploidy, resulting from uncontrolled mitotic division, is believed to confer cells evolutionary advantage, malignant potential and resistance to chemotherapy (Lopez-Saavedra and Herrera, 2010). Since it facilitates the acquisition of oncogenes and/or the loss of tumour suppressor genes, its connection with tumorigenesis was early anticipated (Foijer, 2010). Nevertheless, whether it is a cause or a consequence of tumorigenesis is still a matter of debate (Holland and Cleveland, 2009). Several pathways drive CIN in human cancers, but mitosis is the most likely opportunity for chromosome loss and gain as a result of defects in sister chromatid cohesion, kinetochore-microtubule attachment and dynamics, SAC activity, as well as an abnormally elevated centrosome number (Foijer, 2010; Foijer et al., 2008; Lopez-Saavedra and Herrera, 2010; Thompson et al., 2010). Mutations in genes encoding for regulators of sister chromatid union were reported, possibly accounting for their premature separation or for abnormal chromosome disjunction during anaphase (Fang and Zhang, 2011; Foijer, 2010; Holland and Cleveland, 2009). Moreover, Separase depletion or overexpression was found to induce tetraploidy, further substantiating the role of cohesion-related elements in CIN (Thompson et al., 2010). Supernumerary centrosomes, arising from the deregulation of their duplication cycle or as a consequence of tetraploidy, favor CIN by increasing the establishment of merotelic interactions. In the presence of a multipolar spindle, the frequency of anaphase lagging chromosomes rises significantly, surpassing the ability of correction machinery to effectively repair them before anaphase onset (Fang and Zhang, 2011; Foijer, 2010; Ganem et al., 2009; Holland and Cleveland, 2009; Thompson et al., 2010). Since perturbations affecting regulators of kinetochore-microtubule attachments (e.g., Aurora B, Kif2b, MCAK and Hec1) stabilize their interactions, they make the correction of attachment errors more difficult, contributing to CIN phenotype (Bakhoum et al., 2009; Fang and Zhang, 2011; Foijer, 2010; Green and Kaplan, 2003; Silkworth et al., 2009; Thompson et al., 2010). Given the SAC essential role in controlling mitotic events and, thus, in maintaining genomic stability, and since many tumor cells are aneuploid, mutations in SAC genes were initially suggested as a possible molecular explanation for tumorigenesis. Mutated SAC genes encoding for altered mitotic checkpoint proteins could explain its inefficiency and, therefore, could allow for precocious chromosome segregation during mitosis, resulting in an asymmetrical distribution of genetic material to the daughter cells. Many studies were carried

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out in order to establish a causal connection between mutations in genes encoding for SAC proteins and tumor development (Marchetti and Venkatachalam, 2010). One of the most illustrative results is the one that links biallelic mutations in the SAC BubR1-encoding gene, Bub1B, with mosaic variegated aneuploidy (MVA) (Chi and Jeang, 2007; Hanks et al., 2004; Lopez-Saavedra and Herrera, 2010; Rio Frio et al., 2010; Suijkerbuijk and Kops, 2008; Suijkerbuijk et al., 2010; Thompson et al., 2010). MVA is a rare autosomal recessive disease that is characterized by a high degree of aneuploidy, mild to severe physical and mental limitations and a strong predisposition to cancer (Hanks et al., 2004; Suijkerbuijk and Kops, 2008; Suijkerbuijk et al., 2010; Thompson et al., 2010; Yen and Kao, 2005). Other studies have identified heterozygous mutations in Bub1 and BubR1 in a panel of 19 aneuploid colorectal cancer cell lines (Cahill et al., 1999). Mutations have also been found in Mad2-encoding gene, both in breast cancer (Percy et al., 2000) and gastric cancer (Kim et al., 2005) cell lines. However, many other attempts surprisingly failed to find SAC mutations, both in human cancer cell lines and in tissue samples from oncologic patients (Yen and Kao, 2005). For instance, none or few mutations were detected in Bub3, BubR1 and Bub1 genes in glioblastoma, breast, lung, bladder and thyroid cancers (Fagin, 2002; Haruki et al., 2001; Myrie et al., 2000; Olesen et al., 2001; Ouyang et al., 2002; Reis et al., 2001; Sato et al., 2000). Bub1 gene mutations were shown to be a rare event in a study using 92 acute myeloid leukemia specimens and 5 hematopoietic cell lines (Lin et al., 2002), in head and neck squamous cell carcinoma and lung cell lines (Yamaguchi et al., 1999), in a series of colorectal, hepatocellular and renal tumors (Shichiri et al., 2002) and in breast and gastric carcinomas (Langerod et al., 2003; Shigeishi et al., 2001). Also, Mad1 gene was found to carry few or no mutations in lymphomas, bladder, breast, gliomas (Tsukasaki et al., 2001) and lung carcinomas (Nomoto et al., 1999), as does Mad2 gene in transitional-cell carcinomas of the bladder, soft-tissue sarcomas, hepatocellular carcinomas (Hernando et al., 2001), breast, lung (Gemma et al., 2001; Percy et al., 2000; Takahashi et al., 1999) and digestive tract (Imai et al., 1999) cancer cells. In addition, no mutations were found in the coding sequences of Mad2 gene in 11 hepatoma cell lines (Sze et al., 2004). In a study of 8 hepatocellular carcinoma cell lines and 50 hepatocellular carcinoma specimens, although some polymorphic base changes were noticed in Bub1, BubR1 and Cdc20, no mutations accountable for SAC impairment were detected neither in these genes nor in Bub3 or Mad2B (Saeki et al., 2002). It should be noted that these studies did not cover all possible known SAC genes, meaning that some of their mutations might be yet to unveil. Furthermore, in the vast majority of cases, the effect of the gene mutation was not studied at the protein level. Further quantitative and subcellular localization assays are thus needed to clarify the actual impact of these mutations (Yen and Kao, 2005). Nevertheless, the low frequency of mutations affecting SAC genes indicates that they are not the main mechanism through which cells may become aneuploid (Lopez-Saavedra and Herrera, 2010; Schvartzman et al., 2010; Thompson et al., 2010). Subsequently, studies concerning expression of SAC components, both at the gene and protein level, have gained attention, suggesting a correlation between altered expression levels, compromised SAC activities and tumorigenesis (Chi and Jeang, 2007; Fang and Zhang, 2011; Holland and Cleveland, 2009; Kops et al., 2005; Suijkerbuijk and Kops, 2008). The SAC efficiency can be easily evaluated as the ability of a given cell population to sustain

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a prolonged mitotic arrest upon exposure to chemical compounds that interfere with microtubule polymerization and dynamics. Such evaluation has been performed in large panels of tumor cell lines, as well as in histological samples collected from numerous patients. SAC impairment was implicated in the resistance to anti-microtubule agents-induced apoptosis in human lung cancers (Masuda et al., 2003), as well as in breast cancer (Yoon et al., 2002) and in head and neck squamous cell lines, in which it may contribute to chromosomal instability (Minhas et al., 2003). Most studies have sought for a molecular explanation for the SAC weakening. Overexpression of SAC components seems to be more frequent (Foijer, 2010; Holland and Cleveland, 2009; Liu et al., 2009). Even subtle deviations in SAC mRNA and protein levels were shown to drive tumorigenesis (Bharadwaj and Yu, 2004). Low BubR1 levels, resulting from biallelic mutations in the Bub1B gene, were associated with chromosome alignment and segregation defects (Suijkerbuijk et al., 2010); a significantly reduced Bub1B expression was ascertained as the causative event of aneuploidy in colorectal adenocarcinomas (Burum-Auensen et al., 2008). On the other hand, Bub1B expression was shown to be high in 25.9% of 27 salivary duct carcinomas, although it had no prognostic significance (Ko et al., 2010). Additionally, BubR1 overexpression was documented in oesophageal squamous cell (Tanaka et al., 2008), thyroid (Wada et al., 2008), hepatocellular (Liu et al., 2009) and squamous cell carcinomas (Hsieh et al., 2010), as well as in lung cancers (Seike et al., 2002), where it has been associated with worse prognosis, carcinogenesis progression and suggested as a possible compensatory mechanism that could represent a potential tumor biomarker (Hsieh et al., 2010). BubR1 overexpression was reported in 50.3% of 181 gastric cancer samples, correlating significantly with aneuploidy, tumor invasiveness, metastasis likelihood and poor prognosis (Ando et al., 2010), and in 68% of 43 gastric carcinomas (Grabsch et al., 2003). BubR1 overexpression was found to be closely related to chromosomal instability in bladder cancer (Yamamoto et al., 2007) and in clear cell kidney carcinomas (Pinto et al., 2008). Although some studies point to diminished expression and aberrant transcription of the hBub1 gene (Lin et al., 2002), its expression was found to be up-regulated in follicular thyroid adenomas when compared to adjacent normal tissues (Wada et al., 2008). Along with that of Bub1B, Bub1 overexpression was also found in a large panel of breast tumor samples and proposed to be implied in the transition of breast tissues from normal to benign tumors (Bieche et al., 2011). Bub1 overexpression was also demonstrated, both at mRNA and protein levels, in salivary gland tumors, where it contributes to abnormal cell proliferation (Shigeishi et al., 2006). Gastric cancers also overexpress Bub1 in 84% of 43 samples under analysis (Grabsch et al., 2004). Similar alterations were detected in Mad2 expression. Mad2l1 gene up-regulation was reported in Familial Adenomatous Polyposis colorectal adenomas, suggesting that this up-regulation is associated with adenomatous polyposis coli (APC) gene mutation and may constitute an early event in colorectal carcinogenesis (Abal et al., 2007). Pronounced Mad2 overexpression has also been documented in advanced differentiated thyroid carcinomas (Wada et al., 2008), salivary duct carcinomas (Ko et al., 2010) and lung cancers, in which it has been correlated with enhanced aggressiveness, shorter survival and identified as a prognostic factor (Kato et al., 2011). Up-regulated Mad2l2 expression, both at gene and protein levels, was found in 21% of 118 colorectal tumor samples, in which it has been suggested to promote mitotic aberrancies, chromosomal instability and reduced patient survival (Rimkus et al., 2007). Also,

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Mad2 protein overexpression, concomitant with that of Aurora A and Aurora B, was observed in aneuploid colorectal adenocarcinomas (Burum-Auensen et al., 2008), as well as in a series of 6 oesophageal squamous cell carcinoma (ESCC) cell lines and 21 ESCC patients, along with that of BubR1 (Tanaka et al., 2008). Inversely, Mad2 protein levels were found to be lower in colon cancer cell lines that had been exposed to deoxycholate, a hydrophobic bile acid associated with cancer risk (Payne et al., 2010). Mad2 decreased levels were suggested to contribute to an escape from cell death, thus leading to tumorigenesis (Payne et al., 2010). Mad2 protein was also shown to be underexpressed in 75% of 8 testicular germ cell tumour (Fung et al., 2007) and in 54.5% of 11 aneuploid hepatoma tumor cell lines that failed to arrest in mitosis (Sze et al., 2004).

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Mouse Models Link SAC Dysfunction, Aneuploidy and Cancer To further investigate the role of the SAC components in checkpoint signaling and in the prevention of chromosomal imbalance, mouse models lacking SAC genes were created (Foijer, 2010; Foijer et al., 2008; Li et al., 2009; Schvartzman et al., 2010; Suijkerbuijk and Kops, 2008; Thompson et al., 2010; Yen and Kao, 2005). Conventional gene knockouts have been constructed for almost all SAC known genes, including Mad1, Mad2, Bub1, BubR1, Bub3 and CENP-E, and hypomorphic alleles for Bub1 and BubR1 have been generated (Holland and Cleveland, 2009). In spite of being compatible with viability and fertility, their downregulation increased cancer susceptibility and caused aneuploidy in mouse embryonic fibroblasts and tissues, in a degree that was dependent on the knocked-out gene and on the extension to which its expression had been decreased (Holland and Cleveland, 2009; LopezSaavedra and Herrera, 2010). Mouse embryonic cells lacking Mad2 displayed a dysfunctional SAC, leading to chromosome missegregation and apoptosis (Dobles et al., 2000). BubR1 (+/) mouse embryonic fibroblasts were proven to defectively activate SAC and to have reduced amounts of Securin and Cdc20, predisposing mice to rapid development of lung and intestinal adenocarcinomas and supporting BubR1 role as a tumor suppressor (Dai et al., 2004). In turn, although there was not a higher susceptibility to tumor formation, perhaps because of the presence of a partially functional SAC, increased aneuploidy and premature sister chromatid segregation were reported in Bub3-haploinsufficient mouse embryonic fibroblasts (Kalitsis et al., 2005). Analysis of mutant mice has shown Bub1 to be essential in preventing malignant cell transformation and in mediating cell death upon chromosome missegregation (Jeganathan et al., 2007).

SAC Components and Perspectives in Anticancer Therapy The developments in the knowledge in cell cycle regulation and control have allowed the conception of several pharmacological approaches aiming at stopping tumor cell

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proliferation. Abnormal cell proliferation is frequently associated with altered expression levels of cell cycle-regulating proteins or with alterations in checkpoint mechanisms. These alterations constitute an advantage not only for tumor cell progression, but also for the acquisition of increasingly aggressive phenotypes (De Falco and De Luca, 2010). Given the role of microtubules and SAC proteins on chromosome segregation and cell division accuracy, these have become the main targets of anti-cancer treatment strategies. Current chemotherapy approaches use microtubule-targeting agents (MTAs). MTAs affect the dynamic equilibrium between microtubule polymers and tubulin heterodimers, and have been used with a considerable degree of success in a wide range of tumors (Bannon and Mc Gee, 2009; Fojo and Menefee, 2007; Zhou and Giannakakou, 2005). Vinca alkaloids and taxanes, like paclitaxel and docetaxel, are amongst the most used drugs in cancer treatment. The first have an inhibitory action in microtubule polymerization, while the latter stabilize microtubules. Their effects are more pronounced in mitotic cells, which explains their classification as anti-mitotic drugs (Yamada and Rao, 2010). Vinca alkaloids were extracted from Vinca rosea (also known as Catharanthus roseus), and promote microtubule depolymerization, which causes a prolonged mitotic arrest and subsequently results in cell death. They are used in solid and hematological tumor treatment (Jordan and Wilson, 2004; Perez, 2009). Taxanes paclitaxel and docetaxel were isolated from Taxus brevifolia and Taxus baccata, respectively. They stabilize microtubule polymerization by interfering with their dynamics, which blocks cell cycle at G2/M phase, leading to cell death. Taxanes are highly efficient, for instance, in lung, breast and head and neck squamous carcinomas. MTAs are used either alone or combined with other cytotoxic agents (Perez, 2009). They act by interfering with mitotic spindle dynamics, thereby inducing SAC activation and mitotic arrest (Zhou and Giannakakou, 2005). However, even though mitotic arrest avoids cell proliferation, it does not necessarily end in cell death (Riffell et al., 2009). Some cells are capable of escaping mitosis in the absence of chromosomal segregation or cell division, in a process termed SAC adaptation or mitotic slippage. These cells exhibit multiple nuclei and polyploidy, both potential tumorigenesis stages. Accordingly, the fates of mitosisarrested cells are still unknown (Riffell et al., 2009). In this respect, small-molecule inhibitors of the APC/C produce a more efficient retention in mitosis than MTAs do, since proteolysis is APC/C-dependent and required for mitotic slippage. For this reason, APC inhibitors may constitute a more powerful strategy to induce and sustain mitotic arrest (Zeng et al., 2010). Since many tumor cells present defective SAC activity, they sometimes do not respond to mitotic errors, which affects MTAs efficacy (Bolanos-Garcia, 2009). Hence, the development and optimization of new therapeutic strategies that target SAC proteins may contribute to a more successful treatment. While a defective SAC may contribute to aneuploidy and CIN (Figure 2), a more expressive suppression of its activity leads to cell death as a result of massive chromosome mis-segregation. In this regard, from all protein kinases validated for this purpose, those that act in the SAC, in particular Bub1, BubR1 and Mps1, constitute main drug targets (Bolanos-Garcia, 2009). Therapeutic approaches could thus make use of the specific targeting of SAC-defective tumor cells. RNAi-mediated Mps1 depletion has allowed for a decrease in tumor cell viability (Janssen et al., 2009; Janssen et al., 2011). Mps1 is a crucial SAC component that monitors chromosome alignment; as such, it influences the stability of kinetochore-microtubule

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interactions (Colombo and Moll, 2010). Its inactivation compromises the SAC, originating alignment errors and decreasing cell viability. In vitro assays have unveiled an orally bioavailable Mps1 small-molecule inhibitor that selectively reduces tumor cell progression (Colombo et al., 2010). When compared to cancer cells, normal cells were significantly less sensitive to Mps1 inhibition, whether through siRNA or by treatment with specific smallmolecule-based inhibitors. If proven to occur in vivo, this difference could illustrate the potential of SAC selective inhibitors as a therapeutic approach (Colombo and Moll, 2010). These results corroborate that Mps1 may constitute a suitable target to preferentially eliminate cancer cells (Colombo et al., 2010; Janssen et al., 2009; Janssen et al., 2011). Inhibitors that specifically interfere with Aurora kinase activity significantly decrease viability of cells in rapid division, resulting in a 98% reduction in tumor volume in nude mice injected with human leukemia cells (Bolanos-Garcia, 2009). In fact, several studies have demonstrated that cells with abnormal Aurora A or Aurora B expression have mitotic spindle defects and do not undergo cytokinesis. For that reason, interfering with Aurora kinase activity has been suggested as a possible strategy for cancer treatment (De Falco and De Luca, 2010; Deep and Agarwal, 2008). Diverse small-molecule Aurora kinase inhibitors are under development in clinical trials; pan-Aurora kinase inhibitor Danusertib (PHA-739358) was the first to be tested in humans (Colombo and Moll, 2010). Apoptotic cell death was observed when BubR1 or Mad2 protein levels were decreased or when BubR1 kinase activity was blocked in human cancer cells. Unless when cytokinesis was also inhibited, apoptotic cell death took place within six cell divisions (Kops et al., 2004; Michel et al., 2004). This can also be explored in order to inhibit tumor cell proliferation (Kops et al., 2004; Michel et al., 2004).

