Molecular Determinants of Radiation Response

Current Cancer Research Series Editor Wafik El-Deiry For other titles published in this series, go to www.springer.co...

Author: Theodore L. DeWeese | Marikki Laiho

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Current Cancer Research

Series Editor Wafik El-Deiry

For other titles published in this series, go to www.springer.com/series/7892

Theodore L. DeWeese Marikki Laiho ●

Editors

Molecular Determinants of Radiation Response

Editors Theodore L. DeWeese Department of Radiation Oncology and Molecular Radiation Sciences Johns Hopkins University School of Medicine, 401 North Broadway Suite 1440 Baltimore, MD 21231 USA [email protected]

Marikki Laiho Department of Radiation Oncology and Molecular Radiation Sciences Johns Hopkins University School of Medicine Baltimore, MD 21231 USA [email protected]

ISBN 978-1-4419-8043-4 e-ISBN 978-1-4419-8044-1 DOI 10.1007/978-1-4419-8044-1 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011922499 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Every day, mammalian cells accumulate an estimated 100,000 lesions in their DNA resulting from exposure to reactive oxygen species, chemical deterioration of their bases, and exposure to exogenous agents such as ultraviolet and ionizing radiation. Cells have evolved complex response mechanisms to recognize and repair this injury in order to maintain genomic integrity in the face of this unrelenting assault. The study of DNA damage response has a long and storied history beginning in the 1940s by scientists like Albert Kelner and Renato Dulbecco. Their work revealed the existence of enzymatic photoreactivation and ultimately laid the foundation for the idea that cells respond to DNA damage and that DNA damage repair does exist. Since that time and with the development of sophisticated molecular techniques, an evolving story regarding the cellular response to DNA injury and its importance has emerged. The spectrum of diseases that have benefited from this contemporary research effort is broad and includes virtually all fields where genotoxic stress from oxidative injury, radiation insult, and chemical exposure plays a role in disease initiation, evolution, and treatment. One of the most important areas where these modern studies have had an impact is in the area of cancer biology. Through these studies, we now have a firmer grasp on the molecular response of the cell to DNA injury and how these responses influence mutagenesis, cell cycle progression, DNA damage signaling, neoplastic transformation, and cancer therapy. This book reviews a number of these important topics and will serve to provide a review of key basic and translational aspects of each. We have divided the book into two general categories for the purpose of organization: (1) Molecular Basis of DNA Damage Responses, and (2) Modulation of Radiation Responses – Opportunities for Therapeutic Exploitation. In the first section, Dr. Redon and colleagues provide a review of the histone variant, H2AX, and the role of this protein in the early response of the cell to DNA injury. Dr. Bunz discusses key proteins involved in ATM-dependent signaling of DNA injury and the importance of this protein in controlling DNA damage repair. Similarly, Dr. Lobrich, and colleagues discuss the role of signaling molecules and regulators of a complex signaling system that modulates cellular progression through the cell cycle following radiation injury and the link to repair of DNA damage. Drs. Shen and Falbo provide an overview on the critical role of chromatin structure and

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proteins associated with chromatin remodeling in the response of the cell to DNA damage and in the maintenance of DNA fidelity. Finally, one of the most versatile model systems that has served to elucidate specific DNA damage response pathways at the organismal level is Caenorhabditis elegans. Data derived from this model have been key to our understanding of data generated in higher organisms, including mammalian cells. In their chapter, Drs. Bailly and Gartner review important data on DNA damage signaling, repair, and cell fate generated using this model system. The book then transitions into a discussion of DNA damage response topics that have therapeutic relevance. First, Dr. Hammond and her colleagues discuss cellular and tumor hypoxia, a critically important microenvironmental condition of many human tumors. They review the molecular underpinnings of the hypoxic state, and how these factors alter radiation-induced DNA injury and repair. Collectively, the chapters by Drs. Vischioni, et al., Freytag, et al., Yazlovitskaya and Hallahan, and Dunn, et al., provide in-depth analyses of molecularly based therapies directed toward a variety of cellular targets, all of which have potential to modulate the response of the cell to radiation-induced DNA damage for therapeutic benefit. This includes discussions on (1) inhibitors of specific proteins known to be involved in DNA strand break repair; (2) development of viral and nonviral gene therapy systems that enhance radiation injury through a variety of DNA-directed mechanisms; (3) identification of pro-survival proteins, induced by radiation in tumor vasculature that can serve as molecular targets for radiation-modifying drugs; and (4) the potential for anti-EGFR agents in combination with radiation to substantially improve the therapeutic benefit of radiation therapy. Next, Dr. Roti Roti, et al., provide a comprehensive review of heat effects on signaling proteins, nuclear matrix-associated proteins and chromatin remodeling, and demonstrate how these heat-induced effects result in substantial alterations in cellular response to radiation. Finally, Dr. Drake reviews the evolving knowledge on the immunological effects of radiation, an area of investigation that has great potential to change the future of cancer care. Together, these chapters are a collection of contemporary works on DNA injury and the cellular response associated with it. While not every topic in the DNA damage response domain could be reviewed in a monograph of this size, we do believe the authors have done an outstanding job in providing timely and relevant discussions on their respective subjects, allowing the reader to become more familiar with the field and where the future lies within it. We firmly believe the information contained in this book underscores the significance of DNA damage response in cancer research and the need for continued investigation in this area in order to make substantive progress toward eliminating the suffering associated with cancer. We would fully concur with the opinion expressed by Dr. Bruce Alberts when he said: “If I were the czar of cancer research, I would give a higher priority to recruiting more of our best young scientists to decipher the detailed mechanisms of both apoptosis and DNA repair, and I would give them the resources to do so” (ref: Alberts, B, Science, 320:19, 2008). We hope that his sentiment and this book will

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provide inspiration to those young scientists seeking to work in an area with great potential and importance for our collective future. The editors wish to acknowledge Ms. Debbie Stankowski for her tireless efforts in collecting and organizing all of the manuscripts from our illustrious contributors. Baltimore, MD

Theodore L. DeWeese Marikki Laiho

Contents

Part I Molecular Basis of the DNA Damage Responses 1 H2AX in DNA Damage Response........................................................... Christophe E. Redon, Jennifer S. Dickey, Asako J. Nakamura, Olga A. Martin, and William M. Bonner

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2 DNA Damage Signaling Downstream of ATM...................................... Fred Bunz

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3 Checkpoint Control Following Radiation Exposure............................. Markus Lobrich, Aaron A. Goodarzi, Tom Stiff, and Penny A. Jeggo

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4 Chromatin Responses to DNA Damage................................................. Karina Falbo and Xuetong Shen

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5 Caenorhabditis elegans Radiation Responses........................................ 101 Aymeric Bailly and Anton Gartner Part II Modulation of Radiation Responses: Opportunities for Therapeutic Exploitation 6 Hypoxia and Modulation of Cellular Radiation Response.................. 127 Ester M. Hammond, Monica Olcina, and Amato J. Giaccia 7 Inhibitors of DNA Repair and Response to Ionising Radiation................................................................................................... 143 Barbara Vischioni, Nils H. Nicolay, Ricky A. Sharma, and Thomas Helleday 8 Gene Therapy and Radiation.................................................................. 173 Svend O. Freytag, Kenneth N. Barton, Farzan Siddiqui, Mohamed Elshaikh, Hans Stricker, and Benjamin Movsas ix

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9 Molecular Targeted Drug Delivery Radiotherapy................................ 187 Eugenia M. Yazlovitskaya and Dennis E. Hallahan 10 EGFR Signaling and Radiation.............................................................. 201 Emily F. Dunn, Shyhmin Huang, and Paul M. Harari 11 Thermal Modulation of Radiation-Induced DNA Damage Responses................................................................................... 227 Joseph L. Roti Roti, Robert P. VanderWaal, and Andrei Laszlo 12 Radiation-Induced Immune Modulation............................................... 251 Charles G. Drake Index.................................................................................................................. 265