Figure 2. Contribution of SAC impairment to aneuploidy. A fully functional SAC ensures accurate chromosome segregation and genomic stability. In cells with weakened SAC, however, occasional missegregations may escape SAC control and lead to aneuploidy.

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A possible strategy consists in Mad2 silencing. siRNA nanoparticle-based strategies were already tested, leading to an unequal division of genetic material and, consequently, to apoptosis in colon carcinoma cells (Kaestner et al., 2011). Assays using nude mice to which Mad2 siRNA containing nanoparticles were systemically administered showed a decrease in tumor growth, suggesting SAC inhibition as a promising concept for anticancer research (Kaestner et al., 2011). However, therapeutic strategies regarding SAC inhibition through RNAi have not been well explored to date (Kaestner et al., 2011).

Conclusion In spite of their satisfactory pre-clinical effectiveness, the clinical efficacy of the compounds that interfere with cell cycle falls short of expectations. SAC inhibitors are not completely efficient by themselves, so they must be tested in combination with standard chemotherapeutic drugs because of their vast clinical application. Therefore, a reasonable anti-cancer approach would be the combination of cell cycle-based agents with conventional chemotherapy, so that the resistance to the drug could be minimized (De Falco and De Luca, 2010; Zhou and Giannakakou, 2005).

Acknowledgments

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This work was supported by grant 02-GCQF-CICS-2011N, from Cooperativa de Ensino Superior Politécnico e Universitário (CESPU), and by grant PTDC/SAU-FCF/100930/2008 from Fundação para a Ciência e Tecnologia (FCT).

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Rio Frio, T. et al., 2010. Homozygous BUB1B mutation and susceptibility to gastrointestinal neoplasia. N Engl J Med, 363(27): 2628-37. Saeki, A. et al., 2002. Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma. Cancer, 94(7): 2047-54. Sato, M. et al., 2000. Infrequent mutation of the hBUB1 and hBUBR1 genes in human lung cancer. Jpn J Cancer Res, 91(5): 504-9. Schafer, K.A., 1998. The cell cycle: a review. Vet Pathol, 35(6): 461-78. Schmidt, M., Medema, R.H., 2006. Exploiting the compromised spindle assembly checkpoint function of tumor cells: dawn on the horizon? Cell Cycle, 5(2): 159-63. Schvartzman, J.M., Sotillo, R., Benezra, R., 2010. Mitotic chromosomal instability and cancer: mouse modelling of the human disease. Nat Rev Cancer, 10(2): 102-15. Seike, M. et al., 2002. The promoter region of the human BUBR1 gene and its expression analysis in lung cancer. Lung Cancer, 38(3): 229-34. Shichiri, M., Yoshinaga, K., Hisatomi, H., Sugihara, K., Hirata, Y., 2002. Genetic and epigenetic inactivation of mitotic checkpoint genes hBUB1 and hBUBR1 and their relationship to survival. Cancer Res, 62(1): 13-7. Shigeishi, H. et al., 2001. No mutations of the Bub1 gene in human gastric carcinomas. Oncol Rep, 8(4): 791-4. Shigeishi, H. et al., 2006. Correlation of human Bub1 expression with tumor-proliferating activity in salivary gland tumors. Oncol Rep, 15(4): 933-8. Silkworth, W.T., Nardi, I.K., Scholl, L.M., Cimini, D., 2009. Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome missegregation in cancer cells. PLoS One, 4(8): e6564. Stegmeier, F. et al., 2007. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature, 446(7138): 876-81. Sudakin, V., Chan, G.K., Yen, T.J., 2001. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J Cell Biol, 154(5): 925-36. Suijkerbuijk, S.J., Kops, G.J., 2008. Preventing aneuploidy: the contribution of mitotic checkpoint proteins. Biochim Biophys Acta, 1786(1): 24-31. Suijkerbuijk, S.J. et al., 2010. Molecular causes for BUBR1 dysfunction in the human cancer predisposition syndrome mosaic variegated aneuploidy. Cancer Res, 70(12): 4891-900. Sze, K.M., Ching, Y.P., Jin, D.Y., Ng, I.O., 2004. Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells. J Biomed Sci, 11(6): 920-7. Takahashi, T. et al., 1999. Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers. Oncogene, 18(30): 4295-300. Tanaka, K. et al., 2008. Mitotic checkpoint genes, hsMAD2 and BubR1, in oesophageal squamous cancer cells and their association with 5-fluorouracil and cisplatin-based radiochemotherapy. Clin Oncol (R Coll Radiol), 20(8): 639-46. Thompson, S.L., Bakhoum, S.F., Compton, D.A., 2010. Mechanisms of chromosomal instability. Curr Biol, 20(6): R285-95. Tsukasaki, K. et al., 2001. Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene, 20(25): 3301-5.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter IV

The Role of Centromere Cohesion and Associated Proteins in Alzheimer’s Disease: A Relation to Aneuploidy? V. P. Bajic1, D. J. Bonda2, L. Zivkovic3, Z. Milicevic4, B. Plecas-Solarovic3, Xiongwei Zhu2 and B. Spremo-Potparevic3 1

Institute of Pharmaceutical Research Galenika, Belgrade, Serbia Department of Pathology, Case Western University, Cleveland, OH, US 3 Faculty of Pharmacy, Institute of Physiology, Department of Biology and Human Genetics, University of Belgrade, Belgrade, Serbia 4 Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences ‘‘Vinca‘‘, University of Belgrade, Belgrade, Serbia

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Abstract Aside from its two characteristic pathologies, amyloid beta (Aβ) containing senile plaques and neurofibrillary tangles (NFT‘s), Alzheimer‘s disease (AD) exhibits a mitotically active phenotype. Despite being in a post-mitotic, quiescent state, affected cells show features of activation of a full cell cycle, such as activation of cyclins and cyclin dependent kinases and activation of cell cycle checkpoint control proteins. Replication of DNA in AD neurons (cyclin B and CDK 1 ) has been successfully correlated with various cytogenetic alterations of neuronal chromosomes, one of which corresponds to the occurrence of tetraploidy in AD neurons. Other groups have found chromosome instability, i.e. premature centromere division (PCD) of the X chromosome in cortical neurons and peripheral blood lymphocytes and bi-nuclear neuronal cells in AD brains. These cytogenetic alterations not only show that replication has occurred in these cells, but that a number of proteins which constitute the centromere complex have become dys-regulated. Thus, this temporal instability of the centromere or premature centromere

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V. P. Bajic, D. J. Bonda, L. Zivkovic et al. division (PCD) in neuronal and peripheral blood cells in AD display that cohesion is altered by an unknown mechanism which consequently may lead to aneuploidy. Until recently there has been no data on the role of proteins controlling the centromere region in postmitotic neurons. New data suggests that cohesin complexes and related proteins that mediate sister–chromatid cohesion in dividing cells may also contribute to gene regulation and other processes in postmitotic cells. Evidence demonstrating that instability of centromere-cohesion dynamics in the early phases of the cell cycle which coincides with re-entry alteration in cortical neurons enables the possibility to further elucidate initial processes leading to AD. We may thus become able to answer the question ―Is Alzheimer‘s disease a consequence of aneuploidy‘‘? In this review, we shall discuss the involvement of cohesion impairment and the related proteins in the involvement of chromosome instability leading to aneuploidy and how these processes are related to AD pathology.

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Introduction Accumulating evidence indicates that AD is characterized by aberrations in the cell cycle regulation within post-mitotic neurons, and that the resulting genomic variability becomes manifest as aneuploidies. An adult human body is composed of at least 1014 cells and 210 different cell types and estimates of genomic variations in somatic cells are on the order of 104-105 DNA lesions per day (Iourov et al., 2010). Most of the lesions are corrected by repair mechanisms, however these processes represent one of the exogenous sources of change in the human genome and may be a cause of different pathologic processes (Jackson and Bartek, 2009). During the numerous cell divisions in an organism, aneuploidy, (i.e. changes in the chromosome number), occurs very often. Chromosome instability, including aneuploidy, is considered as one of the mechanisms of aging at the cellular level (Yurov et al., 2009; Yurov et al., 2010; Finkel et al., 2007). Numerous authors suggest that aneuploidy is a relevant factor in neurodegenerative changes in Alzheimer's disease (Potter, 1991; Potter, 2008; KormannBortolotto et al., 1993; Migliore et al., 1997; Žekanovski and Wojda, 2009). Early studies have shown that the median frequency of aneuploidy in neurons of adult, healthy individuals is approximately 10% which corresponds to a frequency of 0.1-0.7% per chromosome pair (Iourov et al., 2006). In a fetal brain this frequency is 2-3 times higher and is probably a consequence of numerous mitotic divisions, while apoptosis is the mechanism by which the organism eradicates aneuploid cells (Yurov et al., 2007). Additionally, it was shown that aneuploid neurons in healthy adult persons are functionally active due to changes in genomic expression when compared to euploid neurons (Kingsbury et al., 2005). In brains of AD subjects the number of aneuploid neurons exceeds 20% (Mosch et al., 2007). However, in piramidal neurons of the hippocampus of AD patients aneuploidy is present in 3-4 % of cells (Yang et al., 2001). Zivkovic et al., (2006) showed hypoploidy and hyperploidy for chromosome 18 in peripheral blood lymphocytes which exhibited a statistically significant increase in the AD group compared to the control one. This increase in spontaneous aneuploidy of chromosome 18 in AD patients which was correlated with prematrure centromere division (PCD) shows that deregulation of the time of centromere separation can be considered as a manifestation of chromosome instability leading to aneuploidy (Figure 1).

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Figure 1. Discrimination between aneuploidy of chromosome 18 via FISH used pL 1.84α repetitive DNA probe for the centromeric region.

Increased aneuploidy of chromosome 17 and 21 has also been shown in peripheral tissue, specifically in cells of the buccal epithelium of AD patients (Thomas and Fenech, 2008). Moreover, utilizing a new molecular cytogenetic technique and and analyzed interphase chromosomes by a specific multicolor banding (ICS-MCB), Iourov et al. (2008) evaluated aneuploidy of chromosome 21 in different parts of the brain of AD patients and corresponding controls. In all investigated regions (cerebral cortex, hippocampus and cerebellum), the percentage of aneuploidy of chromosome 21 in AD was significantly higher than in controls. In the control samples the average frequency of aneuploidy of chromosome 21 was 0.7%, but in the samples of AD brains aneuploidy of chromosome 21 was present in over 20% of cells (Iourov et al., 2008). The relationship of Down syndrome (DS) and AD, described two decades ago by Potter et al. (1991) further supports the involvement of aneuploidy in neurodegenetation. It is known that DS patients experience rapid changes in the brain around age 30, which histologically can not be distinguished from those found in AD ( Petronis, 1999; Stanton and Coetzee, 2004; Granic et al., 2010). Studies show overrepresented DS cases in families with AD, as well as AD cases in families with DS children. Also, repeated non-disjunction of chromosomes 21, X and 18 in women who are clinically normal but who have Down offspring have twice the chance to develop AD ( Fitzgerald et al., 1986; Migliore et al., 2006; Zivkovic L et al., 2006; Coppede F. et al., 2007). Since it is known that the gene for APP is located on chromosome 21 ( on the position q21), it is likely that chromosome 2, the particular anuploidy responsible for DS, leads to increased synthesis of this protein, and accordingly, to more production of Aβ peptid that is responsible for pathological changes in the AD brain (Oyama et al., 1994; Patterson and Costa, 2005). These findings suggest that aneuploidies in sporadic AD are a consequence of chromosome instability or premature centromere division (PCD), which mostly affect chromosome X and 21 (Bajic et al., 2008; Spremo-Potparevic et al., 2008; Potter, 2008). Based on our current knowledge of PCD (Figure 2.), we postulate that centromere instability (aneuploidy) of neuronal cells increases over time, leading to mitotic catastrophe and premature cell death. Mitotic catastrophe, which has been recognized as one of the earliest

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events in neuronal degeneration, may in fact be sufficient to initiate the neurodegenerative cascade (Ogawa et al., 2003).

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Figure 2. Schematic representation of FISH visualization of PCD using a specific probe to the centromeric region of susceptible chromosomes, such as X, 21,17 and 18 ( ).

A number of spindle assembly checkpoint proteins (Matsuura et al., 2006; Kienitz et al., 2005), proteins related to cohesion (Hoque and Ishikawa, 2002; Diaz-Martinez et al, 2007) and a specific cyclin, (CDK 11) (Dongli et al., 2007; Bajic et al., 2011), can express PCD phenotype in various cells. When inhibited the finding that PCD (Spremo-Potparevic et al., 2008) and tetraploidy (Yang et al., 2003) is related to the alteration of spindle assembly checkpoint control is strengthened by the notion that mitotic catastrophe can occur after failed mitosis, during the activation of the polyploidy checkpoint, in a partially p53-dependent fashion. The spindle-assembly checkpoint and the postmitotic checkpoint, (i.e., p-53-induced prevention od DNA replication) have a fundamental role in safeguarding chromosomal stability (Chan et al., 2008), thus inhibiting aneuploidy. Alzheimer‘s disease could therefore be the end product of aneuploidic events encompassing chromosome 21, X and 17, especially in neuronal progenitors. This mechanism could also be triggered by the Aβ42 release from neuronal and glial cells at the initial stage of AD pathology (Granic et al., 2010 and it could spread the AD phenotype to susceptible brain areas. It is also possible that aneuploidy may be a result of the mature neurons cell-cycle reentry in response to environmental stimuli (Zekanovsky and Wojda, 2009).

Alteration of Cohesion and Cohesion Related Proteins in Alzheimers Disease Lead to Aneuploidy? Recently, an extensive review by Frank and Tsai. (2009) showed that the activities of a cycling cell and those of a postmitotic cell are not so different. That is the unique physiological operations taking place in neurons have been ascribed to ― core cell cycle regulators‘‘ that are key regulators in cell division. Functions of these cell cycle regulators include neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and

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synaptic maturation and plasticity. In this review, we will focus our attention on cohesion and cohesion related proteins in reference to their neuronal functions and how their alterations may have an impact in AD to the process of aneuploidy in AD. The centromere and chromatid arms are held by a protein called cohesin (Nasmyth, 2005; Nasmyth and Haering, 2009). Cohesin is essential for chromosome segregation and DNA damage repair. Cohesin contains two SMC subunits (structural maintenance of cohesion), SMC1 and SMC3, and two non-SMC subunits, Scc1 (Rad21) and Scc3. The activity of cohesion is also dependent on interactions with regulatory factors, including PDS5, Scc2, Scc4 and ECO 1 (Nasmyth and Haering, 2005). Cohesin is loaded onto chromosomes before S phase and establishes cohesion between the duplicated chromosomes (sister chromatids) during DNA replication. This regulated linkage is released in preparation for chromosome segregation through a well-defined mechanism invloving the phosphorylation and proteolytic cleavage of the non-SMC cohesin subunit Scc1/Rad 21. In contrast, the mechanisms that underlie the loading and assembly of cohesin onto chromosomes are poorly understood (Nasmyth, 2005). Cohesin are expressed in differentiated postmitotic cells. In vertebrates, cohesin binds to chromatin at the end of mitosis, long before cohesion is established in the next cell cycle suggesting that cohesin may also be involved with unreplicated DNA, independent of its role in cohesion (Wendt et al., 2008). The cohesion complex is now seen as a way for centromeres to influence expression (Dorsett, 2007), regulation of time directly through the clock gene paralogue TIM-1 (Timeless 1) (Chan et al., 2003), homologue repair (Mckay et al., 1996) and check point control (Fukagawa et al., 2004). Cohesion is maintained during interphase and the cell cycle by a number of proteins (Nasmyth and Haering, 2009) but the most abundant proteins in sister chromatid separation are securins, separins and cohesions. Securin accumulation during interphase and binding to separin prevents premature separin activation. During the normal cell cycle anaphase promoting complex (APC) eventually degrades securing, thus activating separin to facilitate chromosome segregation. Accordingly, loss of securin, the inhibitor to separin function, would lead to constitutive separin activation, enabling cohesion removal from heterochromatin regions and resulting in PCD (Nasmyth et al., 2000). Mice lacking securin show aberrant cell cycle progression and premature centromere division (Wang et al., 2001). Temporal instability perceived as PCD in neuronal and peripheral blood cells in AD indicates that cohesion is altered by an unknown mechanism that may lead to aneuploidy (Bajic et al., 2008; Barbero JL, 2011). Furthermore, certain human diseases, such as Cornelia de Lange syndrome have been linked to mutation in cohesion and in proteins that regulate cohesion. These mutations do not cause obvious defects in cell proliferation, and imply that the resulting developmental abnormalities are not caused by defects in cohesion. Rather, evidence points to a role for cohesin outside of chromatid cohesion ( Dorsett, 2007; Liu and Krantz, 2009). Cimini and Degrasi, (2005) showed that the persistence of centromere/kinetochoremicrotubule misattachments through mitosis is a major cause of chromosome mis-segregation and aneuploidy. The same authors report that a number core centromere-kinetochore proteins (CENP A;CENP-I; CENP-E), anaphase promoting complex (APC) and metaphase-anaphase check point proteins (Mad2, BubR1) result in chromosome mis-segragation and aneuploidy.

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Cohesin and centromere associated proteins are therefore a major source of aneuploidy in dividing cells (Cimini and Degrasi, 2005). Still, the persistent activity in post mitotic neurons shows us the importance of cohesion and cohesion related proteins for proper function of neuronal cells and maintenance-of postmitotic state. In neurogenesis neurons acquire an aneuploidic state which that relates to the variability of the neuronal complex. Neuronal aneuploid cells are functionally active (Kingsbury et al., 2005; Zekanowski and Wojda, 2009), but how this state of aneuploidy relates to and possibly alters the secondary roles of cohesion (such as the role of Rad 21 in axon pruning (Pauli et al., 2008)) is still not known.

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Cohesin Associated Proteins and Chromosome Stability The pituitary tumor transforming gene (PTTG) was cloned initially from a rat pituitary tumor. Structural homology suggested that PTTG may be a mammalian securin (an essential protein in cohesion regulation of centromeres) and this has been confirmed by its involvement in regulating sister chromatid separation during mitosis (Wang et al., 2001; Jallepali et al., 2001). Overexpression of PTTG activates the expression of p53 and modulates its function, with this action of PTTG being mediated through the regulation of c-myc expression. PTTG also up-regulates the activity of the bax promoter and increases the expression of bax through modulation of p53 expression. PTTG can regulate apoptosis in both a p53-dependent and a p53-independent manner. Jallepali et al. (2001) have shown that PTTG in mice and securin appears to be critical for the maintenance of chromosome stability and cell cycle progression. Proteins that regulate the spindle assemble checkpoint, such as Mad2 in cooperation with securin and cyclin B prevent aneuploidy during meiosis I in mouse oocytes (Homer et al., 2005). By using a hybrid model for exploring chromosome 21 instability and RNAi processing Fukagawa et al., (2004) showed that Dicer-deficient cells express mitotic defects and that many cells died in interphase by apoptosis. The data showed that cells died of aberrant localization of cohesion protein Rad 21 with chromosomes expressing PCD and Bub 1 inactivation (Fukagawa et al., 2004). Inactivation of Bub 1 shows that the check point pathway has a defect. If the mitotic checkpoint is defective, the affected cells should progress through anaphase. However, cells cannot enter anaphase because the sister chromatids and centromeres have separated prematurely and the chromosomes are not aligned at the metaphase plate leaving the cells with multiple spindles. It worth noting that dicer signal profile in mature cerebellar granule and hippocampal neurons was observed to increase in mature and morphologically differentiated astrocytes (Barbato et al., 2007). Both, hippocampal neurons and astrocytes are vulnerable in Alzheimer‘s disease showing ectopic expression of ‗‘ cell cycle core proteins‘‘ (Bonda et al., 2010; McShea et al., 2007; Nagy, 2000). Hebert et al. (2010) showed that the absence of DICER, a member of the miRNA gene network, is accompanied by a mixed neurodegenerative phenotype. Neuronal loss was observed in the hippocampus with predominance to the cortex. These findings coincide with

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tau hyperphosphorylation suggesting that the miRNA network may contribute to neurodegenerative phenotype in Alzheimer‘s disease (Hebert et al., 2010). Leland et al. (2009) showed that abnormal Bub 1 function results in premature separation of chromosomes into separated sister chromatides, a mechanism that has been proposed to be involved in the genesis of the majority of human germ cell aneuploidy. In relation to Down syndrome and Alzheimer‘s disease and aging, new evidence has been presented by Chiang et al. (2010) that weakened centromere cohesion is the leading cause of aneuploidy in oocytes. This finding suggests a mechanism underlying Down syndrome and late stage AD. For the influence of neurogenesis on the problem of aneuploidy see for review Zekanovski and Wojda, (2009) and Arendt et al. (2010). The fact that AD patients demonstrated temporal dysfunction of centromere segregation and increased aneuploidy peripherally and centrally (Spremo-Potparevic et al., 2004; 2008; Zivkovic et al., 2006, Miglore et al, 1997; 2006; Granic et al., 2010; Arendt et al, 2010) suggests to us that cohesin and cohesion related proteins, provide an interesting and crucial role in the pathway to neuronal cell death that characterizes the disease. The anaphase-promoting complex (APC) is tethered to cohesion in processes of chromosome segregation and separation in mitotic cells and new data show functional activity of APC in post-mitotic neurons in which alterations of this protein may lead to aneuploidy (Cimini and Degrassi, 2005). APC or cyclosome is an E3 ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome and functions by regulating cell cycle transitions in proliferating cells. As recently revealed, APC has novel roles in postmitotic neurons (Alemida et al., 2005; Teng and Tang, 2005; Stegmuler and Bonni, 2005; Aulia and Tang, 2006; Kim et al., 2009; Puram et al., 2010; Yang et al., 2010). APC/C triggers the onset of anaphase, inducing the destruction of sister chromatid cohesion by ubiquinating securin. Securin is an inhibitory chaperone of a thiol protease called separase. Separase once activated cleaves the Scc 1 or Rad 21 subunit of the multi protein complex cohesin that holds sister chromatides together (see Figure 3)

Figure 3. A schematic presentation on the role of APC/C in the separation and segregation of a chromosome. Aneuploidy: Etiology, Disorders and Risk Factors : Etiology, Disorders and Risk Factors, Nova Science Publishers, Incorporated, 2012. ProQuest

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APC is regulated by its activator Cdh-1, which is abundantly found in postmitotic neurons. Cdh 1-APC and Cdc20-APC have emerged as key regulators of diverse aspects of neuronal connectivity, from axon and dendrite morphogenesis to synapse remolding and development (Gieffers et al., 1999; Puram et al., 2010; Yang et al., 2010). This shows that APC has multiple roles at different cellular locations and appear not to be connected to cell cycle regulation. Auila et al. (2006) shows that APC may have a role in suppression of cyclin B in postmitotic neurons, thus preventing terminally differentiated neurons to re-enter the cell cycle. This role maybe impaired in Alzheimer‘s disease, i.e. it provides an explanation for the mechanism of cyclin B1 reactivation in AD (Yang et al., 2003; Almeida et al., 2005). Wirth et al. (2004) demonstated that inactivation of APC/C subunit Apc2 in quiescent hepathocytes may stimulate re-entry into the cell cycle (without any mitogenic stimulus), thereby leading to hepatic failure. Another interesting observation from Wirth et al., (2004) is that they found that 50% of cells where still quiescent even though APC/C has been deleted. They concluded that ―This finding implies that the APC/C has a crucial role in quiescent hepatocytes, namely, to restrain their re-entry into the cell cycle‖. Wirth et al. (2004) suggested that APC/C could perform this function either by suppressing the production of extracellular mitogens or more directly by suppressing accumulation of intracellular proteins capable of promoting proliferative growth. Abolition of the APC/C causes a major change in the state of quiescent hepatocytes such that they are more readily stimulated to embark on cell proliferation. Their readiness to enter the cell cycle allows them to do so either spontaneously or in response to mitogenic signals. In this regard we may hypothesize that APC/C not only regulates the cell cycle re-entry in hepatocytes, but also in doing so in neuronal cells by stabilizing cohesion dynamics in order to prevent aneuploidy and the overproduction of Aβ.

CDK 11 A New Cell Cycle Cyclin in Cohesion Dynamics and APP For some time there has been a notion that altered cell cycle events in vulnerable neurons precede the occurrence of amyloid-β (Aβ) and neurofibrillary tangles in AD (Vincent et al., 1996; Vincent et al., 1997; Smith, 1998; Herrup et al., 2010), leading to the degeneration of select neuronal populations in the hippocampus and other cortical brain regions. Ectopic expression of a number of mitosis-specific proteins, cyclins and cycline depedent kinases, have been reported in susceptible neurons in AD (Arendt et al., 1996; McShea et al., 1997; Zhu et al., 2000; Raina et al., 2004; Lee et al., 2009). Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11p110) throughout the cell cycle, a 58-kDa protein (CDK11p58) that is specifically translated from an internal ribosome entry site and expressed only in the G2/M phase of the cell cycle, and a 46kDa protein (CDK11p46) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis (Bajic et al., 2008). CDK11 has been demonstrated to be adversely affected in AD. Moreover, expression of CDK 11 is increased in M17 neuroblastoma cells that are transfected with the Swedish mutant human gene when compared to the empty vector. CDK 11 increased expression was

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also correlated to increasing levels of toxic forms of Aβ ( Bajic et al, 2011). A number of researchers have found that, when inhibited, CDK 11 can have a devastating effect on the animal or cell culture phenotype. Corresponding knock out mice do not survive and cells depleted of CDK 11 show cytogenetic abnormality, such as PCD (Dongli et al., 2007). Dongli et al., (2007) showed that depletion of CDK 11 delocalizes the cohesion protein Rad 21. A number of authors have reported that cells with depleted Rad 21 show centromere instability (Chen et al., 2002). Rad 21, also called Scc1 (sister chromatide cohesion protein 1) is part of the mitotic complex. Rad 21 has been demonstrated to be cleaved by caspase proteins in order to trigger sister chromatid separation during apoptotic signaling (Chen et al, 2002). Our lab has found that Rad 21 may show dependence on the presence of the Swedish mutation in M17 cells (Bajic et al., 2010). Using immunoflourescence Rad 21 and CDK 11 are found to localize in the nuclei of M17 cells, M17 APP and M17 APP Swedish mutant type. Based on Western blot and densitometry, Rad 21 expression is decreased in M17 APPswed mutant, whereas CDK 11 is increased (Bajic et al., 2011). The decrease is also found in Tg + 2576 mice for Rad21 but not for CDK 11. Alterations of expression levels of Rad 21 and CDK 11 indicate that cohesion in postmitotic neurons maybe linked to expression of APP. Additionally, the fact that in AD the neurons that express CDK11 in the cytoplasm remain viable, suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, presents an intriguing novel function of the APP signaling pathway in AD.

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Conclusion The aneuploid phenotype in Alzheimer‘s disease (Mosch et al., 2007) has lead us to the conclusion that the AD cell has impaired regulation of cohesion and may have an activated cell cycle checkpoint pathway that renders it ―ahead of its time‘‘. Still, mechanisms that regulate the AD cell do not favor abrogation of the G2/M phase. That is, although various mitotic markers are upregulated in vulnerable neurons in AD and DNA in these neurons is successfully replicated (i.e., proceeds to S phase), no evidence of actual mitosis has ever been found. This suggests that neuronal cells are arrested at a point (s) prior to the actual event of cellular division and therefore must either complete the cycle or die. Alteration of centromere-cohesion dynamics may play an intrinsic role in cell death by mitotic catastrophe (Bajic et al., 2008). Ultimately, these curious chromosome alterations (aneuploidy) in neurons and peripheral blood lymphocytes of AD, believed to be a result of aberrant cohesion processing warrant further investigation. Ultimately, the goal is the identification of a novel biomarker (Gustav-Rothenberg et al., 2010) for better prevention strategies and possible new pathways for pharmacological intervention.

Acknowledgment Authors thank the Ministry for Science and Education of Serbia for financial support (project No 173034).

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Chapter V

Cohesins, Genomic Stability, and Cancer José L. Barbero* Cell Proliferation and Development Program, Chromosome Dynamics in Meiosis Laboratory. Centro de Investigaciones Biológicas (CSIC), Madrid, Spain

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Summary The multifunctional protein complex named cohesin remodels the chromatin structure in different essential cellular processes such as chromosome segregation, during cell division in mitosis and meiosis, heterochromatin formation, DNA-repair, DNA replication initiation, and regulation of gene expression (Figure 1). The control of the association/dissociation of cohesin complexes to chromatin is carried out by an increasing number of denominated cohesin-interacting proteins or cohesin-regulators. Malfunctions of cohesin complexes and/or its regulators lead to cell death, chromosomal instability, aneuploidy, or developmental syndromes, such as Cornelia de Lange and Roberts syndrome/SC phocomelia, which have been denominated cohesinopathies. In addition, the number of studies and results that implicate cohesin and cohesin-regulators in different human cancers and in mechanisms of resistance to anti-tumor therapy have grown. These findings show that the control of cohesin metabolism during cell life is essential to correct DNA dynamics and to preserve cellular euploidy and genome stability.