Contributors

Aymeric Bailly Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK Kenneth N. Barton Department of Radiation Oncology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA William M. Bonner Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892, USA Fred Bunz Associate Professor, Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, David H. Koch Cancer Research Building (CRB2), Room 453, 1550 Orleans Street, CRB II, Room 462, Baltimore, MD 21231, USA Jennifer S. Dickey Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Charles G. Drake Johns Hopkins Kimmel Cancer Center, 1650 Orleans Street, Baltimore, MD 21231, USA Emily F. Dunn Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, K4/332, 600 Highland Avenue, Madison, WI 53792, USA Mohamed Elshaikh Department of Radiation Oncology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA xi

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Contributors

Karina Falbo Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, MDA SP/RD, 1808 Park Road 1-C, Box 389 Smithville, TX 78957, USA Svend O. Freytag Department of Radiation Oncology, Henry Ford Health System, One Ford Place, 5D, Detroit, MI 48202, USA Anton Gartner Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK Amato J. Giaccia Department of Radiation Oncology, Center for Clinical Sciences Research, Stanford University, Stanford, CA 94303-5152, USA Aaron A. Goodarzi Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RQ, UK Dennis E. Hallahan Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park Avenue, Suite 200, St. Louis, MO 63130, USA Ester M. Hammond The Cancer Research UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Churchill Hospital, Oxford OX3 7DQ, UK Paul M. Harari Department of Human Oncology, University of Wisconsin, 600 Highland Avenue, K4/336, Madison, WI 53792, USA Thomas Helleday Cancer Research UK-Medical Research Council, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, OX3 7DQ, UK and Department of Genetics Microbiology and Toxicology Stockholm University, Stockholm S-106 91, Sweden Shyhmin Huang Department of Human Oncology, University of Wisconsin, 600 Highland Avenue, K4/336, Madison, WI 53792, USA Penny A. Jeggo Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RQ, UK

Contributors

Andrei Laszlo Radiation and Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA Markus Lobrich Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt 64287, Hesse, Germany Olga A. Martin Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Benjamin Movsas Department of Radiation Oncology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA Asako J. Nakamura Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Nils H. Nicolay Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK Monica Olcina The Cancer Research UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Churchill Hospital, Oxford OX3 7DQ, UK Christophe E. Redon Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Joseph L. Roti Roti Radiation and Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA Ricky A. Sharma Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK Xuetong Shen Department of Carcinogenesis, The University of Texas, MD Anderson Cancer Center, MDA SP/RD, Unit 116, 1808 Park Road 1-C, Box 389, Smithville, TX 78957, USA

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Contributors

Farzan Siddiqui Department of Radiation Oncology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA Hans Stricker Department of Radiation Oncology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA Tom Stiff Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RQ, UK Robert P. VanderWaal Radiation and Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA Barbara Vischioni Division of Radiation Oncology, European Institute of Oncology and University of Milan, Milan, Italy Eugenia M. Yazlovitskaya Department of Radiation Oncology, Vanderbilt University School of Medicine, B-902 Vanderbilt Clinic, 1301 22nd Ave South, Nashville, TN 37232-5671, USA

Part I Molecular Basis of the DNA Damage Responses

Chapter 1

H2AX in DNA Damage Response Christophe E. Redon, Jennifer S. Dickey, Asako J. Nakamura, Olga A. Martin, and William M. Bonner

Abstract Histone H2AX is a tumor suppressor, helping to preserve genome integrity. It does this by becoming massively and quickly phosphorylated at the sites of nascent DNA double-strand breaks (DSBs) in chromatin. In this chapter, we discuss the state of current knowledge concerning the various aspects of H2AX metabolism, including DSB formation, H2AX phosphorylation to form foci, foci size, and foci stoichiometry with DSBs. While H2AX is essential for efficient DSB repair, it is not essential for basic DSB repair. Mice lacking H2AX are viable under sterile conditions. However, H2AX plays an essential role in two processes necessary for long-term animal survival: immune system development and male fertility. We discuss the phosphorylation of H2AX during telomere dysfunction, interrupted replication/transcription, virus infection, and apoptosis. Phosphorylated H2AX foci serve as platforms for the recruitment of DNA repair and chromatin remodeling factors as well as factors involved in the cell-cycle checkpoint. Phosphorylated H2AX foci are also useful to detect DSBs related to cancer, senescence, and the radiation-induced bystander effect. We conclude by discussing clinical applications which are also coming to the forefront. These include applications in radiation biodosimetry, individual radiosensitivity, efficiency of chemotherapeutic agents to damage cancer and normal cells, and the evaluation of environmental toxins. Keywords g-H2AX foci • Double-strand break repair • Ionizing radiation • Cancer • Clinical applications • Environmental toxins

W.M. Bonner (*) Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_1, © Springer Science+Business Media, LLC 2011

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1.1 Introduction Damage to cellular constituents including DNA is a routine risk of cellular existence, and cells maintain an impressive array of pathways dedicated to repairing DNA lesions and hopefully restoring the damaged DNA to its original sequence. While most single-stranded lesions are easily repaired, DNA doublestrand breaks (DSBs) present special challenges. DSBs compromise the redundancy built into the DNA molecule so that restoring the DNA to its original sequence or even to accurately rejoin the broken fragment is not certain. Also, the broken chromatin ends may rejoin with fragments other than their original partners, leading to chromosomal rearrangements. In addition, if DSBs persist into mitosis, fragments of chromatids may be lost during anaphase, leading to an unbalanced genome. The DNA DSB, therefore, is considered one of the most serious types of DNA damage. One mechanism that has evolved in eukaryotes involves the formation of foci at DSB sites, which serve to concentrate many molecules of various types of proteins involved in DNA repair, chromatin remodeling, and cell-cycle control (McKinnon and Caldecott 2007; Bonner et al. 2008). The foundation of these foci involves the phosphorylation of a histone H2A isoform, H2AX, on a conserved C-terminal sequence centered on an omega-4-serine residue (Rogakou et al. 1998). Upon formation of a DSB, many H2AX molecules, hundreds to thousands in mammals and dozens in budding yeast, become phosphorylated within minutes in the chromatin flanking the DSB site. H2AX phosphorylated on the omega-4-serine is named g-H2AX (Rogakou et al. 1998, 1999; Bonner et al. 2008). In this chapter, we discuss the various aspects of g-H2AX involvement in DNA damage repair and possible applications of the response for human health.

1.2 DSB Formation and H2AX Phosphorylation The genetic information of eukaryotic cells is maintained on DNA fibers, which are organized and compacted into nucleosomes, the fundamental chromatin subunit (Campos and Reinberg 2009) (Fig. 1.1a left panel). In humans, this compaction enables ~2 m of DNA fibers to fit inside a 0.01 mm diameter nucleus. The nucleosome comprises 145 bp DNA and a core histone octamer containing two molecules of each of the four core histone species, H2A, H2B, H3, and H4. Each nucleosome is separated by a minimum of 20 bp of DNA (linker DNA) that is associated with histone H1 (Redon et al. 2002; Campos and Reinberg 2009). H2AX accounts for about 10% of the total H2A protein in normal human fibroblasts (Rogakou et al. 1998). Besides phosphorylation of H2AX on omega-4 serine, it contains two more known phosphorylation sites, an N-terminal serine and a C-terminal tyrosine (Pantazis and Bonner 1981; Cook et al. 2009; Krishnan et al. 2009). The designation g-H2AX is independent of the phosphorylation state of these other two sites.

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As opposed to most core histones that are synthesized during S phase, H2AX synthesis is both DNA replication-dependent and -independent (Wu et al. 1982). There is a single H2AX gene in mammals, which is structured like replication-linked histone genes. The H2AX gene lacks introns and contains a stem–loop motif just downstream of the translation termination site, which serves as a transcription termination site; however, there is also a considerable amount of read-through transcription to a polyA site about 1 Kb further downstream. The result is a protein synthesized at a basal level throughout the cell cycle and in quiescent cells with a peak during S-phase (Wu et al. 1983). The cell-cycle-independent synthesis of H2AX may serve its function in DNA repair as DNA lesions can happen in any stage of the cell cycle. Thus, newly synthesized H2AX would be available as part of the cellular DNA damage response.

1.2.1 Characteristics of g -H2AX Focal Formation and Removal H2AX, in common with the other histone species, is modified posttranslationally by acetylation, biotinylation, methylation, phosphorylation, and ubiquitylation (Pantazis and Bonner 1981; Redon et al. 2002; Chew et al. 2006; Ikura et al. 2007; Pinto and Flaus 2010). Most of these modifications happen on both N-terminal and C-terminal tails and have specific functions in the cell life such as DNA metabolism, DNA condensation, or DNA damage repair. While H2AX and other H2A species share highly conserved core structure, H2AX also contains a unique C-terminal omega-4-serine residue, also highly conserved through evolution (Redon et al. 2002; Martin et al. 2003; Dickey et al. 2009a) (Fig. 1.1a right panel). However, there is a linker sequence connecting these two conserved regions, which does not appear to be conserved either in sequence or length. The variability of the linker length may be related to the variations in internucleosomal lengths of organisms through evolution (Pinto and Flaus 2010). Three related PIK-kinases, ATM, ATR, and DNA-PK, are primarily responsible for H2AX phosphorylation on the omega-4-serine (Bonner et al. 2008). An anti-g-H2AX antibody raised to the H2AX phosphorylated C-terminal peptide CKATQAS(PO4)QEY in humans revealed that the g-H2AX molecules are localized at DNA DSB sites (Rogakou et al. 1999).

1.2.1.1 Size of g -H2AX Foci When the maximal extent of H2AX phosphorylation is measured by 2D gel electrophoresis on cell types with different fractions of H2AX relative to total H2A, it is the fraction of phosphorylated H2AX relative to total H2AX that remains relatively constant regardless of the relative H2AX content, from 2% in lymphocytes and HeLa to 20% in SF268 (Rogakou et al. 1998). This fraction is ~0.03% g-H2AX per DSB (Redon et al. 2003). If H2AX is randomly distributed in the chromatin, 0.03%

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corresponds to ~2 Mbp in mammals. In haploid yeast, which contain 1/400th the amount of the mammalian DNA complement, and in which the H2AX homolog comprises ~95% of the total H2A, stoichiometric measurements also result in a value ~0.03% of the H2A(X) phosphorylated per DSB. In contrast to these stoichiometric determinations, immunocytochemistry suggests that g-H2AX molecules cover much more than 2 Mbp DNA (Fig. 1.1b). Measurements in muntjac and human mitotic cells have revealed that g-H2AX foci formed postirradiation cover ~20 Mbp or more of chromatin (Rogakou et al. 1999). The two different estimates of focus size from stoichiometry vs. immunocytochemistry, 2 vs. 20 Mbp, are consistent with a model in which only ~10% of the H2AX is phosphorylated at any

Fig. 1.1 H2AX and g-H2AX foci. (a) Left panel: H2AX is an element of the nucleosome, the fundamental repeating unit of chromatin. The nucleosome core particles consist of approximately 147-bp DNA wrapped around a histone octamer containing two of each core histones H2A, H2B, H3, and H4 and connected by a “linker DNA” of 20 nucleotides minimum length. Linker histone (H1 and its isoforms), binding the “linker DNA,” sits at the top of the nucleosome at the DNA entry and exit points (to simplify, H1 tails are not represented). Because H2AX represents 2–20% of H2A isoforms, a nucleosome could in principle contain either H2A or H2AX or both H2A and H2AX polypeptides. Both H2A and H2AX are composed of a N-terminal tail and a central globular domain containing the lysine 119, which is ubiquitylated during the processes of DNA repair and histone exchange. H2AX contains an exclusive C-terminal tail, consisting of two evolutionarily conserved motifs (light gray and black) separated by a linker varying in sequence and length between animal and/or plant species (dark gray) (right panel). H2AX C-terminal conserved motif (in black) contains the omega-4 serine that is phosphorylated upon DNA DSB formation (asterisk) to form g-H2AX. (b) Model for g-H2AX focus in mammal and yeast. Upper panel: Stoichiometric studies show that ~2,000 H2AX molecules are phosphorylated at a DSB site in human and ~50 in yeast (red bars). However, microscopy and chromatin immunoprecipitation have shown that the g-H2AX molecules are distributed over a substantially larger region than expected. These observations suggest that ~10% of H2AX in the focus are phosphorylated at any time (green bars). Lower panel: g-H2AX formation in yeast and human lymphocytes 30 min after 0.4 and 200 Gy, respectively. Yeasts insets were approximately scaled to lymphocytes to show the relative g-H2AX focus sizes between species (left image). The right image is a enlargement of the inset from the left image. (c) Schematic representation of the g-H2AX foci distribution being dependent on the nature of irradiation. While g-H2AX foci are spread in cell nuclei irradiated with low-LET (X-rays, g-rays), g-H2AX foci are spaced closely together along the particle track in nuclei after high-LET irradiation. The track is a direct visualization of the ionizing radiation track led by a particle traveling through a cell nucleus. (d) Heterochromatin is refractory for g-H2AX foci formation. Lymphocytes were irradiated with 6 Gy of g-irradiation and harvested 5 min later. The left image shows a representative single optical slice of g-H2AX foci distribution in lymphocyte after g-irradiation, while the g-H2AX signal was subtracted in the middle image. Heterochromatin masses are represented by strong PI staining. To better visualize the position of g-H2AX foci in the nucleus, g-H2AX were replaced with dotted line (left image). Note that g-H2AX foci preferentially localize in the low PI staining regions (euchromatin) and/or at the border of heterochromatin masses. (e) Are all DSBs labeled by g-H2AX? g-H2AX detection in blood white cells 30 min after 0.6 Gy g-irradiation of blood sample. In opposite to lymphocytes (Ly.) where cells show a robust g-H2AX foci formation, most neutrophils (Ne.) are deprived of g-H2AX staining. This observation could lead to consider that in some cell types, DSB formation does not lead to g-H2AX focus formation. (f) g-H2AX focus spreading on Muntiacus muntjak mitotic chromosomes at the indicated time after 0.6 Gy-g-IR. (Part F modified with permission from Rogakou et al. (1999), The Rockfeller University Press). g-H2AX: green; DNA: red

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one time in the focal region flanking the DSB site. A similar situation exists in budding yeast (Shroff et al. 2004). Thus, each g-H2AX focus appears to subsume very similar fractions of the genome in different species (Fig. 1.1b). 1.2.1.2 DSB/g -H2AX Stoichiometry One fundamental question about using g-H2AX as a marker for DSB induction is whether all DSBs formed by irradiation result in g-H2AX focal formation. Specifically, how well do DSB numbers calculated from enumerating g-H2AX foci correlate with those from pulsed-field gel electrophoresis measurements (PFGE). Estimates of DSB numbers from PFGE have varied in the range 3.6–9.7 DSBs per 1,000 Mbp per Gy (Whitaker et al. 1995), which translates to 23–62 per G1 human cell (6,400 Mbp) per Gy, but most values clustered in the 30–40 DSBs per G1 human cell (6,400 Mbp) per Gy (Lobrich et al. 1994, 1995; Cedervall et al. 1995). Variations in this number can result from the cell type, the oxygen content of the cell and tissue, and the type of radiation (low LET vs. high LET). Rothkamm and Lobrich (2003) investigated the induction of DSBs in primary human fibroblasts in the G1 phase of the cell cycle by examining both g-H2AX formation and by PFGE. An average of 36 g-H2AX foci per Gy per cell was visible after 3 min irradiation using doses of 0.2 or 2 Gy, while PFGE studies performed in the dose range between 10 and 80 Gy gave 39 DSBs per Gy per cell. Thus, their study presented the evidence that the numbers of g-H2AX foci are almost the same as the number of DSBs. 1.2.1.3 Spatial Distribution of g -H2AX Foci The pattern of g-H2AX foci observed in nuclei after radiation exposure is not uniform. First, it is dependent on the type of radiation. Several studies have shown that exposure of cells to high-LET particles is more damaging than to low-LET radiation (photons, X-rays/g-rays) (Goodhead and Nikjoo 1989; Cucinotta et al. 2000, 2003). Immunofluorescent detection of g-H2AX reveals that the foci appear to be homogeneously distributed throughout the nuclei following low-LET irradiation (Redon et al. 2009). In contrast, high-LET irradiation produces streaks of g-H2AX foci along the tracks of individual particle traversals (Fig. 1.1c) (Desai et al. 2005). The repair of DSBs is much slower when induced by high-LET than by low-LET (Jenner et al. 1993), reflecting the complexity of DSBs induced by such radiation. Immunofluorescence of g-H2AX confirmed observations made previously with pulsed-field electrophoresis by showing a slower disappearance of g-H2AX foci after high-LET than after low-LET irradiation (Leatherbarrow et al. 2006). Second, g-H2AX focal formation in nuclei is dependent on the level of chromatin compaction, regardless of the radiation type. Several studies in both yeast and mammalian cells have shown that H2AX is refractory to phosphorylation in compacted chromatin