Keywords. Aneuploidy, cancer, cell cycle control, chromosome segregation, chromosome stability, cohesin, cohesin–regulators, cohesinopathies, DNA-repair, sister chromatid cohesion

*

Corresponding author: José L. Barbero, Cell Proliferation and Development, Chromosome Dynamics, Centro de Investigaciones Biológicas (CSIC), C/ Ramiro de Maeztu 9, E-28040 Madrid. SPAIN, e-mail: [email protected]. Tel.: +34 918373112 ext 4311. Fax: +34 915360432.

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Figure 1. Cohesin metabolism. Scheme of the main cell life processes in which cohesins are involved.

Introduction Cohesin complexes were first characterized during the research for proteins maintaining sister chromatids together after DNA replication and until metaphase/anaphase transition. The canonical cohesin complex consists in four subunits named SMC1, SMC3 (from Structural Maintenance of Chromosomes), SCC1 and SCC3 (from Sister Chromatid Cohesion) (Figure 2) [for a review see 1]. These four subunits are able to form a ring-like structure, which could modulate different local chromatin conformations depending on the interactions with diverse cohesin-interacting proteins. In this sense, cohesin complexes preserve sister chromatid cohesion during cell division in mitosis and meiosis by binding along the arm and centromeres of chromosomes [2]. Cohesin complexes are recruited to sites of double strand breaks (DSB) to contribute to DNA repair of these damaged regions of chromosomes [3]. Cohesins have an architectural function cooperating to organize chromatin loops at DNA replication factories [4]. The expression of a large number of genes is regulated by the interaction between enhancer and promoter and this interaction is in turn controlled by a specific chromatin cohesin-dependent structure [5]. These functions are mediated by a large number of proteins, which regulate the spatial-temporal loading, location, and dynamic of

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cohesin complexes. Examples of cohesin-interacting proteins during different cohesin functions are: The adherins SCC2 and SCC4 (Sister Chromatid Cohesion) in chromosome segregation during mitosis and meiosis, the cohesion establishment/maintenance proteins ESCO1 and ESCO2 acetyltransferases, PDS5A and B (Precocious Dissociation of Sister), WAPL (Wings Apart-Like), SORORIN, SGO1 and SGO2 (Shugoshins ―the guardian of the spirit‖ in japanesse) and the cohesion removal proteins PLK1 (Polo Like Kinase 1) and Aurora B and securin/separase complex. Cohesin mediates transcriptional insulation during gene expression control by interacting with CCCTC-binding factor (CTCF), a zinc-finger protein required for transcriptional insulation [6,7]. More recently, it has been shown that cohesin and Mediator, a transcriptional co-activator, physically connect the enhancers and core promoters during gene activation in mouse embryonic stem cells [8]. Cohesin complexes interact with Pre-RC complex during DNA replication and promote replication fork progression [9]. During DNA-repair in laser-induced DNA damage, cohesin interacts with the DNA DSB repair factor Mre11/Rad50 [10]. Some of these cohesin-interacting proteins are function-specific, such as CTCF for gene insulation, but those that are involved in the loading of the cohesin complex, establishing and maintaining the stable association to chromatin are required for multiple cohesin functions. Therefore, the yeast ESCO1/2 acetyltransferase homolog, named Eco1, is required for DNA replication, for DNA damage repair, and for sister chromatid cohesion [11]; Scc2/Scc4 adherin complex, responsible for cohesin complex loading, is also essential for the multiple roles of cohesins. Mutations in genes encoding for the human homologues SCC2 and ESCO2 produce the developmental disorders denominated Cornelia de Lange syndrome (CdLS; OMIM: 122470, 300590, 610759) and Roberts syndrome/SC phocomelia (RBS; OMIM: 268300), respectively. In view of the different and essential cellular tasks in which cohesins are involved, it is not surprising that the alterations of the normal cohesin metabolism were intimately related with the generation and progression of human tumors. In the last three years, the role of cohesins in developmental human syndromes, cohesinophaties, has been extensively reviewed in the related scientific literature [for three revisions in this field, see 12-14]. The present review focuses on the emergent links between cohesin networks and cancer.

Cohesins, Chromosome Instability and Cancer Genome instability is considered a hallmark of most of cancer cells. Perhaps the two cellular mechanisms more critical for chromosomal stability in which cohesins are involved are DNA-damage repair and chromosome segregation during cell division. In the next two sections, I will center on the cohesins and cohesin-interacting proteins involved in these two cellular important processes and their links with tumor formation and development.

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Figure 2. Cohesin ring complex and cohesin core subunits. Ring model of cohesin complex in which SMC1 and SMC3 form a ring structure maintained also by the interactions with the non-SMC subunits SCC1 and SCC3. The cohesin subunits SMC1, SCC1, and SCC3 present different homologues in superior eukaryotes. The subunits SMC1 , RAD21L1, REC8 and STAG3 (in red) are meiosis-specific cohesins.

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Cohesins, DNA-Damage Repair and Cancer Different endogenous and exogenous agents could constantly damage the DNA structure. Cells have mechanisms that ensure the correct genetic material repair before they continue the cell cycle. The initial result on the participation of cohesins in theses mechanisms was already reported before cohesins were known to mediate sister chromatid cohesion; the Scc1 ortholog from Schizosaccharomyces pombe was first identified as a protein whose mutation causes sensitivity to radiation because damaged DNA cannot be properly repaired and, thus, called rad21 (from Radiation-sensitivity) [15]. Following experiments, essentially in budding yeast, showed that cohesin mutants are defective in repair damaged DNA and provided evidence that DNA repair depends on the function of cohesin to mediate sister chromatid cohesion. In addition, studies of Rad21-depleted chicken cells have shown that vertebrate cohesin also functions in both segregation and repair [16]. This cohesin requirement could be explained because DNA DSB are preferentially repaired by recombination between sister chromatids and the cohesion between them would facilitate this process. The necessity of sister chromatid cohesion for DSB repair was also supported by the discovery of two findings: On one hand, the absence of functional cohesin loading proteins Scc2/Scc4 complex abolished DSB cohesin accumulation and provoked defective DNA repair [17] and on the other hand, yeast mutants for Eco1 cohesion-establishment protein are sensitive to DNA damaging agents that cause DSB, such as X rays or bleomycin [18].

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In addition to the machinery of chromosome cohesion, other specific DNA-damage repair proteins are necessary for the cohesin localization to DSB sites (Figure 3). A component of the DNA-damage sensing complex MRX (Mre11/Rad50/Xsr2) is required for cohesin assembly around the DSB. Phosphorylation of histone H2AX by Mec1/Tel1 generates what is known as H2AX. Yeast strains expressing a non-phosphorylatable H2AX fails to recruit cohesin, thus suggesting that H2AX may act as a signal for cohesin assembly [19]. Interestingly, some modifications by phosphorylation of Smc1 and Smc3 cohesin subunits are carried out by specific kinases following IR and UV damage. Mutations in Smc1 or Smc3 that prevent phosphorylation result in abrogated DNA damage responses [20].

Figure 3. Cohesins and DNA-damage repair. After the generation of DNA damage by different agents, DNA-repair mechanisms are triggered including the recruitment and accumulation of cohesin complexes to the double strand break (DSB) sites to reinforce sister chromatid cohesion in this region.

These molecular mechanisms, fundamentally characterized in experiments carried out in yeast, have been supported and reinforced in the last few years by studies in animal models and in human diseases. Different cell lines isolated from Cornelia de Lange patients displayed a reduced capacity to tolerate DNA damage [21]. Rad21 has been related with cancer chemotherapy for years; inhibition of Rad21 expression by RNA interference in human breast cancer cells enhanced the cytotoxicity of etoposide and bleomycin in these cells [22]. In agreement with this result, Xu et al. (2010) reported that overexpression of Rad21 gives resistance to chemotherapy in high grade luminal, basal, and HER2 breast cancers [23]. Deletion of the mouse‘s Rad21 gene results in early embryonic lethality, but heterozygous

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Rad21+/- mice were obtained by breeding. Rad21+/- mice were viable and developed to apparently normal adulthood without morphological defects. However, Rad21+/- animals have enhanced sensitivity to whole body irradiation, suggesting that Rad21 gene dosage is critical for ionizing radiation (IR) response [24]. The three members of the STAG cohesin family (STAG1, STAG2, and STAG3) have also been implicated in cancer. The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel–Lindau disease and the VHL tumor suppressor gene (TSG) which is inactive in most sporadic clear cell RCC. To identify candidate genes for renal tumorigenesis, Foster et al. [25] characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. The gene encoding for the human cohesin subunit STAG1 maps within close proximity to the breakpoints and thus it is a candidate gene involved in RCC. In array studies searching for genome alterations in a series of 167 malignant myeloid diseases, Rocquain et al. (2010) [26] found recurrent deletions of Rad21 and STAG2 genes, indicating that cohesin components are new players in leukemogenesis. Other interesting cohesin-related players involved in DSB repair is the Smc5/Smc6 complex. This complex is recruited to regions around the break and it binds to regions that span at least 25Kb on each side. The function of Smc5/Smc6 complex in the repair of DNA breaks, like that of cohesin, is to promote sister chromatid recombination. For a review about the functions of this complex, see reference 27.

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Cohesins, Chromosome Segregation and Cancer There are different proteins that control the dynamic of cohesin complexes during chromosome segregation in mitosis and meiosis (Figure 4). As an introduction to this section, the most relevant characteristics of these cohesin-regulators will be commented on, because their mechanisms and biological roles have been reviewed extensively [28-30].

Figure 4. Cohesins and chromosome segregation. Representation of the different steps in the control of sister chromatid cohesion during chromosome segregation and the cohesin-interacting proteins regulating the cohesin complex loading, cohesion establishment and maintenance and cohesion dissolution at the metaphase/anaphase transition.

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The loading of cohesin complexes to chromosomes depends on the Scc2/Scc4 adherin complex. This loading is not sufficient for the cohesion function in chromosome segregation and the Eco1/Ctf7p acetyltransferase is required for the establishment of cohesion. A substrate of Eco1 acetylase is SMC3 and this cohesin subunit is acetylated in an Eco1dependent manner during replication to promote sister chromatid cohesion. Two cohesinregulators, Rad61/WAPL and PDS5, are also involved in the opening/closing of the cohesin ring by interactions with different cohesin subunits. These cofactors are necessary for cohesin complex dynamics, but are not considered components of the canonical cohesin complex. One of these cofactors is PDS5, which interacts with human STAG1- and STAG2-containing complexes in somatic cells. Two vertebrate PDS5 proteins, PDS5A and PDS5B, have been characterized. They are not required for cohesin association to chromosomes, but are needed for maintaining cohesion. Another interesting cohesin-regulator is the product of the previously identified Drosophila wings apart-like (WAPL) gene, involved in heterochromatin organization. Human WAPL regulates the resolution of sister chromatid cohesion and promotes cohesin complex removal by direct interaction with the RAD21 and SA/STAG cohesin subunits. Sororin is a protein, which has been implicated in centromere cohesion. It was first identified in a screen for substrates of Anaphase Promoting Complex (APC) in vertebrates and, to date, no homologues have been described in other organisms. Different results in somatic cells suggested that sororin interacts with the cohesin complex and it is essential for the maintenance of sister chromatid cohesion. Sororin is ubiquitinized and degraded after sister chromatid cohesion is dissolved. More recently, it was reported that sororin is also needed for efficient repair of DNA double-strand breaks in G2 and for maintaining the stably chromatin-bound cohesin in G2, suggesting a crucial cohesin-regulator role for this protein. The release of cohesin complexes from chromatin at the metaphase/anaphase transition is mediated by separase, a specific cysteine protease, that cleaves the SCC1 subunit of the cohesin complex, destabilizing cohesion and allowing chromatid segregation. Before anaphase, separase remains inactivated by binding to its specific inhibitor securin. In metazoa, dissociation of cohesin complexes from chromatin proceeds in a two-step manner. In a first step, the bulk of cohesin complexes is removed from chromosome arms during prophase by a separase-independent pathway, in which phosphorylation of the STAG2 subunit by Aurora B and Polo-like kinases triggers the removal of arm cohesins. Cohesin complexes remain essentially at centromeres until the chromosomes are correctly bi-oriented and the spindle assembly checkpoint is satisfied in metaphase. Activation of the anaphase promoting complex/cyclosome (APC/C) leads to ubiquitination and degradation of securin, allowing cleavage of SCC1/RAD21 from centromeric cohesin complexes by separase and triggering the onset of anaphase.

Cohesin Complex Subunits The study of 11 somatic mutations in 132 human colorectal cancers identified 6 of them mapping to 3 cohesin (SMC1a, SMC3, and STAG3) genes and 4 to a cohesin-regulator SCC2 gene [31]. Colorectal cancer cells are characterized by chromosomal instability, resulting in

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chromosome gain or loss. It is possible to argue that abnormal cohesin pathway activity leads to chromosome missegregation and chromosome instability. This hypothesis is supported by the observation that colorectal cancer cells exhibit up to 100-fold higher rates of missegregation than normal cells [32]. In addition, using a microcell mediated chromosome transfer and expression microarray analysis, Notaridou et al. [33] identified the cohesin STAG3 gene as one of the nine genes associated with functional suppression of tumorogenicity in ovarian cancer cell lines (AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1, and STAG3) and as a candidate gene associated with risk and development of epithelial ovarian cancer. So far, we have shown different studies that link cancer development with disorders in the four core cohesin subunit genes, but there is also experimental data linking cohesin-interacting proteins and tumorigenesis.

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Cohesin-Regulators: Cohesion Establishment and Maintaining Based on the loss of PDS5B in many cancers and on its mutations in germ lines provoking birth defects, Denes et al. [34] hypothesized that PDS5B plays a role in stem cell differentiation and in embryonal carcinoma. They showed that PDS5B knockdown disrupted Oct4, Nanog, and SOX2 patterns, in addition to others stem cell differentiation mechanisms. Their results suggested that the link between the PDS5B-related birth defects that show Cornelia de Lange syndrome and cancer is a disrupted early stem cell differentiation program. On the other hand, the other PDS5 member, PDS5A, is overexpressed in high-grade gliomas, which are characterized by a high degree of chromosomal instability and aneuploidy [35], linking again loss of chromosome cohesion with genomic instability. WAPL was also characterized as an oncogene in uterine cervical cancer and it is induced by human papillomavirus (HPV) E6 and E7 oncoproteins. WAPL overexpression induces apparition of multinucleated cells and increases the number of chromatid breaks in the cell, contributing to molecular mechanisms of tumor progression from HPV-infected cells to cervical carcinoma [36]. Later, these authors reported that human WAPL gene encodes a large number of spliced variants and that the expression patterns of these variants could have diagnostic potential for cervical lesions [37]. Cell division cycle associated 5 (CDCA5) protein, also known as sororin, has been recently identified as an up-regulated gene in most lung cancers using a cDNA array containing 27,648 genes or expressed sequence tags [38]. Sororin is phosphorylated by extracellular signal-regulated kinase (ERK) at Ser79 and Ser209 in vivo. The suppression of sororin expression by siRNAs or the inhibition of the interaction between sororin and ERK inhibited the growth of lung cancer cells, indicating a functional role of activation of CDCA5/sororin in lung cell cancer proliferation [38]. To investigate the putative role of the centromere cohesion guardian shugoshin 1 (SGO1) in human colorectal cancer, Iwazimi et al. [39] performed SGO1 knockdown using an shRNA expression vector. Human SGO1 knockdown cells proliferated slowly and presented marked chromosomal instability (CIN) in the form of aneuploidy. Other characteristics of these transfected cells were increased centrosome amplification, the presence of binucleated cells, and mitotic catastrophes. The results of this study showed that SGO1 down-regulation leads

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to CIN in human colorectal cancer cells and it could be a molecule involved in the CIN pathway found in colorectal cancer progression.