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(heterochromatin), occurring preferentially in open chromatin (euchromatin) (Cowell et al. 2007; Kim et al. 2007; Goodarzi et al. 2009). Thus g-H2AX focal formation is observed more often in euchromatin and at the euchromatin/heterochromatin border than in heterochromatin masses (Fig. 1.1d). The repair of these DSBs linked to heterochromatin regions (10–25% of total DSBs) requires ATM/ KAP-1 signaling for repair (Goodarzi et al. 2008). Third, not all cell types respond similarly with g-H2AX focal formation. Although comparison of irradiation-induced H2AX foci in different animal tissues, cell cultures, and primary cells from different individuals indicate a high degree of the assay reproducibility and it’s main dependence on the dose rather than cell type (Martin et al. 2004; Rube et al. 2008; Redon et al. 2009), there are some exceptions. Cellular DNA content needs to be taken into account. In normal human lymphocytes, an average of 15 g-H2AX foci per Gy are formed (Redon et al. 2009), which is low compared to estimates usually reported for cultured cells (Yasui 2004; Kato et al. 2006; Leatherbarrow et al. 2006). However, G0 lymphocytes also possess less DNA per cell on average than do proliferating cells. Kato et al. (2008) showed that twice as many g-H2AX foci are observed in metaphase cells relative to G1 cells at any given dose. In contrast to lymphocytes, neutrophils exhibit a poor g-H2AX response, showing few or no foci in response to irradiation (Fig. 1.1e). This observation correlates with the findings that unlike lymphocytes, mature neutrophils are unable to repair DNA breaks induced by g-irradiation, perhaps due to their lack of proteins involved in DNA repair (Terai et al. 1991; Ajmani et al. 1995; Kurosawa et al. 2003). Since the postmitotic life span of neutrophils is ~2 days, DNA repair and genomic integrity may not be a priority. However, together, these results indicate that if a cell is capable of responding to DNA damage, then most if not all DSBs induce g-H2AX focus formation. 1.2.1.4 Temporal Distribution of g -H2AX Foci Mass staining and immunocytochemistry both show an increase in g-H2AX amounts relative to radiation dose, and both show a rapid increase in g-H2AX levels for 30 min after exposure followed by a slower decline (Rogakou et al. 1999) (Fig. 1.1f). Short-term analysis of fixed cells reveals the presence of radiation-induced g-H2AX foci in cells 1–2 min after irradiation, and 10 min postexposure, the foci are usually well formed and contain DSB repair factors such as 53BP1, MRE11, and RAD50, indicating that active repair is in progress (Paull et al. 2000; Martin et al. 2008). Numbers of irradiation-induced foci decrease as DSBs are rejoined. The majority of foci disappear by 8 h postirradiation (Rogakou et al. 1999; Martin et al. 2002, 2004; Cucinotta et al. 2008; Redon et al. 2009). However, a substantial number of g-H2AX foci are detectable from 48 h to 7 days after radiation exposures greater than 1 Gy (Redon et al. 2009; Bhogal et al. 2010). It has been suggested that these residual g-H2AX foci may be a critical factor determining cell survival (Banath et al. 2010). While their exact properties are obscure, these residual irradiation-induced foci can be attributed to incomplete or stalled repair of more complex DSB lesion,

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faulty rejoining of DSBs, lethal DNA lesions, persistent chromatin alterations, apoptosis, activity of several kinases and phosphatases, and checkpoint signaling (Goodhead 1994; Nikjoo et al. 2001; Banath et al. 2010).

1.2.1.5 Removal of g -H2AX Foci The mechanism of g-H2AX removal after DSB repair is still unclear. Several protein species including the PP2A and the PP4 family of phosphatases and WIP1 are involved in g-H2AX dephosphorylation (Chowdhury et al. 2005, 2008; Keogh et al. 2006; Macurek et al. 2010). Chromatin remodeling complexes such as TIP60 in human cells and INO80-C and SWR-C in yeast cells also contribute to g-H2AX removal from chromatin by histone exchange (Thiriet and Hayes 2005). The association between TIP60 and UBC13 is enhanced after DNA damage and correlates with H2AX acetylation on lysine 5, which is necessary for H2AX ubiquitylation by UBC13 and release from chromatin (Kusch et al. 2004; Ikura et al. 2007). It is suggested that both direct dephosphorylation of g-H2AX and its removal by histone exchange occurs simultaneously distant from the DSB site and near the DSB site, respectively (Chowdhury et al. 2005). Histone removal during repair would allow some specific factors to access and to rejoin DNA directly at the DSB site. This observation is correlated with the finding that g-H2AX loss is significant at the break site where DNA becomes single stranded by resection (Shroff et al. 2004). Nucleosome-displaced g-H2AX could also be dephosphorylated by phosphatase(s). Thus, in budding yeast Pph3 is thought to dephosphorylate g-H2AX only after its removal from chromatin (Keogh et al. 2006).

1.2.1.6 Changes in g -H2AX Kinetics DSB repair deficiencies during senescence and aging and indirect cellular responses such as bystander effects can compromise typical g-H2AX phosphorylation/dephosphorylation kinetics. For example, radiation-induced g-H2AX foci and DSB repair complexes form substantially more slowly in cells from older healthy donors and even more slowly in cells from Werner syndrome (premature aging) patients. Cells from younger Werner syndrome patients recruit DNA repair proteins to g-H2AX foci at rates similar to older healthy donors (Martin et al. 2008). This suggests that an ageassociated decline in the integrity of the genome may be due to the decreased speed with which aging cells can assemble DNA repair machinery at a damage site. One possible consequence of slower focal formation is incorrect repairs, producing cells more prone to cancer. DNA repair and maintenance protein deficiencies also lead to altered g-H2AX responses including delayed repair kinetics and/or higher g-H2AX focal formation (Smilenov et al. 2005; Lavin 2008; Rube et al. 2008; Hamilton 2009). These altered focal kinetics may form the basis for clinical or environmental applications of the g-H2AX assay and are discussed below.

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1.2.2 g -H2AX Formation as an Essential Step in Biological Processes H2AX is phosphorylated not only in response to DNA damage induction by external agents but also in response to programmed biological process. In fact, H2AX-null mice live normally in a germ-free environment, and while they are hypersensitive to irradiation, there are two other defects preventing them from surviving in the wild (Celeste et al. 2002).

1.2.2.1 Immune System Development H2AX-null mice are incapable of performing class switch recombination, leading to compromised immunity. In addition, these mice exhibit lower numbers of lymphocytes that have undergone V(D)J recombination (Celeste et al. 2002). Since these breaks are programmed in immune system development, their presence would not be expected to be stressful, but they do make the cell more susceptible to chromosome translocations and B-cell malignancies when incorrectly performed.

1.2.2.2 Male Fertility Male H2AX-null mice are sterile (Celeste et al. 2002). Meiosis is characterized by the formation of programmed DSBs catalyzed by SPO11, which triggers the initiation of homologous recombination (Li and Ma 2006). During homologous recombination in meiosis I, g-H2AX is present in the chromatin and disappears as the homologous chromosomes pair in late pachytene (Celeste et al. 2002). It is important to note that H2AX-null male mice show the normal appearance of SPO11-dependent RAD51 foci. In contrast, female H2AX-null mice remain fertile. This indicates that the deficiency is male-specific and that H2AX is not essential for homologous recombination during meiosis I. SPO11-dependent g-H2AX foci disappear as the homologous chromosomes pair in late pachytene (Bellani et al. 2005). However, the X-Y chromosome pair retains g-H2AX staining even after they have condensed to form the XY body (also known as the sex body). In contrast to SPO11-dependent g-H2AX formation by ATM, XY bodyinduced phosphorylation of H2AX is regulated by ATR (Bellani et al. 2005). In H2AX-null male mice, there is a higher incidence of the X and Y chromosomes failing to pair during meiosis I, and often these two chromosomes are found distant from each other in pachytene spermatocytes (Celeste et al. 2002). The unique feature of the X and Y chromosomes is that they share a rather short pseudoautosomal region between them, which is used for pairing. Apparently, H2AX makes the pairing process more efficient, which is critical only for the X-Y pairing. Thus, it is not known whether g-H2AX is being induced by a DSB somewhere in the XY body or whether in this case, g-H2AX may be responding to a different stimulus.

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1.2.3 g -H2AX Formation in Other Biological Processes 1.2.3.1 Telomere Dysfunction Telomeres, the repetitive DNA sequence motifs and specialized proteins that cap the ends of linear chromosomes, are essential for chromosomal stability. However, since the DNA polymerase cannot duplicate the DNA to the ends of the double helix during replication, telomeric DNA consequently becomes shorter with each cell division in human somatic cells (de Lange 2004). After many cell divisions, the telomeric DNA becomes too short to bind the basic capping structure and becomes an eroded telomere. The eroded telomere structure is a type of double-stranded DNA damage, which also activates g-H2AX focus formation (Takai et al. 2003). In somatic cells, which lack telomerase, the enzyme that adds telomeric DNA repeats, eroded telomeres persistently induce formation of g-H2AX foci and may be highly susceptible to chromosomal rearrangements, similar to more canonical DSBs.

1.2.3.2 Replication/Transcription Accidental DSBs can be generated from DNA single-stranded breaks (SSBs) during replication and/or transcription. DNA SSBs are produced by a variety of exogenous and endogenous sources including radiation, ROS, chemicals, defective DNA repair processes, and inhibited enzymes such as DNA topoisomerase I (top1) or topoisomerase II (top2) (Bonner et al. 2008). Top1 is a ubiquitous enzyme that removes DNA supercoiling by producing top1 cleavage complexes. The cleavage complexes are generally widely distributed throughout the genome and are transient. However, when the rapid resealing of the TOP1 cleavage complex is inhibited by common DNA base alterations (abasic sites, mismatches, oxidized bases, carcinogenic adducts, and UV lesions) or by the top1 inhibitors, DNA polymerase (or RNA polymerase II during transcription) is stalled at the site of the lesion (Pommier et al. 2006). This results in the formation of a DNA DSB and the appearance of a g-H2AX focus. Likewise, the stabilization of top2-DNA covalent complexes by a top2 poison such as etoposide or doxorubicin also may generate DNA DSBs (Banath and Olive 2003).

1.2.3.3 Virus Infection g-H2AX formation by virus have been reported and is due to both viral DNA integration as well as the hijacking of cellular processes involved in DNA metabolism by virus oncoproteins (Duensing and Munger 2002; Daniel et al. 2004; Xie and Scully 2007). Viral infection can create DNA lesions in the host cell, which activate the DNA damage response (DDR) pathway. H2AX is phosphorylated at the sites of retroviral infection, but g-H2AX is not required for the repair itself (resealing)

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of these integration sites, supporting the notion that H2AX would be involved in anchoring broken DNA together (Daniel et al. 2004). The murine g-herpesvirus 68 (gHV68) actively induces g-H2AX through expression of the viral kinase, orf36, which plays a vital role for viral replication in infected animals (Tarakanova et al. 2007). The role of H2AX for g-HV68 infection is shown by the fact that, 1 week postinfection, gHV68 amounts in H2AX−/− macrophages were almost 100-fold lower than in H2AX+/+ hosts. 1.2.3.4 Apoptosis Apoptosis is a tightly regulated multistep pathway that is responsible for cell death during development as well as in response to cellular stress. Apoptosis leads to a variety of morphological changes in a cell, including loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and DNA fragmentation. H2AX phosphorylation occurs during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments but before the appearance of internucleosomal DNA fragments (Rogakou et al. 2000). g-H2AX formation is primarily due to the action of DNA-PK and JNK-1 kinases (Lu et al. 2006; Mukherjee et al. 2006; Sluss and Davis 2006). During apoptosis, g-H2AX phosphorylation is usually robust, showing a pan-nuclear staining or an “apoptotic g-H2AX ring” staining (Mukherjee et al. 2006; Solier and Pommier 2009). In addition to phosphorylation at S139, the phosphorylation of another H2AX C-terminal residue, tyrosine 142, has a role in apoptosis (Cook et al. 2009; Stucki 2009) (Fig. 1.2a). This phosphorylation, generated by the WSTF kinase, is constitutive, and dephosphorylation by the EYA1/3 phosphatases occurs after DNA damage. Tyrosine 142 dephosphorylation is required for proper DNA repair by allowing g-H2AX formation and its binding to MDC1, both mechanisms indispensable for a correct DNA damage response. If for some reason after DNA damage, tyrosine 142 dephosphorylation is aborted, the cell response will change to apoptosis.

1.3 g -H2AX in DNA DSB Repair H2AX is a multirole player in the DNA damage response. First, H2AX phosphorylation creates a zone around the DSB site that facilitates the concentration of DNA repair and signaling proteins that will participate in DNA resection and cell-cycle checkpoint (Celeste et al. 2002; Bonner et al. 2008). Second, g-H2AX recruits cohesins to promote sister chromatid-dependent recombinational repair (Unal et al. 2004; Xie et al. 2004). Third, g-H2AX participates directly, or by recruiting other factors, to chromatin remodeling to support DSB repair (van Attikum and Gasser 2005). Finally, preventing the dissociation of broken ends is thought to be another task of g-H2AX (Bassing and Alt 2004).

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Fig. 1.2 g-H2AX formation and role in recruitments of DNA repair proteins. (a) H2AX in the crossroad between cell survival and cell death in response to DNA damage. In addition to the phosphorylation occurring at serine 139 in response to DNA damage, the phosphorylation of another H2AX C-terminal residue, tyrosine 142, modulates apoptosis. WSTF phosphorylates Tyr 142 constitutively, while dephosphorylation by EYA1/3 occurs after DNA damage. During the DNA repair response, MDC1 binds to phosphoserine 139, binding that requires dephosphorylation of phosphotyrosine 142. In the case of the programmed cell death, the phosphotyrosine is not dephosphorylated and results in both the inhibition of repair factors binding to g-H2AX and the recruitment of proapoptotic factors such as the JNK-1 kinase. (b) Several kinases are involved in g-H2AX formation. If they have a redundant role in H2AX modification, their primary role in H2AX phosphorylation is dependent on the origin of the DNA damage. ATM is the primary kinase for H2AX phosphorylation after radiationinduced DNA damage (left) while ATR role is preponderant after replication stress-induced DSBs (middle). Finally both DNA-PK and JNK-1 kinases are involved in H2AX phosphorylation in response to DNA fragmentation in apoptosis (left). (c) g-H2AX plays a role in the