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Cohesin-Regulators: Cohesion Dissolution The complex separase and its inhibitor securin are responsible for the total dissolution of sister chromatid cohesion in anaphase (Figure 4). Mammalian securin gene was originally isolated in 1997 and characterized as a pituitary tumor-transforming gene (Pttg1), which encodes the PTTG protein from rat pituitary tumor cells [40]. PTTG/securin is highly expressed in various tumors and it can induce human cellular transformation. PTTG/securin is associated with more aggressive tumor behavior and has been identified as one of 17 key signature genes associated with metastatic disease [41]. In addition, a PTTG binding factor (PBF) was identified through its interaction with PTTG and it was characterized as a protooncogene that is upregulated in several cancers [42]. PTTG1/securin is also overexpressed in hepatocellular carcinoma. Chronic infection with the hepatitis B virus (HBV) is the main causal factor for hepatocellular carcinoma and the viral protein HBx plays an essential role in the pathogenesis of hepatic tumors. To investigate the putative correlation between the abnormal expression of PTTG1 and the tumorigenic mechanism of HBx, Molina-Jiménez et al. [43] analyzed the PTTG1 expression in biopsies from patients chronically infected with HBV in different disease stages and from HBx transgenic mouse models. These authors found that HBx viral protein promotes an accumulation of PTTG1 by inhibition of PTTG1 ubiquitination and degradation. The molecular mechanisms by which HBx carried out this inhibition is currently under research. Separase is the endopeptidase that cleaves Rad21 and Rec8 during the metaphase/anaphase transition, provoking the separation of sister chromatids. Overexpression of separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In a mouse mammary transplant model, induction of separase expression in the transplanted FSK3 cells for 3– 4 weeks results in the formation of aneuploid tumors in the mammary gland [44]. In a later study, Meyer et al. [45] showed that separase is significantly overexpressed in osteosarcoma, breast, and prostate tumor specimens. There is a strong correlation of tumor status with the localization of separase into the nucleus throughout all stages of the cell cycle. Additionally, overexpression of separase transcript strongly correlates with a high incidence of relapse, metastasis, and a lower 5-year overall survival rate in breast and prostate cancer patients, suggesting that separase is an oncogene [45]. Polo-like kinase 1 (Plk-1) and Aurora B are two kinases that have as substrate cohesins and other proteins involved in chromosome segregation. PlK1 is overexpressed in various human cancers, and this is mostly associated with a poor prognosis [46]. The first data to associate PLK1 with neoplastic growth was generated by studies showing that PLK1 concentrations are also increased in primary cancer tissues [47]. This prompted a number of studies that subsequently demonstrated that PLK1 is overexpressed in a broad spectrum of human tumors compared with normal controls. Furthermore, some reports have indicated that PLK1 expression is a reliable marker for identifying a high risk of metastasis [48]. More recently, Ito el al. [49] described the post-transcriptional regulation of Plk1 expression by

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RNA interference mediated by miR-593* and Plk1 downregulation in EC cells decreases cell proliferation in vitro via G2/M cell cycle arrest, and drastically suppresses tumor formation in vivo. Aurora B kinase is implicated in different essential functions during chromosome segregation to preserve genomic stability. Examples of these functions are: Sister chromatid cohesion, chromosome condensation, mitotic spindle assembly, syntelic chromosome attachments, and spindle assembly checkpoint (for a Review see reference 50). Aurora B is overexpressed in cancer cells, and an increased level of Aurora-B correlates with advanced stages of colorectal cancer. Overexpression of Aurora-B results in multi-nucleation and polyploidy in human cells [51] and, in addition, it has been reported that Aurora-B overexpression induces chromosome lagging in metaphase, chromosome segregation error, and errors in cytokinesis, and thus suggesting a direct link between Aurora-B and carcinogenesis [52]. These findings and the crucial roles of Aurora B and Plk1 in chromosome dynamics during cell cycle have led many to consider these two kinases as important targets for cancer therapy [53-55].

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Concluding Remarks Cohesin complex, which was characterized as a ring protein complex that maintained sister chromatids together during chromosome segregation, is now considered a real architect of chromatin structure during critical dynamic DNA processes. In many cases, these processes are designed to safeguard the stability of genetic material and its proper distribution to the daughter cells. Thus, it is not surprising that when there are errors/problems in the cohesin complex metabolism related with this guardian function, one of the likely results was the development of a tumor. During the last 2-3 years, an increasing number of scientific works showed that cohesin functions are also mediated by cohesin-interacting proteins. There are two kinds of cohesin-interacting proteins: those that regulate different aspects of the cohesin metabolism and are necessary for several functions (such as Scc2/Scc4, Eco1), and those that contribute to one cohesin specific role by a spatial-temporal interaction with cohesin complex (such as CTCF, MEDIATOR). In addition, post-translational modifications, such as acetylation and phosphorylation, in specific residues of cohesin subunits, are also required for specific cohesin functions, suggesting the putative existence of a cohesin code similar to well established histone code. The future research on the molecular mechanisms of both the cohesin-interacting proteins and the specific cohesin post-translational modifications, and on the alterations in the cohesin network during pathological conditions is crucial in determining the relationship between this interesting ring protein complex and the formation/development of tumors in humans.

Acknowledgments I apologize to all colleagues whose contributions have not been referenced due to space restrictions. This work was supported by the Spanish Ministerio de Ciencia e Innovación

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(grant BFU2009-08975/BMC), CSIC (grant PIE-201120E020) and the Comunidad de Madrid (grant P-BIO-0189-2006).

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[39] Iwaizumi M, Shinmura K, Mori H, Yamada H, Suzuki M, Kitayama Y, Igarashi H, Nakamura T, Suzuki H, Watanabe Y, Hishida A, Ikuma M, Sugimura H. (2009) Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer. Gut. 58: 249-260. [40] Pei L, Melmed S. (1997) Isolation and characterization of a pituitary tumortransforming gene (PTTG). Mol. Endocrinol. 11:433-441. [41] Ramaswamy S, Ross KN, Lander ES, Golub TR. (2003) A molecular signature of metastasis in primary solid tumors. Nature Genet. 33:49-54. [42] Smith VE, Franklyn JA, McCabe CJ. (2010) Pituitary tumor-transforming gene and its binding factor in endocrine cancer. Expert rev. Mol. Med. 12,e38. [43] Molina-Jiménez F, Benedicto I, Murata M, Martín-Vílchez S, Seki T, Antonio PintorToro J, Tortolero M, Moreno-Otero R, Okazaki K, Koike K, Barbero JL, Matsuzaki K, Majano PL, López-Cabrera M. (2010) Expression of pituitary tumor-transforming gene 1 (PTTG1)/securin in hepatitis B virus (HBV)-associated liver diseases: evidence for an HBV X protein-mediated inhibition of PTTG1 ubiquitination and degradation. Hepatology 51:777-787. [44] Zhang N, Ge G, Meyer R, Sethi S, Basu D, Pradhan S, Zhao YJ, Li XN, Cai WW, ElNaggar AK, Baladandayuthapani V, Kittrell FS, Rao PH, Medina D, Pati D. (2008) Overexpression of Separase induces aneuploidy and mammary tumorigenesis. Proc. Natl. Acad. Sci. 105:13033-13038. [45] Meyer R, Fofanov V, Panigrahi A, Merchant F, Zhang N, Pati D. (2009) Overexpression and mislocalization of the chromosomal segregation protein separase in multiple human cancers. Clin. Cancer Res. 15:2703-2710. [46] Strebhardt K, Ullrich A. (2006) Targeting polo-like kinase 1 for cancer therapy. Nat. Rev. Cancer 6:321-330. [47] Holtrich U, Wolf G, Bräuninger A, Karn T, Böhme B, Rübsamen-Waigmann H, Strebhardt K. (1994) Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors. Proc. Natl. Acad. Sci. 91:1736.1740. [48] Dai W, Li Y, Ouyang B, Pan H, Reissmann P, Li J, Wiest J, Stambrook P, Gluckman JL, Noffsinger A, Bejarano P. (2000) PRK, a cell cycle gene localized to 8p21, is downregulated in head and neck cancer. Gnes Chromosomes Cancer 27:332-336. [49] Ito T, Sato F, Kan T, Cheng Y, David S, Agarwal R, Paun BC, Jin Z, Olaru AV, Hamilton JP, Selaru FM, Yang J, Matsumura N, Shimizu K, Abraham JM, Shimada Y, Mori Y, Meltzer SJ. (2010) Polo-like kinase 1 regulates cell proliferation and is targeted by miR-593* in esophageal cancer. Int. J. Cancer [Epub ahead of print]. [50] Vader G, Lens SM. (2008) The Aurora kinase family in cell division and cancer. Biochem. Biphys. Acta 1786: 60-72. [51] Tatsuka M, Katayama H, Ota T, Tanaka T, Odashima S, Suzuki F, Terada Y. (1998) Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1like midbody-associated protein mitotic kinase in human cancer cells. Cancer Res. 58:4811-4816. [52] Ota T, Suto S, Katayama H, Han ZB, Suzuki F, Maeda M, Tanino M, Terada Y, Tatsuka M. (2002) Increased mitotic phosphorylation of histone H3 attributable to

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AIM-1/Aurora-B overexpression contributes to chromosome number instability. Cancer Res. 62:5168-5177. [53] de Cárcer G, Pérez de Castro I, Malumbres M. (2007) Targeting cell cycle kinases for cancer therapy. Curr. Med. Chem 14:969-985. [54] Strebhardt K. (2010) Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy. Nat. Rev. Drug Discov. 37:159-172. [55] Libertini S, Abagnale A, Passaro C, Botta G, Portella G. (2010) AuroraA and B kinases targets of novel anticancer drugs. Recent. Pat. Anticancer Drug Discov. 5:219241.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter VI

Adult Neurogenesis and Aneuploidy in Etiology, Pathogenesis and Pathology of Alzheimer’s Disease Philippe Taupin* School of Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland

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Alzheimer‘s disease (AD) is a neurodegenerative disease. The disease is characterized by widespread neurodegeneration, amyloid deposits, neurofibrillary tangles and aneuploidy. Patients with AD elicit learning and memory deficits and anosmia. Adult neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) of mammals. Neurogenesis is enhanced in the hippocampus and is reduced in the subventricular zone (SVZ) of patients with AD. On the one hand, enhanced neurogenesis in the brain patients with AD suggests a regenerative attempt to compensate for the neuronal loss. On the other hand, reduced neurogenesis would contribute to pathological processes associated with the disease. Aneuploidy is an underlying of the pathogenesis of AD, such as neurodegeneration. Neurogenesis has the potential to generate aneuploid neuronal cells in the adult brain. The generation of aneuploid neuronal cells in the brain of patients with AD would contribute to the etiology and pathogenesis of the disease. Hence, adult neurogenesis would contribute to the pathology of AD, the understanding and contribution of which remain to be elucidated and determined.

*

Corresponding author: Philippe Taupin. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Tel. 353 (0)1 700 - 5284. Fax 353 (0)1 700 - 5412. Email [email protected].

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Introduction AD affects 35 million of individuals worldwide and is the most common form of dementia among elderly [1]. There are two forms of the disease. The early onset form of AD (EOAD) is diagnosed before age 65. It is a rare form of the disease and is primarily an inherited disease. The late onset form of AD (LOAD) is diagnosed after age 65. LOAD accounts for over 93 percent of all cases of AD and it is primarily a sporadic form of the disease [2]. AD is characterized by amyloid deposits and neurofibrillary tangles in the brain, the histopathological hallmarks of the disease. Genetic mutations and genetic, acquired and environmental risk factors have been identified as causative for AD [3]. The brain of patients with AD elicits a high level of aneuploidy. Studies have reported that neurogenesis is enhanced in the hippocampus and is reduced in the SVZ of patients with AD. Hence, neurogenesis in the adult brain would contribute to the pathology of AD. In the chapter, we will discuss the contribution of aneuploidy and adult neurogenesis to the etiology and pathogenesis of AD.

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Amyloid Plaques, Neurofibrillary Tangles and Causative Factors of Alzheimer’s Disease Amyloid plaques and neurofibrillary tangles are present throughout the brain of patients with AD, particularly in the regions of degeneration such as the entorhinal cortex and hippocampus [4]. Amyloid plaques are composed of extracellular deposits of beta-amyloid peptide and of alpha 1-antichymotrypsin, a protease inhibitor. Amyloid plaques are composed primarily of the 42 amino acid form of beta-amyloid peptide. Protein beta-amyloid originates from the post-transcriptional maturation of the amyloid precursor protein (APP) [5]. Under certain conditions, such as the presence of specific mutations in the genes coding for APP, presenilin 1 (PSEN-1) or PSEN-2, or certain risk factors, the maturation of the APP gene results in an excessive production of the 42 amino acid beta-amyloid peptide and in the formation of amyloid deposits [6,7]. Amyloid deposits may be a causative factor of AD. Neurofibrillary tangles are intracellular deposits of hyperphosphorylated microtubuleassociated phosphoprotein tau protein [8]. The hyperphosphorylation of tau protein results in the formation of neurofibrillary tangles and cell death [9,10]. Genetic mutations in the genes of APP, of PSEN-1 and of PSEN-2 have been identified as causative for EOAD, whereas genetic, acquired and environmental risk factors are the main causative factors for LOAD [11,12]. Aging, the presence of certain alleles in the genetic makeup of the individuals, such as the presence of the apolipoprotein E varepsilon 4 allele (ApoE), hypertension, diabetes and oxidative stress are the main genetic, acquired and environmental risk factors causative factors identified for LOAD [13-15]. AD is associated with neurodegeneration in areas of the brain that are vital to memory and other cognitive abilities, such as the entorhinal cortex and the hippocampus. It is also associated with neurodegeneration in areas related to olfaction, such as the olfactory bulb [16-

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.

18]. As the disease progresses, other regions of the brain are affected, leading to severe incapacity and death. There is still no cure for AD. The advent of adult neurogenesis and NSC research opens new avenues and opportunities for treating AD.

Aneuploidy in Alzheimer’s Disease

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Individuals with AD have a high level of aneuploid cells, particularly lymphocytes and nerve cells [19]. Four to 10% of nerve cells in regions of degeneration in the brain of patients with AD express proteins of the cell cycle and are aneuploid [20,21]. The nondisjunction of chromosomes during cell division is at the origin of aneuploid cells, such as lymphocytes, in patients with AD, whereas cell cycle re-entry and DNA duplication, without cell division, is at the origin of nerve cells that are aneuploid in the brain of AD patients [22]. Aneuploidy nerve cells in the brain of patients with AD are fated to die. They may live in this state for months undergoing a slow death process [23]. Aneuploidy nerve cells would be an underlying of the neurodegenerative process in AD and contribute to the pathogenesis of the disease. The genes for APP, ApoE, PSEN-1, PSEN-2 and tau protein are located on chromosomes 21, 19, 1, 14 and 17 respectively [24]. Aneuploidy for these chromosomes would contribute to the development and pathogenesis of AD, by the over expression of those genes involved in AD. Such over-expression would trigger a cascade of events promoting the development of the disease, such as the formation of amyloid plaques and neurofibrillary tangles and neurodegeneration [25]. Patients with AD elicit aneuploidy particularly for chromosome 21 [19]. Aneuploidy for chromosome 21 in individuals with AD contributes to the pathogenesis of the disease by promoting the formation of amyloid plaques and neurodegeneration.