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Fig. 1.2 (continued) recruitment of 53BP1 and BRCA1. (1) Following DSB formation, the initial response consists in the binding of the MRN complex (Mre11-Rad50-Nbs1) to the DSB ends and the ATM enrollment and activation. The outcome of ATM activation results in the subsequent phosphorylation of several DNA repair factors including H2AX phosphorylation on serine 139 (g-H2AX). MDC1 is then recruited at the DNA breaks by binding to g-H2AX via its C-Terminal BRCT domains. The MRN-ATM complex is also recruited at the DSB sites via the binding of NBS1 to the CK2-dependent constitutive phosphorylation of MDC1. The increased ATM enrollment results in a feedback loop with accrued phosphorylation of several DNA repair factors including MDC1, NBS1, and H2AX. (2) The next step in the response to DSB formation consists in chromatin remodeling. ATM phosphorylation of MDC1 allows the recruitment of the RNF8-UBC13 E3 ubiquitin ligase complex and consecutively H2A and H2AX ubiquitylation. H2A and H2AX modification, in turn, mobilize RNF168-UBC13, the other E3 ubiquitin ligase complex, amplifying the ubiquitin–histone conjugates at the DSB sites. (3) Subsequently, the ubiquitylation of both H2A and H2AX provides docking sites for RAP80, a component of the BRCA1-A complex, resulting in BRCA1 recruitment via the adaptor protein ABRA1. De-ubiquitylation of histones can occur by the action of the BRCA1-A complex-associated deubiquitylating enzyme BRCC36. It is not known if at this point BRCA1 itself participates in additional H2A or H2AX ubiquitylation. Finally, histone modifications, including histone ubiquitylation, also cause an alteration in chromatin remodeling, allowing exposure of previously hidden methylated H3 and H4 histone tails. 53BP1 TUDOR domains, then, bind unmasked methylated H3 and H4, allocating 53BP1 recruitment to DSB sites

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Following DSB induction, ATM, ATR, and DNA-PK, three members of the phosphatidylinositol-3 kinase (PIKKs) family with specialized and overlapping functions, are activated and phosphorylate proteins involved in DNA repair including H2AX (Bonner et al. 2008) (Fig. 1.2b). If the three PIKK kinases can potentially phosphorylate H2AX, the kinase responsible for H2AX phosphorylation at a given time is dependent on the manner of damage induction (Bonner et al. 2008). DSBs induced by irradiation primarily recruit ATM, while replication-induced DSBs activate primarily ATR via replication protein A (RPA). DNA-PK, which was shown to be responsible for H2AX phosphorylation during apoptosis, is also responsible for H2AX modification along with both ATM and ATR but less frequently.

1.3.1 g -H2AX and the Recruitment of DNA Repair and Chromatin Remodeling Factors In the case of irradiation-induced DSBs, the ends are recognized by the Mre11Rad50-NBS1 (MRN) complex, leading to ATM dimer recruitment. Autophosphory lation and activation results in ATM monomerization (Durocher and Jackson 2001; Shiloh 2001; Yang et al. 2003). Upon activation, ATM phosphorylates hundreds of H2AX molecules that are necessary for the accumulation and the retention at the DNA break sites of numerous proteins involved in DNA repair and signaling (Shiloh 2003). The g-H2AX formation is the starting point for a hierarchical and timely regulated recruitment cascade leading to the accumulation of mediator proteins, including BRCA1 and 53BP1, at the DSB sites (Fig. 1.2c). BRCA1 and 53BP1 play key roles in genome integrity by modulating checkpoints and DNA repair (Fernandez-Capetillo et al. 2002; FitzGerald et al. 2009; Huen et al. 2010). Though H2AX plays an important role in BRCA1 mobilization in response to DNA damage, the H2AX null mutant exhibits less severe phenotype than the BRCA1 mutant (Celeste et al. 2002). This can be explained by the fact that a portion of BRCA1 is also recruited to the DSB sites by another mechanism, independent of H2AX phosphorylation, through its interaction with MRN (Greenberg 2008). The fraction of BRCA1 accumulating at the DSB site in a g-H2AX dependent manner is found in the BRCA1-A complex (BRCA1/BARD1/ABRA1/BRCC36/RAP80 and recently discovered MERIT40/BRE/NBA1) (Wang et al. 2009; Solyom et al. 2010). Its recruitment at the DNA break happens in a succession of protein modification and protein–protein recognition steps described below.

1.3.1.1 BRCA1-A Complex The early response to DSB formation consists of the binding of MDC1, another mediator, to g-H2AX through its BRCT domains (Stucki et al. 2005). MDC1 contributes to the propagation of the DNA damage repair signal by recruiting more MRN

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complex (via NBS1) and ATM (Fig. 1.2c panel 1). The MDC1-MRN complex is dependent on a serine phosphorylated in a cluster of conserved repeat motifs by casein kinase 2 (Lukas et al. 2004; Chapman and Jackson 2008). Enhanced recruitment of ATM leads in turn to escalating H2AX phosphorylation and MDC1 recruitment to several megabases around the DSB site. This signal amplification loop also promotes MDC1 phosphorylation by ATM. Phosphorylated MDC1 then interacts with RNF8 (Huen et al. 2007), which, along with the E2 enzyme UBC13, catalyzes the addition of Lys63-linked polyubiquitin chains to both H2A and H2AX (Mailand et al. 2007). Histone ubiquitylation by RNF8/UBC13 triggers the recruitment of the RNF168/UBC13 complex via the C-terminal MIU domain of RNF168 (Doil et al. 2009; Stewart et al. 2009). Mutations in the E3 ubiquitin ligase RNF168 have been recently linked to the DNA repair deficiency disorder RIDDLE syndrome (Stewart 2009). RIDDLE (Radiosensitivity, Immunodeficiency, Dysmorphic features, and learning difficulties) syndrome is a human immunodeficiency disorder associated with defective DSB repair (Stewart et al. 2007). The RNF168/UBC13 complex is then involved in the ubiquitylation amplification of substrates such as H2A and H2AX (Fig. 1.2c panel 2). Ubiquitinated H2A and H2AX provide docking sites for the UIM domains of ubiquitin ligase RAP80, which in turn, recruits ABRA1, a mediator protein that also binds simultaneously BRCA1/BARD1 and BRCC36 (Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007).

1.3.1.2 53BP1 53BP1 accumulation at DSB sites is also dependent on H2AX phosphorylation. The role of 53BP1 in the cell-cycle checkpoint may be explained by its ability to interact with p53 via its BRCT motifs, a phenomenon that would allow its phosphorylation/ activation by ATM at the DSB site (FitzGerald et al. 2009). 53BP1 also contains two tandem Tudor domains that bind methylated histones H3 and H4 with high affinity for H4K20Me20 and that are necessary for its accumulation at DSB sites (FitzGerald et al. 2009). Like BRCA1, 53BP1 accumulation at DSB sites is dependent on RNF8 (Mailand et al. 2007). However, in contrast to BRCA1, the relationship between RNF8 and 53BP1 is not well understood. It is thought that in response to DSB formation, the accumulation of DNA damage response factors and the subsequent histone modifications result in chromatin and nucleosome remodeling, which expose H4 N-terminal tails, revealing H4K20me2 and allowing 53BP1 accumulation (Mailand et al. 2007) (Fig. 1.2c panel 3).