Adult Neurogenesis and Neural Stem Cells Neurogenesis occurs in the adult mammalian brain, primarily in the dentate gyrus (DG) of the hippocampus and in the SVZ, in various species including humans [26,27]. In the DG, newly generated neuronal cells migrate to the granule cell layer, where they differentiate into granule-like cells. They extend axonal projections to the CA3 region of the Ammon‘s horn, where they establish synaptic contacts with their target cells [28,29]. Newly generated neuronal cells of the DG establish mossy fiber-like synapses with their target cells of the CA3 region [29]. In the SVZ, newly generated neuronal cells migrate through the rostro-migratory stream to the olfactory bulb [26]. NSCs are the self-renewing multipotent cells that generate the main phenotypes of the nervous system, nerve cells, astrocytes and oligodendrocytes. Newly generated neuronal cells in the adult brain would originate from NSCs. In support of this contention, self-renewing multipotent NSCs have been isolated and characterized from the adult brain of mammals [30,31]. Because of their potential to generate the main phenotypes of the nervous system, NSCs have the potential to treat and cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases such as AD. The confirmation that adult neurogenesis

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occurs in the adult brain and that NSCs reside in the adult CNS reveals that the adult CNS may be amenable to repair. The stimulation of endogenous neural progenitor or stem cells of the adult brain and the transplantation of adult-derived neural progenitor and stem cells are being considered for repairing and restoring the damaged or injured nerve pathways. For neurodegenerative diseases for which the neurodegeneration is widespread, such as AD, neural progenitor and stem cells may be administered systemically, by intravenous injection, to migrate and reach the degenerated and diseased areas of the CNS [32,33].

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Neurogenesis and Aneuploid Newly Generated Neuronal Cells in Alzheimer’s Disease Studies report that the expression of doublecortin, a marker of immature neuronal cells, is enhanced in the hippocampus of the brain of patients with LOAD [34]. The expression of markers of neural progenitor and stem cells, such as nestin and Musashi1, is reduced in the SVZ of patients with AD [35]. This suggests that neurogenesis is enhanced in the hippocampus of the brain of AD patients, particularly LOAD, and reduced in the SVZ of patients with AD. Enhanced neurogenesis in the hippocampus would represent a regenerative attempt to compensate for the neuronal loss in the AD brain. Reduced neurogenesis in the SVZ of AD brain would underlie the compromised olfaction associated with the disease [36]. This shows that adult neurogenesis and NSCs may be involved in the pathology and pathogenesis of AD, particularly LOAD. However, the techniques and protocols primarily used in these studies are the sources of pitfalls and limitations [37,38]. The involvement and contribution of adult neurogenesis and NSCs to the pathology and pathogenesis of AD remain therefore to be elucidated and confirmed. Cells that are the most likely to become aneuploid are dividing cells. Hence, the process of adult neurogenesis has the potential to generate populations of cells that are aneuploid, particularly in the neurogenic regions [25]. The nondisjunction of chromosomes in dividing neural progenitor and stem cells of the adult brain would lead to aneuploid neural progenitor cells that may not proceed with their developmental program and to aneuploid neuronal cells. Aneuploid adult neural progenitor cells and aneuploid newly generated adult neuronal cells may be fated to die or may survive for extended period of time. Aneuploidy for chromosomes carrying genes involved in AD, such as the genes for ApoE, APP, PSEN-1, PSEN-2 and tau protein, in neural progenitor cells and newly generated neuronal cells in the adult brain would contribute to the formation of amyloid plaques and neurofibrillary tangles and to neurodegeneration, particularly in the neurogenic regions of the adult brain such as the hippocampus. Causative factors of AD, such as the hyperphosphorylation of tau proteins and oxidative stress, are involved in the segregation and migration of chromosomes during cells division and promote aneuploidy [39]. These causative factors may promote the generation of aneuploid adult neural progenitor cells and of aneuploid newly generated adult neuronal cells in the brain of individuals with AD. Aneuploid adult neural progenitor cells and aneuploid newly generated adult neuronal cells may contribute to the etiology and pathogenesis of AD. This is particularly in the

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hippocampus, a region involved in learning and memory and particularly affected in patients with AD.

Conclusion and Perspectives The confirmation, that adult neurogenesis occurs in the adult brain and NSCs reside in the adult CNS, opens new avenues and opportunities for treating neurodegenerative diseases and particularly AD. Neurogenesis is modulated in the brain of patients with AD. The contribution of adult neurogenesis to the pathology and pathogenesis of AD remains to be fully understood and determined. It may be involved in regenerative attempts or in pathological processes. Aneuploidy contributes to the etiology and pathogenesis of AD. Aneuploid nerve cells in the adult brain may originate from cell cycle re-entry and DNA duplication, without cell division and from the nondisjunction of chromosomes during cell division during the process of adult neurogenesis, particularly in the neurogenic regions. Aneuploid newly generated neuronal cells may contribute to the etiology and pathogenesis of AD particularly in the hippocampus, a region primarily affected in patients with AD. Future investigations will aim at determining and understanding the involvement and contribution of adult neurogenesis to the pathology and pathogenesis of AD and to identify and characterize aneuploid newly generated neuronal cells in the brain of patients with AD. Future directions will aim at bringing adult NSCs to therapy for the treatment of AD.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter VII

Aneuploidy in Cultured Human Multipotent Mesenchymal Stromal Cells V. A. Nikitina, E. S. Voronina, L. D. Katosova and N. P. Bochkov Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Russia

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Abstract The guarantee of safety is a main condition to clinical application of cell transplantation. During the cultivation procedures chromosome abnormalities can arise which can lead to long term consequences of cell therapy. The aneuploid cell frequency (nullisomy, monosomy and trisomy) in the interphase nuclei of mesenchymal stromal cells (MSC) had analyzed on autosomes 6, 8, 11 and sex chromosomes. The phenotype of MSCs on early and late passages had no difference. In spite of decrease proliferation possibilities by 11-12 passage, cells kept typical MSC immunophenotype. The aneuploid cell clone formation appeared in some cultures. Two cultures of MSC from bone marrow had clones of aneuploidy cells: with trisomy 8 and monosomy X. In two cultures of MSC from adipose tissue clones with monosomy 6 were revealed at the late passages. The results show potential of genetic transformation and selection of MSCs with abnormal karyotype during cultivation in vitro. The results substantiate the need for more profound study of stem cell genetics and development of quality control system for the cell therapy.

Keywords: Multipotent mesenchymal stromal cells (MSC), aneuploidy, clone formation

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Introduction Mesenchymal multipotent stromal cells (MSC) are widely used in tissue engineering and regenerative medicine [1]. Numerous studies in biology of MSCs have demonstrated its regenerative, immunomodulating properties and its ability for differentiation and transdifferentiation. The amount of MSCs taken from body (bone marrow, adipose tissue, umbilical blood and other sources) is insufficient for therapeutic use, so it is essential to cultivate MSCs in vitro. A long-term passaging of MSC cultures may result in generation of genetically aberrant cells, its positive selection in population and contamination of transplantation material [2]. Literary sources concerning chromosomal instability and cell transformation due to long-term cultivation in vitro give evidence of necessity of cytogenetic control of MSCs [2,3,4,5]. Aneuploidy frequency is one of characteristics of genome stability and is necessary to evaluate the risk of cell therapy. The purpose of the present study is to evaluate the frequency of aneuploidy in cultured human MSCs.

Materials and Methods

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Cell Extraction and Cultivation Samples of adipose tissue of frontal abdominal wall were obtained as a result of liposuction and other surgical procedures. Bone marrow was extracted in order to homogeneous transplantation to patients with hematologic diseases. All donors had signed informed consent. Clonogenic cultivation of bone marrow stromal fibroblasts was held by A.J. Friedenstein et al. [6]. Mononuclear cells from bone marrow and adipose tissue were plated into ventilated cell culture flasks containing DMEM medium with 10-20% fetal bovine serum and 1% of insulin-transferrin-selenite. After 24-48 hours non-adherent cells were removed while medium rotation. Hereafter cultures were incubated till 75-90% confluency was attained. Afterwards cells were removed by trypsin solution and re-plated into new flasks. Cells were cultivated at 37°C, absolute humidity, 5% CO2 in the air. Beginning from the third passage MSC cultures had a monomorphic shape and consisted of spindle-shaped cells.

MSCs Characterization Multiplicity of cell growth was counted as ratio of cell quantity obtained in the passage to cell quantity planted in the previous passage. Surface antigenes were determined with use of panel of antibodies containing CD14, CD19, CD34, CD45, CD73, CD90, CD105 and HLADR. The relative amount (in percent) of cells in the MSC culture expressing given antigen was measured. Osteogenic differentiation was inducted by adding 7х10-3 M of βglycerophosphate, 1х10-8 M of dexamethasone and 2х10-4 M of ascorbic acid into the medium. Adipogenic differentiation of stromal progenitor cells was inducted by adding 1х107 M of dexamethasone and 1х10-9 M of insulin into the medium. Osteogenic progenitor cells

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were identified in alkaline leukocyte phosphatase activity test (Merck). Sudan (black or red) was used to identify adipogenic progenitor cells.

Cytogenetic Analysis

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Cytogenetic analysis was carried out on 2nd-3rd day after the last passage. Cells were incubated for 8-10 minutes in hypotonic solution and fixated by methanol - glacial acetic acid mixture (3:1). Centromere-specific DNA-probes for chromosomes X (DXZ1), Y (DYZ3), 6 (D6Z1), 8 (D8Z1), 11 (D11Z1) (Abbott, Vysis) were used for interphase FISH-analysis. Denaturation, hybridization and purification were done according to standard protocol. For contrast dyeing of nuclei DAPI was used. Two hybridizations for each culture on any passage were done evaluating two or three chromosomes simultaneously. About 500-1000 interphase nuclei were analyzed in each probe. The frequency of monosomy and trisomy was examined for each analyzed chromosome. The frequency of aneuploidy was counted as sum of monosomy and trisomy frequencies for each chromosome. All the MSC cultures were divided on two groups. In the first one, MSCs on the early passages were described (2nd-5th passage, approximately 3-10 cell divisions). In the second group cultures on the late passages were described (10th -15th passage, approximately 17-20 cell divisions). According to our calculations [7] one cell division on the early passages continued about 66 hours and 165 hours on the late passages. Clone formation was evaluated in relation with previous statistical calculations [7]. Definition of immunophenotype and cytogenetic study for majority of MSC cultures was done twice – on 2nd-5th and 10th-15th passages. Statistica software was used for statistical analysis.

Results and Discussion Proliferative activity of MSCs from bone marrow decreased after 10 passages. Multiplicity of cell increase was 5.88±0.84 between 3rd and 4th passage, 4.81±0.33 between 5th and 6th, 2.63±0.11 between 7th and 9th and 2.03±0.08 between 10th and 12th passages. After cultivation of MSCs during 3-4 passages high CD90, CD105, CD73 expression (above 60%) was observed. Less than 5% of stromal cells had CD45, CD34, CD19 antigens. As the result of increase of cultivation time the amount of CD90 expressing cells had decreased, hemopoietic cells and monocytes contamination had vanished. Forty one cultures from different passages were analyzed, 13 – adipose tissue derived (adMSC) and 28 – from bone marrow (bmMSC). Data on mean frequency of monosomy and trisomy of chromosomes 6, 8, 11 and X (in cultures with female karyotype) of adMSC and bmMSC on different stages of cultivation is presented in Table 1. The frequencies of monosomy and trisomy of different chromosomes of bmMSC did not differ (p>0.05) (Tab. 1) and were 1% and 0.3% respectively. Early passages of adMSC had statistically relevant difference between monosomy 8 and 11 (p=0.02) and monosomy 8 and X (p=0.03) frequencies. The comparison of frequencies of mono- and

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trisomy of chromosomes 6, 8, 11 and X of MSCs from different tissues revealed that frequency of monosomy 8 in adMSC from early passages was higher than observed in bmMSC (p=0.02). Apparently, revealed differences are connected with high frequency of monosomy 8 in adMSCs. This data does not allow us to make conclusion about individual participation of these chromosomes in mutagenesis in different tissues, since the differences were revealed only for separate types of aneuploidy. To reveal the regularity of chromosome segregation the accumulation of data on aneuploidy frequency is required. Table 1. Aneuploidy frequency in MSC cultures Chromosome

Aneuploidy type

6

monosomy trisomy n monosomy trisomy n monosomy trisomy n monosomy trisomy n

8

11

X

MSC from bone marrow passage passage 2-5 10-15 0.87±0.17 0.78±0.15 0.30±0.09 0.28±0.08 17635 11801 0.74±0.10 0.99±0.14** 0.22±0.07 0.16±0.05 19874 12687 1.03±0.16 1.11±0.17 0.24±0.06 0.33±0.08 23055 11027 1.30±0.31 0.58±0.18 0.21±0.10 0.15±0.03 7995 7114

MSC from adipose tissue passage passage 2-5 10-15 1.49±0.38 1.18±0.43 0.30±0.09 0.42±0.17 13031 3520 1.52±1.00 1.80±0.38*,** 0.28±0.10 0.40±0.14 13031 3520 0.65±0.27 0.77±0.18* 0.13±0.04 0.29±0.22 13108 3327 0.87±0.38 0.75±0.19* 0.19±0.08 0.20±0.13 12100 3459

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n – number of analyzed cells. * - significant difference between monosomy 8 frequency and frequencies of monosomy 11 (p=0.02) and monosomy X (p=0.03) in adMSC (2-5 passage). ** - significant difference between monosomy 8 frequencies in adMSC and bmMSC (p=0.02).

Aneuploidy frequency mean values of sex chromosomes with male phenotype of bmMSC are presented in the Table 2. The frequency of spontaneous loss of Y chromosome in bmMSC was higher than observed for X chromosome (p=0.03). This can be explained by the rarity of nullisomy X that is due to loss of the only X chromosome or hybridization artifact. Table 2. Mean frequency of sex chromosomes aneuploidy in MSC cultures from male bone marrow Passage 2-5 n 10-15 n

Aneuploidy type nullisomy disomy nullisomy disomy

X chromosome 0.12 0.04* 0.96 0.18 17566 0.02 0.02 2.09 0.50** 5408

Y chromosome 0.39 0.12* 0.69 0.14 15459 0.31 0.20 1.72 0.47** 5408

n – number of analyzed cells. * - significant difference between nullisomy X and Y frequencies (p=0.03). ** - increase of disomy X and Y frequencies by 10-15 passage (p=0.01).

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There was no significant difference between X and Y chromosome disomies (p>0.05). Statistically relevant increase in amount of cells with two X and Y chromosome signals in comparison between late and early passages was observed (Tab. 2). Aneuploidy frequencies of other chromosomes did not changed during cultivation process (p>0.05), except cases when clones arrived. Frequency of trisomy was lower than monosomy for all chromosomes. As described above [7], there are two mechanisms of monosomy formation: chromosome nondisjunction and anaphase lag during mitosis. Table 3. Clone characterization N

Passage

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MSC from bone marrow 1 4 6 12 2 4 10 MSC from adipose tissue 3 12 4 10

Clone characterization

Portion of aberrant cells, %

nuc ish(D8Z1 nuc ish(D8Z1 nuc ish(D8Z1 nuc ish(DXZ1 nuc ish(DXZ1

24 34 16 12 92

3)[314]/(D8Z1 2)[1005] 3)[348]/(D8Z1 2)[657] 3)[163]/(D8Z1 2)[856] 1)[48]/(DXZ1 2)[345] 1)[954]/(DXZ1 2)[67]

nuc ish(D6Z1 1)[141]/(D6Z1 2)[569] nuc ish(D6Z1 1)[104]/(D6Z1 2)[385]

20 21

In some cultures clones with abnormal karyotype were observed (Table 3). There were two abnormal clones in bmMSC cultures: clone with trisomy 8 and monosomy X. Culture with trisomy 8 was explored three times. In 4th passage 24% of all analyzed cells had extra chromosome 8. In 6th passage their amount increased to 34% and in 12th passage it decreased to 16%. Conditions of cultivation and culture passage were identical, that is why changes in size of clone may indicate the different rate of its fission during cultivation and that proliferation and aging in aneuploid cells are faster than in diploid ones. In other case the culture of bmMSC of healthy woman had clone with one X chromosome. The amount of such cells in 4th passage was 12% and increased to 91% in 10th passage. Two adMSC cultures on late passages had two cell populations: one with monosomy 6 and another with diploid karyotype. Apparently, genetic aberrations in these cultures appeared during cultivation, unlike bmMSC defect clones, which were detected in early passages after 3-10 cell divisions. The source of abnormal clone in these cultures could be aberrant cells of donor, which had a selective advantage in proliferation. Morphology and immunophenotype of all MSCs, including mosaic ones, corresponded to requirements of International Society for Cellular Therapy [8].

Conclusion Thereby, described mean frequencies of mono- and trisomies characterize spontaneous level of aneuploidy in MSCs from different tissues. In the majority of MSC cultures the

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frequency of aneuploidy does not change during cultivation. During cultivation bmMSC from male donors the amount of cells with two X or Y chromosomes increases in late passages. In four MSC cultures (approximately 10%) clones of aneuploid cells were detected. Furthermore, bmMSC clones were detected in early passages and persisted till late stages of cultivation, meanwhile adMSC clones appeared after 10th passage. The source of these clones could be abnormal donor cells or aberrant cells which appeared in vitro during extraction or on the early stages of cultivation. Fast growth of aneuploid cells testifies about their selective proliferative advantage. It was shown, for if initial level of aneuploidy equals to 5%, then the 20-30% acceleration of cell cycle will increase the portion of aneuploid cells to 20-40% during few cell divisions [7]. Use of cell transplantats which contain clones of aberrant cells may result in malignant transformation or other complications of cell therapy. Hence there is opinion the cultures with karyotypic changes are not dangerous for transplantation. Apparently, clones with different chromosome aberrations are different in it potential hazard. So, trisomy 8 is one of the most common aberrations in the setting of malignant myeloid diseases [9]. The biological role of trisomy 8 in pathogenesis of tumors may be related with increase in amount of copies and expression enhancement of c-myc oncogene, which is located in long arm of chromosome 8. Developing of fast complex approaches for detection, evaluation of proliferation rate, genetic stability and oncogenicity of abnormal clones will help to avoid undesired effects of cell therapy. We are very grateful to the employees of Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology and ZAO ―ReMeTex‖ for collaboration and granted MSC cultures.

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References [1] [2]

[3] [4]

[5]

[6]

Bernardo ME, Pagliara D, Locatelli F. Mesenchymal stromal cell therapy: a revolution in Regenerative Medicine? Bone Marrow Transplantation, 2011 P. 1–8. Bochkov N.P., Nikitina V.A., Voronina E.S., Kuleshov N.P. Methodological guidelines for genetic safety testing of cell transplants. Bulletin of experimental biology and medicine, 2009, V. 148(4), P. 677-683. Duesberg P., Li R. Multistep carcinogenesis: a chain reaction of aneuploidizations. Cell Cycle. 2003, V. 2(3), P. 202-210. Meza-Zepeda L.A., Noer A., Dahl J.A., Micci F., Myklebost O., Collas P. Highresolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence. Journal of Cellular and Molecular Medicine, 2008, V. 12(2), P. 553-563. Rubio D., Garcia S., De la Cueva T., Paz M.F., Lloyd A.C., Bernad A., Garcia-Castro J. Human mesenchymal stem cell transformation is associated with a mesenchymalepithelial transition. Experimental Cell Research, 2008, V. 314(4), P. 691-698. Friedenstein A.J., Chailakhyan R.K. Stromal cells of bone marrow and hemopoietic microenvironment. Archive of Pathology, 1982, N 10, P. 3-11.

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Bochkov N.P., Vinogradova M.S., Volkov I.K., Voronina E.S., Kuleshov N.P. Statistical analysis of clone formation in human stem cell cultures. Cell Technologies in Biology and Medicine, 2011, N 2, P. 63-66. Horwitz E.M., Prather W.R. Cytokines as the major mechanism of mesenchymal stem cell clinical activity: expanding the spectrum of cell therapy. The Israel Medical Association Journal, 2009, V. 11(4), P. 209-211. Wolman S.R., Gundacker H., Appelbaum F.R., Slovak M.L. Impact of trisomy 8 (+8) on clinical presentation, treatment response, and survival in acute myeloid leukemia: a Southwest Oncology Group study. Blood, 2002, V. 100(1), P. 29-35.

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In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9 Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter VIII

Somatic and Germ Cell Spontaneous Aneuploidy Level in Healthy Fertile People N. Zotova, E. Markova, V. Artukhova and A. Svetlakov Center for Reproductive Medicine, Krasnoyark, Russia

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Abstract In this chapter we are presenting an investigation of spontaneous aneuploidy level in somatic and germ cells of healthy fertile men and women analyzed by FISH technique. We performed molecular-cytogenetic investigation of blood cells on 28 fertile healthy persons, among them sperm cells from 10 men and polar bodies (PBs) from 6 women of reproductive age. Cultivated lymphocytes and fixed sperm were analyzed by FISH for five chromosomes (13, 18, 21, Х, and Y). The first and second PBs aneuploidy were analyzed during IVF (in vitro fertilization) plus PGD program with oocyte donation by FISH for chromosomes 13, 18, 21. In addition to standard statistics, limits and confidence intervals were scored. A total of 28,000 lymphocytes and 10,000 sperm were scored for five chromosomes, 54 first and 54 second PBs - for three chromosomes. We found differences in results, with direct and indirect labeling, and scored an absolute error of FISH technique. The average lymphocytes aneuploidy level for all chromosomes (13, 18, 21, Х, and Y) was 1.73±0.20% for women and 1.05±0.08% for men (P=0.014). The average sperm aneuploidy for five chromosomes was 1.17±0.14%, the limit was 2.82%. We determined limits of mutation detection for each chromosome separately, and for all five chromosomes in lymphocytes and sperm. The total lymphocyte limits were 3.40% for women (four chromosomes) and 1.44% for men (five chromosomes). We investigated correlations between aneuploidy in somatic and germ cells, finding a mild association between frequencies of monosomy in the 13th and 18th chromosomes in sperm and lymphocytes. Also, we revealed correlation between sperm aneuploidy and semen quality, r=0.644. Aneuploidy frequency in PBs of women was extremely variable

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N. Zotova, E. Markova, V. Artukhova et al. (0-40%). We did not find any correlations between polar bodies aneuploidy and aneuploidy levels in lymphocytes.

Introduction

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Fluorescent in situ hybridization (FISH) is widely applied at the moment for diagnostics and monitoring of aneuploidy. Commercial chromosome-specific DNA probes make FISH an available technique for clinical cytogenetical analysis. But normal parameters of aneuploidy in different types of cells are not developed enough. Normal karyotype does not exclude low level aneuploidy in somatic cells and gamete aneuploidy. Low level false-positive diagnostics of monosomy and trisomy may result from technical artifacts, such as unspecific probe binding with DNA, non-optimal conditions for hybridization or detection of hybridization signals, or inaccurate criteria for signal scoring [27]. Identification of aneuploidy in blood cells may depend on the type of DNA probes applied. Sex chromosomes are frequently involved in low level mosaicism, and chromosomes 13, 18, and 21 are the autosomes most frequently involved in newborn aneuploidy [12]. The probes for these chromosomes are commonly applied for aneuploidy diagnostics. Aneuploidy is a result of missegregation of replicated chromosomes between daughter cells. There are a number of molecular mechanisms inducing aneuploidy [10, 28,]. It may be interesting to explore correlation between aneuploidy in somatic cells and germ cells as mechanisms of human aneuploidy. It is suggested that mechanisms underlying somatic and germinal missegregation may be associated [46].

Blood Cell Spontaneous Aneuploidy Spontaneous aneuploidy level in lymphocytes is necessary as a control to assess low level aneuploidy and mosaicism in patients with infertility in reproductive and genetic practice. FISH method may be effectively applied to estimate minimal hyper- or hypohaploid clones and low level or hidden mosaicism [6, 42, 53]. It is important for reproductive cytogenetics because cases of gonosome mosaicism are common [64, 51]. Sometimes, when a sex chromosome abnormality is seen in a low percentage of cells, some specialists suspect it to be an artefact [51]. The level of aneuploidy in blood cells may increase spontaneously or be induced by environmental chemical agents. There are reports that augmented aneuploidy level is related to drugs taking (analgetics, antibiotics, oral contraceptives, or spermicides) [1]. The risk of chromosome aberrations increased with age [56], and in patients with lymphoma and chronic hepatitis C [16]. It was demonstrated that cell cultivation may also induce aneuploidy [29, 40]. Sometimes chromosome translocations may lead to missegregation of uninvolved chromosomes. An increased aneuploidy may be observed for chromosomes not involved in the rearrangement, which known as an interchromosomal effect (ICE) [20].

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Polar Bodies Aneuploidy Polar bodies (PBs) are the by-products of female meiosis, which allow indirect analyzing of human oocyte karyotype during preimplantation genetic diagnosis (PGD). PBs predict the resulting genotype of the maternal contribution to the embryos. Neither the 1st PB, which is extruded as a result of the first meiotic division, nor the 2nd PB, which is extruded following the second meiotic division, have any known biological value for preimplantation and postimplantation development of the embryo [63]. The PB diagnosis of common aneuploidies by FISH was suggested to be useful for detection of oocytes with common chromosomal trisomies in IVF patients [60]. Errors in meiosis I of the oocyte contribute to 80% of the aneupliodies seen in embryos. The most common abnormality in PB1 was a missing chromatid, resulting in trisomy in the embryo (51%), and in PB2 missing and extra signals were of similar rates (39 and 44%, respectively) in the oocytes of patients with a mean age of 38 years undergoing FISH analysis for five chromosomes [62, 5]. Using PBs analysis increased the effectiveness of PGD programs and allowed to avoid embryo mosaicism [37]. Studies of PBs aneuploidy for chromosomes 13, 16, 18, 21, and 22 reported high rates of aneuploidy (from 32.1 to 52.1%), which could reflect the effect of maternal aging on aneuploidy occurrence. However, overestimates due to limitations of the multi-FISH technique, fragmentation and in vitro ageing of polar bodies, or low quality of chromosome spreading cannot be excluded [60]. Thus, the authors have indicated that the reported incidences of chromosome abnormalities could be reduced to 28%. As it is reported the frequency of aneuploidy in human oocytes ranges between 12.0 and 37.3% [5]. Maternal aging is an essential factor in analysing occurrence of aneuploidy in female gametes. Numerous other genetic or environmental risk factors have been suggested. These include maternal smoking, oral contraceptives, irradiation, diabetes, folate metabolism, polymorphism, or allelic combination [43]. The oocyte aneuploidy could lead to embryo aneuplpoidy and chromosome disorders in newborns, unsuccessful IVF cycles, and pregnancy miscarriages [25]. There are a lot of technical moments of single cell analysis: defective fixation results in low quality nuclei making diagnoses difficult, unreliable, [34, 58] and yielding an increased number of completely non-informative nuclei [52]. In addition to technical difficulties of concern, issues also arise in the interpretation of the fluorescent signals. Several pitfalls have been described in the interpretation of FISH signals in interphase nuclei, including overlapping signals, split signals, weak or faded signals and cross-hybridization or polymorphisms in the target sequence of the probe [30, 8, 33].

Sperm Aneuploidy Advanced maternal age is known to be a primary risk factor for transmitted aneuploidy, less is known about paternal risk factors. Several paternal risk factors for sperm aneuploidy, including advanced age, cancer chemotherapy, suicide attempts by use of high-dose diazepam, cigarette smoking, and exposure to air pollution [46] were shown.

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Survey of sperm aneuploidy demonstrates a great variability of data. M. Guttenbach with colleagues (1997) reported that sperm aneuploidy of healthy men for chromosomes 1, 7, 10, 17, X, and Y was 1 — 4% and hypohaploidy prevailed over hyperhaploidy (3.3% and 1.7%, respectively) [17]. In different studies, sperm aneuploidy of healthy normozoospermic donors varied from 0 to 7.7% (for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y), in some cases it could be 30.2% (for chromosomes 18, X, and Y) [39, 7, 54]. Numerous reports show that there is interchromosomal and interindividual variability in the baseline frequencies of specific sperm aneuploidies. Some men may have significantly higher frequencies for some aneuploidies than for others. Few studies have actually evaluated multiple semen samples from the same individuals to determine whether the observed variations are due to technical factors or sporadic events in individuals, or are time-stable characteristics of the individuals [55]. Some studies have been performed to explore whether low sperm parameters (concentration, motility, morphology) is associated with increased sperm aneuploidy rates in infertile patients [7]. Some investigations show a negative correlation of sperm concentration with aneuploidy level in men with abnormal semen parameters [41, 49].

Aim

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The aim of our work was to assess control parameters of aneuploidy level in somatic and germ cells of healthy people and evaluate the limits of aneuploidy detection for five chromosomes (13, 18, 21, Х, and Y). We also estimated significant differences and correlations between investigated parameters.

Materials and Methods Investigated groups Cytogenetic and molecular-cytogenetic investigation of blood cells from 28 fertile healthy persons were performed: 10 men (the average age 32.00±2.28 years) and 18 women (the average age 28.47±1.00 years). For men, sperm FISH were made. For 6 women, polar bodies analysis was performed. All persons had normal karyotype and all men were normozoospermic and satisfied the criteria set for gamete donors issued by ESHRE [9]: age 20—35 years, somatic health, a healthy child, no phenotypic deviations.

Karyotyping and Blood Cells Preparation Blood was collected in Vacuette tubes with Li-heparin. Standard chromosome analysis was performed on metaphase chromosomes of lymphocytes stimulated with PHA and cultivated for 72 h. Chromosome staining was done by G-banding technique. 12 metaphase

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plates were analyzed [47]. Chromosome images and karyograms were analyzed with Band View software (Applied Spectral Imaging, United States). Aneuploidy in non-cultivated cells was assessed in leukocyte fraction isolated from blood by centrifugation in ficoll-urografin gradient density. Cultivated and non-cultivated cells were fixed under equal conditions. One sample was cultivated in 5 repeats to estimate the error of the method. Non-cultivated cells were treated with trypsin or pepsin.

Semen Preparation Semen analysis was performed according to WHO criteria [65]. Sperm fixation for FISH was made according to standard protocols with decondensation of nuclei by 0.25M detiotreitol solution.

Polar Bodies Preparation

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The first and the second PBs aneuploidies were analyzed during IVF (in vitro fertilization) plus ICSI (itracytoplasmatic sperm injection), plus PGD program with oocyte donation. This procedure was performed for oocyte donors with the consent of donors and patients. Females underwent ovarian stimulation according to standard protocols with gonadotrophins. Oocytes were received by transvaginal follicular aspiration. Both first and second polar bodies were removed simultaneously after fertilization. PBs were treated with H2O as hypothony and fixed on slides [22].

FISH Analysis Lymphocytes and sperm were analyzed by FISH for five chromosomes: 13, 18, 21, Х, and Y). Fixed PBs were studied using fluorescent probes for three chromosomes: 13, 18, and 21. For FISH assay, we applied DNA-probes with direct labeling: CEP18 (Spectrum Aqua), CEP X (Spectrum Blue), CEP Y (Spectrum Gold), LSI 13 (Spectrum Red), LSI 21 (Spectrum Green) (Abbott Molecular Inc., United States). Ten women were analyzed by FISH with indirect labeling centromere specific probes by reporter molecules (digoxigenin-11-dUTP, DXZ1, and biotin-16-dUTP, D18Z1), provided by the Cytogenetics Laboratory of the Institute of Medical Genetics (Tomsk, Russia) with subsequent immunochemical detection by avidin-FITC (Sigma, Germany) and antidigoxigenin-rhodamine (Boehringer Mannheim, Germany). Hybridization was performed according to the manufacturer‘s instructions. Immunochemical detection was carried out as described [56]. Samples were stained with Vectashield or DAPI and studied under microscope Olympus BX 51, with required filter set and magnification 600x. Images were analyzed with FISH View software (Applied Spectral Imaging, United Sates). 1000 nuclei per case were counted. Hybridization efficacy was estimated as not less than 85% of signal-positive cells [3].