1.3.1.3 Chromatin Remodeling Complexes H2AX phosphorylation is also implicated in stimulating the binding to chromatin or enhancing the activity of chromatin remodeling complexes such as the human histone acetyltransferase, TIP60 complex, and its yeast counterpart the NuA4 complex

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(Thiriet and Hayes 2005; van Attikum and Gasser 2005). These complexes are involved in the DNA damage response by allowing both histone modifications and chromatin relaxation, two mechanisms necessary for further DNA repair factor recruitment and/or histone exchange/removal (Thiriet and Hayes 2005). NuA4 accumulates in the vicinity of the DSB, and genetic evidences indicate that both of INO80-C and SWR-C complexes are involved in DNA damage repair (Thiriet and Hayes 2005). Chromatin remodeling complexes also play a role after DNA is repaired by restoring the chromatin environment to its predamaged condition (i.e., removal of g-H2AX from chromatin after repair, for example) (Thiriet and Hayes 2005). Studies in yeast demonstrated that Arp4, a protein found in the NuA4, INO80-C and SWR-C (Thiriet and Hayes 2005; van Attikum and Gasser 2005) chromatin remodeling complexes, binds to the yeast g-H2AX homolog in vitro (Downs et al. 2004). Morrison et al. (2004) showed that INO80-C can also be recruited to DSB sites by interaction between another one of its components, Nhp10, and the g-H2AX yeast homolog (Morrison et al. 2004). The Arp4 homolog in mammals, Baf53, is found in several chromatin remodeling complexes such as Tip60 and SWI-SNF (Lee et al. 2007). In humans, the SWI/SNF chromatin remodeling complex is targeted to DNA lesions by its interaction with BRIT1/MCPH1 (Peng and Lin 2009a, b; Peng et al. 2009), a protein linked to a disease gene called microcephalin and identified in numerous cancers (Bartek 2006). In addition to its role in chromatin condensation and tumor suppression, BRIT1/MCPH1 participates in the accumulation of numerous proteins at the DSB sites and has been shown to control the G2–M checkpoint, through its ability to regulate Chk1 and BRCA1 transcriptional levels (Lin et al. 2005; Wood et al. 2007). As it was suggested that the C-terminal BRCT domains of BRIT1/MCPH1 interact with g-H2AX (Wood et al. 2007), the recruitment of SWI/SNF at the DSB sites could be dependent on H2AX phosphorylation.

1.3.1.4 Cohesins In mammalian cells, the formation of g-H2AX is important for homologous recombination between sister chromatids (Xie et al. 2004). Decreased intersister chromatid distances after DSB induction suggest that cohesin loading at DSBs elevates sister-chromatid cohesion (Dodson and Morrison 2009) and could facilitate accurate homologous recombination. Cohesins are responsible for chromatid cohesion, holding sister chromatids together from the time they are replicated to the metaphase–anaphase transition (Unal et al. 2004). Following DNA damage formation, cohesins are loaded to the DSB sites in a g-H2AX-dependent manner (Unal et al. 2004).

1.3.2 g -H2AX and the Cell-Cycle Checkpoint Cell-cycle checkpoints halt progression of cells through the replication cycle if damage is present. The G2/M checkpoint prevents cells with DNA damage from passing through mitosis, since chromatid fragments that lack a centromere may not attach to

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the spindle and not be pulled toward a pole (Fig. 1.1f). If DNA damage cannot be fixed, cells undergo programmed cell death in which H2AX also plays a role (Lu et al. 2006; Mukherjee et al. 2006). In mammalian cells, H2AX was shown to be essential for proper functioning of the G2/M checkpoint after low amounts of DNA damage. This observation can be related to the finding that g-H2AX is involved in BRCA1 and 53BP1 recruitment to the break site (as discussed in the previous paragraph). For example, Fernandez-Capetillo et al. (2002) showed that 1 h after cells are exposed to various amounts of ionizing radiation and mitotic cells are isolated, MEFs from control mice exhibit greater than 75% inhibition in passing through mitosis at doses greater than 0.5 Gy. However, H2AX-null MEFs exhibit little if any inhibition at this dose. Both ATM, which phosphorylates H2AX, and 53BP1, whose recruitment is dependent on g-H2AX, are also essential for proper checkpoints after 0.5 Gy. Studies with yeast show that the g-H2AX homolog is important for prolonged G2/M checkpoint arrest in response to radiation (Nakamura et al. 2004). The role of the yeast g-H2AX homolog in the cell-cycle checkpoint is linked to the fact that g-H2AX is necessary for both Crb2 (the yeast 53BP1 homolog) phosphorylation and foci formation.

1.3.3 Complexity of Repair Foci Containing g -H2AX and Other Factors Following DSB and subsequent g-H2AX formation, more than 50 DDR proteins are recruited and/or modified at the break sites. It is unknown if more DDR proteins located in the g-H2AX foci remain to be discovered, and the spatial organization of these proteins in the repair foci is still largely unknown. However, a few studies in both yeast and mammalian cells have examined the structure of g-H2AX foci, and their observations confirm a time-dependent and hierarchical distribution of DDR proteins (Nakamura et al. 2010). Both g-H2AX and MDC1 were found to span large domains flanking DSBs sites (with MDC1 localizing as doublets in foci), while 53BP1 and the components of the MRN complexes were restricted to single regions, shown to correspond to DSB sites in yeast (Shroff et al. 2004). In yeast, deposition of g-H2AX and cohesins following DSB formation was similarly found to span large domains flanking DSB breaks (Unal et al. 2004). DDR protein distribution in g-H2AX foci seems to be related to their function in DNA repair. Thus, MDC1 and perhaps cohesins, which play primary roles in homologous recombination and/or sister chromatid recombination, are distributed over several areas, while 53BP1 and the MRN complex, which have a more implicit role in nonhomologous end joining, are distributed over smaller areas perhaps more central to the break. In addition to their spatial complexity, g-H2AX foci also show a temporal complexity with DDR proteins recruitment exhibiting both dependent and independent behavior during the cell cycle. Thus, g-H2AX foci change their composition through the cell cycle, with 53BP1, NBS1, and MRE11 dissociating from the foci at G2/mitosis to return at early G1, while MDC1 remains at g-H2AX foci through the entire cell cycle (Nelson et al. 2009; Nakamura et al. 2010).

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1.4 g -H2AX as a Marker g-H2AX focal formation in response to DSB induction is a highly amplified process and, as discussed earlier, is capable of detecting individual DSBs. This sensitivity enables researchers to study DNA DSBs at physiological levels of damage. As such, g-H2AX focal formation has the potential to become a useful in vivo biodosimeter for processes that result in DSB formation. This includes not only processes that result in frank DSBs but also those that may indirectly result in DSBs. Some of these are discussed below. In addition, there are several areas in which clinical applications of g-H2AX focal formation are being developed.

1.4.1 Biological Processes 1.4.1.1 Cancer Elevated levels of g-H2AX are present in different human cancer model systems, including cervical cancer cells, melanoma cells, colon carcinomas, fibrosarcoma, osteosarcoma, glioma, and neuroblastoma cells, as well as in tumor biopsies compared to normal primary cells or normal tissues adjacent to tumors (Banath et al. 2004; Warters et al. 2005; Martin and Bonner 2006; Yu et al. 2006). These results suggest that an increased level of DNA damage is a general characteristic of cancer development (Bartkova et al. 2005; Gorgoulis et al. 2005). Moreover, an increase in g-H2AX content has been found in colonocytes from patients with ulcerative colitis that predisposes them to colorectal cancer (Risques et al. 2008). Damaged telomeres make up the majority of endogenous DNA double-strand damage in tumor cells, and the numbers of telomere-associated g-H2AX foci correlate inversely with the cells’ telomerase activity (Nakamura et al. 2009). The origin of nontelomeric g-H2AX foci in these cells remains an open question. These foci may be caused by specific attributes of cancer cells such as excess reactive oxygen production and accumulation of DNA repair and cell-cycle checkpoint defects (Lavin 2008; Hamilton 2009; Martin et al. 2010).