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Scoring Criteria of FISH Signals In each cell, signals of all chromosomes were counted using scoring criteria with some modifications [35, 3, 56]. 1. Single signals of typical size and intensity were interpreted as the number of chromosome copies. 2. Hybridization signals were verified with other filters. If the signal was registered with other filters, it was regarded as unspecific. 3. If a cell had a single signal, but its intensity was twice as high, it was considered to be an overlapping of two signals located in different planes; it was verified by turning micro screw. 4. Two adjacent signals were interpreted as different chromosomes if a signal of the same size can be positioned between them. 5. If a nucleus had one typical signal and the other one doubled (two closely located labels) it was interpreted as a normal variant. 6. Chromosome monosomy was testified by each filter to avoid signal overlap.

Statistical Analysis Statistical treatment of the results was carried out with Statistica 7.0 software. Mean

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values ( x ) and their errors (m), as well as minimums (min) and maximums (max), were calculated. Statistical significance was tested using nonparametric Wilkinson‘s and MannWhitney U-tests. Correlations were analysed by Kendall-tau test. The limit of mutation detection (lim) as well as 95% confidence interval (CI) were calculated by the formula [27], used in molecular and cytogenetic investigations [40]. The method absolute error was calculated as an error of mean arithmetical value of one sample repeats. The method relative error was calculated as a ratio of the absolute error to a mean value.

Results and Discussion Comparison of Cultivated and Uncultivated Cells as well as Direct and Indirect Labeling To investigate the aneuploidy level in blood cells of 12 healthy women and to consider the possible influence of FISH-probes type and in vitro cultivating effect, cultivated and noncultivated cells were assessed by FISH-technique and with two types of DNA probes. 48,000 cells were totally analyzed. Hypo- and hyperdiploid signals were identified in interphase nuclei in tested samples. Chromosome abnormalities were monosomy and trisomy. Chromosome nullisomy was not registered. The figure depicts FISH-images of aneuploid nuclei of cultivated and non-cultivated cells, with application of direct labeled DNA-probes. Mean values of aneuploidy frequency and limits with 95% CI are presented in Table 1.

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Table 1. Aneuploidy level (%) for chromosomes 13, 18, 21, and X in cultivated and uncultivated blood cells ( x ±m; min-max; lim±CI) Chromosome disturbance Monosomy 13

Trisomy 13

Monosomy 18

Trisomy 18

Monosomy 21

Trisomy 21

Trisomy 13, 21

Probes with direct labeling Uncultivated cells, Cultivated cells, n=12,000 n=12,000 0.35±0.05 0.17±0.05 0.10-0.70 0.10-0.47 0.73±0.18 0.47±0.14 0.33±0.04 0.54±0.11 0.10-0.60 0.20-1.30 0.64±0,15 1.40±0,40 0.19±0.03 0.21±0.06 0.10-0.30 0.10-0.50 0.34±0.07 0.59±0.18 0.13±0.03 0.16±0.04 0.10-0.20 0.10-0.40 0.24±0.05 0.41±0.11 0.13±0.02 0.23±0.05 0.10-0.20 0.10-0.50 0.23±0.05 0.54±0.15 0.14±0.03 0.18±0.04 0.10-0.30 0.10-0.50 0.32±0.09 0.49±0.14 0.01±0.00 -

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Monosomy Х

0.17±0.03 0.10-0.40 0.40±0.11 Trisomy Х 0.11±0.01 0.10-0.20 0.19±0.03 Total aneuploidy 1.13±0.08 0.50-1.60 1.80±0.32 Total aneuploidy for 0.22±0.04 X-chromosome 0.10-0.50 0.65±0.14

0.27±0.08 0.10-1.00 0.92±0.30 0.18±0.04 0.10-0.40 0.41±0.11 1.66±0.22 0.60-3.47 3.40±0.82 0.40±0.09 0.10-1.10 1.13±0.34

Probes with indirect labeling Uncultivated cells, Cultivated cells, n=12,000 n=12,000 ND

ND

ND

ND

ND

ND

0.48±0.08 0.20-1.00 1.08±0.28 0.38±0.06 0.10-0.07 0.87±0.23

ND

ND

ND

ND

ND

ND

0.57±0.15 0.10-2.10 1.77±0.57 0.25±0.03 0.10-0.40 0.52±0.13

0.34±0.06 0.10-0.70 0.85±0.24 0.51±0.06 0.20-1.00 1.01±0.24

NA

NA

0.82±0.15 0.30-2.30 1.98±0.55

0.85±0.09 0.40-1.30 1.55±0.33

Note: ND – no data, NA – not applicable.

In non-cultivated cells, FISH analysis, with direct labeled DNA probes, showed that monosomy and trisomy frequency for different autosomes varied between 0.1-0.7% and 0.10.6%, respectively; in cultivated cells, the variation was 0.1-0.5% and 0.1-1.3%, respectively. The frequencies of X-chromosome hypo- and hyperploidy does not exceed those observed for autosomes. The total frequency of aneuploidy for particular chromosome was assessed as sum of monosomy and trysomy. This did not exceed 1%. In experiments with direct labeled DNA-

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probes, the total frequency of aneuploidy for 4 chromosomes (13, 18, 21, and X) was 1.7% in cultivated cells. The frequency of aneuploidy estimated with indirect labeled DNA-probes for 2 chromosomes (18 and X) was 1.7%. The frequency of X-chromosome aneuploidy determined by indirectly labeled DNA-probes was found to be 2.1 times higher in cultivated cells (P=0.026) and 3.7 times higher in uncultivated cells (P=0.002). This compared to the results obtained with directly labeled probes. Both trisomy and monosomy contributes to the increased frequency. The results show that the aneuploidy frequency in cells with one or three signals depends on DNA-probes applied for FISH-assay. The frequency of aneuploid cells detected with indirectly labeled DNA-probes was higher than with direct labeling. It can be assumed that additional step of immunochemical treatment increases the number of artifact signals [67]. Aneuploidy frequency, estimated by directly labeled DNA-probes, was higher in cultivated cells. The total aneuploidy level for 4 chromosomes enhanced 1.5 times (P=0.041). No significant differences between particular chromosomeswere observed except chromosome 13. The level of chromosome 13 monosomy was significantly lower (P=0.028). In experiments with indirectly labeled DNA-probes, the difference between cultivated and uncultivated cells was found only for X-chromosome trisomy (P=0.001). Cell populations of cultivated and uncultivated blood may be different. In uncultivated blood, leukocyte fraction is characterized by prevailing segmento-nuclear neutrophils and less number of lymphocytes (25-40%). In blood cell cultures, PHA mostly stimulates Tlymphocyte proliferation, which becomes predominate (70%) in culture population [47]. Therefore, modified leukocyte composition may affect the number of hyper- and hypodiploid cells. Both cultivated and uncultivated cells may be applied for the successful analysis of aneuploidy frequency. However, to assess low level aneuploidy it is necessary to consider the cultivation effect on compared groups and control levels of minimal frequency. The calculated limit for each chromosome abnormality, estimated by FISH with directly labeled DNA probes, was 0.19-0.73% for uncultivated cells. The mean aneuploidy levels, and limits for X-chromosome and chromosome 18 evaluated by probes with indirect labeling, were high. The limit for X-chromosome detection was higher in cultivated than in uncultivated cells: 0.41% with direct probe labeling and 1.01% with indirect labeling (0.19 and 0.52%, respectively, in uncultivated cells). The total limit of mutation detection for all 4 chromosomes estimated with direct probe labeling was 3.4% and 4.22% with CI 95%. It is interesting to note that X-chromosome aneuploidy is of higher occurrence than autosome aneuploidy. There are indications that the frequency of aneuplody increases with female age, and clones with X-chromosome loss prevail over other cell clones [44, 48]. We did not observe X-monosomy dominance over other chromosome abnormalities. Our study included rather young fertile women without reproductive disorders and therefore, Xchromosome loss was not typical. Cell cultivation influences the level of aneuploidy, however, it does not affect the frequency of 4 chromosome aneuploidy. It was demonstrated that aneuploidy frequency depends on cultivation period: in 72-h cultures it was higher than in 48-h cultures of healthy donor blood [44]. Other research shows that there was no significant differences in numerical rearrangements of individual chromosomes assayed with indirectly labeled DNA probes in

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cultured and uncultured leucocytes [56]. In patients subject to injurious environmental factors, the aneuploidy frequency of 6 chromosomes in cultivated cells was higher (2.98%) than in uncultivated cells (1.96%). Potential artefact may be manifested in cultivated cells with low level mosaicism: cells with particular karyotype may have advantages or disadvantages for proliferation in culture. FISH-analysis of uncultured cells performed on the original cell population is more adequate. Calculated values of upper limit of aneuploidy detection for each chromosome may be utilized as control values for low level aneuploidy to make a conclusion about mosaicism. The limits of hyper-and hypodiploid cells detection are the lowest values for monosomy and trisomy diagnostics. Distinguishing between low level mosaicism and control values is important for diagnostics of mosaic forms of Turner`s syndrome and X-chromosome trisomy in women, as well as for research of hidden gonosome and low level mosaicism in reproductive genetics. Hidden mosaicism, poorly identified by routine cytogenetic analysis, and low level mosaicism (aneuploid clone less than 10%) may be associated with reproductive disorders [32, 66]. In spite of high resolution of FISH, it is necessary to know aneuploidy limit values to make reliable conclusions. To estimate FISH method error, which is necessary to determine low level aneuploidy, a single blood sample was divided into 5 aliquots cultivated separately (Table 2). The experiment has been carried out with the sample with increased aneuploidy level. The cultivation conditions were equal; FISH signals were counted by the same researcher. FISHassay was performed with directly labeled DNA-probes. Totally 5,000 cells were assessed.

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Table 2. Aneuploidy frequency in blood lymphocytes of a female by 5-colour FISH in 5 redefines Chromosome disturbance

Aneuploidy frequency (%) 1

2

3

4

5

x ±m

Monosomy X Trisomy X Monosomy 13 Trisomy 13 Monosomy 18 Trisomy 18 Monosomy 21 Trisomy 21 Total aneuploidy

0.80 0.60 0.30 0.30 0.60 0.20 0.30 0.10 3.20

0.90 0.30 0.30 0.40 0.60 0.10 0.60 0.40 3.60

1.00 0.20 0.10 0.70 0.60 0.10 0.30 0.30 3.30

0.90 0.60 0 0.50 0.30 0.10 0.20 0.30 2.90

0.90 0.30 0.30 0.30 0.40 0 0.30 0.40 2.90

0.90±0.03 0.40±0.08 0.20±0.06 0.44±0.07 0.50±0.06 0.10±0.03 0.34±0.07 0.30±0.05 3.18±0.13

It was found that aneuploidy mean values for 4 chromosomes with direct labeling may deviate up to 0.13%, i.e. 1.3 cells per 1,000. No significant differences between the repeats were observed. We concludethat using cultivated cells and FISH-probes with direct labeling is the most convenient. For this reason, we applied the scheme to analyze aneuploidy in sperm and lymphocytes of men. We considered that aneuploidy level lower than 1.3% is covered by the resolution ability of the method andcould be ignored.

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Table 3. Aneuploidy level (%) for chromosomes 13, 18, 21 X, and Y in blood cells and sperm of 10 men ( x ±m; min-max; lim±CI) Lymphocytes N=10000 Chromosome disturbance Monosomy 13

Trisomy 13

Monosomy 18

Trisomy 18

Monosomy 21

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Trisomy 21

Disomy X

Sperm N=10000

x ±m Min – Мax Lim ± CI 0.10 ± 0.07 0.10 – 0.30 0.68 ± 0.26 0.20 ± 0.00 0.20 – 0.20 0.20 ± 0.00 0.10 ± 0.04 0.10 – 0.40 0.67 ± 0.25 0.13 ± 0.03 0.10 – 0.40 0.63 ± 0.22 0.12 ± 0.02 0.10 – 0.20 0.35 ± 0.10 0.18 ± 0.04 0.10 – 0.40 0.74 ± 0.25 0.20 ± 0.03 0.10 – 0.40 0.69 ± 0.22

Chromosome disturbance Nullisomy 13

Disomy 13

Nullisomy 18

Disomy 18

Nullisomy 21

Disomy 21

Disomy XY

Disomy X

Nullisomy Y

0.16 ± 0.02 0.10 – 0.20 0.43 ± 0.12

Disomy Y

Nullisomy X, Y

Total aneuploidy (13, 18, 21, X and Y) Total aneuploidy for sex chromosomes

0.98 ± 0.07 0.80 – 1.50 2.19 ± 0.54 0.30 ± 0.03 0.20 – 0.40 0.80 ± 0.22

Total aneuploidy (13, 18, 21, X and Y) Total aneuploidy for sex chromosomes

x ±m Min – Мax Lim ± CI 0.13 ± 0.03 0.10 – 0.20 0.42 ± 0.13 0.27 ± 0.04 0.10 – 0.40 0.83 ±0.25 0.17 ± 0.07 0.10 – 0.30 0.75 ± 0.26 0.13 ± 0.02 0.10 – 0.20 0.37 ± 0.11 0.13 ± 0.03 0.10 – 0.20 0.38 ± 0.11 0.28 ± 0.03 0.10 – 0.40 0.74 ± 0.21 0.18 ± 0.03 0.10 – 0.30 0.53 ± 0.16 0.17 ± 0.04 0.10 – 0.40 0.73 ± 0.25 0.15 ± 0.02 0.10 – 0.20 0.42 ±0.12 0.23 ± 0.09 0.10 – 0.40 1.00 ± 0.34 1.17 ± 0.14 0.50 – 2.10 3.44 ± 1.01 0.43 ± 0.09 0.10 – 0.80 1.73 ± 0.58

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We estimated the value of error which may be the same both for FISH-assay and for other quantitative methods. The value of random error (0.13% in absolute units and 4.08% in relative units) characterizes the method accuracy and is necessary to judge measurement values. The accuracy of mean values is satisfactory if the relative error does not exceed 3-5% [26]. According to the current recommendations of International Standard Organization (ISO) for measurement accuracy evaluation, it relies on absolute, rather than on relative, values [2]. The values of mutation detection limits and the method errors characterize the parameters used in the method: FISH-assay with direct labeling of 4 chromosomes in cultivated blood cells. As the analysis is multicomponent, various sources of errors are possible. In our experiments, random errors resulting from cultivation, fixation, washing, and detection weretaken into account. Evaluation of aneuploidy level in the control group and values of random errors unavoidable for the method showed that with aneuoploidy frequency for each chromosome, not more than 1% the value of random error, is about ten times less. According to our data, aneuploidy values 0.5-1 per 1,000 cells are doubtful because they are lower, or overlap, the method resolution.

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Sperm and Lymphocyte Aneuploidy in Men Human sperm cells are quite easy to obtain, to investigate their aneuploidy level and to compare it with blood cells aneuploidy. We explored 10 healthy fertile men. Cultivated blood cells were used for aneuploidy analysis. Table 3 presents mean aneuploidy level in blood cells and sperm, and limits of mutation detection with 95% confidence intervals for investigated individuals. According to our results, monosomy and trisomy levels for autosomes varied from 0.1 to 0.4%. Nullisomy and disomy levels were in the same limits. Total aneuploidy level for five chromosomes (13, 18, 21, Х, and Y) in lymphocytes was 0.98%, in sperm – 1.17% (not significant differences). It means that meiotic errors happen more frequently than mitotic. Scored limits for each chromosome disturbance varied from 0.20 to 0.99% for autosomes. Maximal aneuploidy level was found for chromosomes 13 and 21: trisomy 21 in lymphocytes and disomy 21 in sperm were 0.18±0.04 and 0.28±0.03, respectively; as well as the limits: 0.74±0.25 and 0.74±0.21 respectively. For identification of possible common mechanisms of mitotic and meiotic errors, we performed correlation analysis of aneuploidy levels in germ and somatic cells. We found mild associations between chromosome 13 monosomy in lymphocytes and chromosome 13 nullisomy in sperm (r=0.609, P