1.4.1.2 Senescence Animals, including humans, age and primary somatic cell lines derived from them undergo only a limited number of divisions. This exhaustion of proliferative potential, called cellular senescence, may protect organisms from cell immortalization and tumorigenesis, thus providing a tumor suppressor function (Prieur and Peeper 2008). Factors thought to play a role in aging at the cellular level include telomere shortening, oxidative stress, accumulating DNA damage, compromised DNA repair

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machinery, and inappropriate gene amplification (Ju et al. 2006; Ozgenc and Loeb 2006; Martin et al. 2008; Zhao et al. 2009; Reddel 2010). It has been shown that g-H2AX foci increase during cellular senescence in culture, whether from proliferation, DNA-damaging agents, or oxidative stress (Nakamura et al. 2008). Likewise, the incidence of foci increases as humans and mice age (Martin et al. 2004, 2008; Hesse et al. 2009). 1.4.1.3 Radiation-Induced Bystander Effect The radiation-induced bystander effect (RIBE) was first described in the early 1990s by Nagasawa and Little when it was noted that when 1% of the cells in a dish were traversed with alpha particles, 30% of the cells displayed increased sister chromatid exchanges (Nagasawa and Little 1992). These results indicated for the first time that cellular damage may be experienced by naïve “bystander” cells when they are in close contact with damaged cells. Since that time, the RIBE has been studied in a number of cellular models as well as in vivo (Mothersill and Seymour 2004; Hei et al. 2008; Prise and O’Sullivan 2009). The RIBE generally manifests itself in the form of DNA damage that leads to various downstream effects. The RIBE has been shown to increase the incidence of apoptosis, point mutations, sister-chromatid exchanges, global genome demethylation, and senescence (Mothersill and Seymour 2004; Hei et al. 2008; Prise and O’Sullivan 2009). As DNA damage, and specifically DNA DSBs, seems to be a precipitating event in many of these manifestations of the RIBE, g-H2AX has been often used as a marker of bystander effect induction and propagation (Zhou et al. 2002; Sokolov et al. 2005, 2007; Martin et al. 2007). Based on these studies, it was found that the DNA damage induced as a result of bystander signaling is distinct in several respects from the DNA damage that might be induced as a result of direct cellular injury (Sokolov et al. 2005; Martin et al. 2007). For instance, bystander-effectinduced DNA damage arises more slowly than that from direct cellular irradiation. When cells are irradiated directly, using either X-rays or alpha particles, DNA DSBs are formed almost immediately. The g-H2AX foci are visible less than 1 min postirradiation and the focal size and incidence peaks by 30 min postirradiation (Bonner et al. 2008). In contrast, it takes several hours for g-H2AX foci to form in bystander cell populations, and focal incidence may peak as late as 8 h postirradiation (Kashino et al. 2004; Sokolov et al. 2005; Martin et al. 2007) (Fig. 1.3a). These kinetic characteristics indicate that a slowly diffusing substance may be contributing to bystander DNA DSB production and that breaks may actually be made indirectly (Kashino et al. 2004; Hei et al. 2008). Likewise, while the g-H2AX foci in directly irradiated cell populations begin to disappear shortly after irradiation as the cellular machinery repairs the breaks, bystander DNA damage can persist for several days (Morgan 2003; Kashino et al. 2004; Sokolov et al. 2005; Martin et al. 2007) (Fig. 1.3a). In some cellular systems, increased DNA damage can be seen in bystander cell populations for as long as

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Fig. 1.3 (a) Upper diagram: Brief description of the bystander effect. When cells are irradiated directly, DSBs are formed immediately and g-H2AX foci appear within minutes. Irradiated cells release factors and ROS that diffuse to unirradiated cells and initiate a delayed response leading to g-H2AX foci formation. g-H2AX foci are represented by green dots. Lower graph: Typical g-H2AX foci kinetics for direct (open circle) and bystander (closed circle) responses to irradiation are shown. Both intensity and timing of the bystander response differ from the direct response. (b) The use of g-H2AX for radiation biodosimetry. Left panel: g-H2AX formation in lymphocytes 30 min after exposure to 0, 0.1, 0.2, 0.6, 1, 1.5 and 3 Gy g-irradiation. Right graph: Linear relationship between the number of g-H2AX foci per cell and the irradiation dose. Red: DNA; Green: g-H2AX

7 days (Martin et al. 2007). These findings indicate either that the DNA damage created by bystander signaling is more difficult to repair than damage induced directly by irradiation or that a feedback mechanism is continually creating new DNA damage long after the original precipitating event is finished. Some of the signaling molecules that have been proposed to function as primary bystander effect mediators, including TGF-b and nitric oxide (NO), induce the transcription of stress response elements that then themselves create increased DNA DSBs (Choy et al. 2007; Shao et al. 2008; Dickey et al. 2009b). For instance, TGF-b has been shown to induce the transcription of nitric oxide synthase (NOS), which leads

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to increased production of NO, and increased DNA DSBs through lipid peroxidation as well as direct DNA damage (Hussain et al. 2003; Zablocka and Janusz 2008). This feedback loop then serves to create DNA DSBs over a longer time than what would be caused by directly damaging the genome with radiation. Other observations show that the bystander effect is a global response that is not restricted to stress-induced irradiation. Ultraviolet exposure, photolytic DNA damage, and exposure to tumorigenic and senescent cells can also cause bystander responses including DNA damage in neighboring cells without the need for any outside damaging stimulus (Dickey et al. 2009b; Pawelec et al. 2009). Identifying the cell populations that are vulnerable to bystander effect signaling, either from radiation or from other forms of stress, remains an active area of investigation. Some research has indicated that actively replicating cells, particularly cells in S-phase, are the most susceptible cells to form g-H2AX foci (BurdakRothkamm et al. 2007; Burdak-Rothkamm et al. 2008; Dickey et al. 2009a, b).

1.4.2 Clinical Applications 1.4.2.1 Biodosimetry and Individual Radiosensitivity Assays predictive of individual radiosensitivity have been the “Holy Grail” of experimental radiation oncologists for decades, but prospects have been limited by the insensitivity of assays, and also the need to use activated and/or transformed lymphocytes. Other issues impeding progress are the cost and labor intensity of clonogenic survival assays, and the delay (8–10 days) in obtaining results to subsequently plan individualized therapy. The literature is replete with failures of predictive assays for both tumors and normal tissues. For example, no correlation has been observed between the radiosensitivity of transformed lymphocytes and primary fibroblasts in radiotherapy patients (Geara et al. 1992). The g-H2AX assay presents a novel approach. Considered to be the most sensitive modern assay for DSB detection (Bonner et al. 2008), it can detect both a single DSB in cell nuclei and the cell responses to radiation doses as low as 1 mGy (Rothkamm and Lobrich 2003). Because of this sensitivity, g-H2AX has been recently identified as a useful biomarker with clinical implications (see below). It is possible to detect irradiation-induced DNA damage in situ in nonactivated human lymphocytes, as well as in tissue biopsies (Qvarnstrom et al. 2004; Lobrich et al. 2005; Redon et al. 2009), hair follicles (unpublished results), or oral cells (Yoon et al. 2009), although in these cells the sensitivity of the assay is generally lower, possibly due to high endogenous g-H2AX level and large number of dying cells. Additionally, radiation dose-dependent responses and persistence of foci make g-H2AX detection a good potential biodosimeter in the event of a major radiation emergency as well as clinical examination or radiotherapy (Olive and Banath 2004; Lobrich et al. 2005; Rothkamm et al. 2007; Geisel et al. 2008; Kuefner et al. 2009; Redon et al. 2009; Bhogal et al. 2010).

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The g-H2AX assay in lymphocytes irradiated in vivo (for CT scan examinations, angiography, and radiotherapy) has been used, showing a good correlation between the g-H2AX focal numbers and the radiation doses received by patients (Qvarnstrom et al. 2004; Lobrich et al. 2005; Sak et al. 2007; Geisel et al. 2008; Kuefner et al. 2009) (Fig. 1.3b). Importantly, the g-H2AX kinetics is similar in human lymphocytes and fibroblasts after ex vivo and in vivo irradiation (Lobrich et al. 2005). The rapid response (maximal number of foci at 30 min postexposure and repair within several hours) gives the base for 1-day assessment of individual sensitivity in a minimally invasive test of primary lymphocytes exposed to ex vivo irradiation, identifying individuals with defective DNA repair. A severely radiosensitive patient with a DSB repair deficiency was identified in a tomography examination study by the g-H2AX focus formation assay and PFGE. That patient had previously shown exceptionally severe side effects after radiotherapy (Lobrich et al. 2005). Thus, feedback on a patient radiosensitivity would allow clinicians to better adjust treatments to individuals, avoiding serious radiotherapy accidents (Abbaszadeh et al. 2009). For this purpose, g-H2AX has been used to identify individuals with ataxia telangiectasia (A-T), a disease predisposing patients to cancer, as well as to identify ataxia telangiectasia heterozygotes, which represent 0.36–1% of the general population (Porcedda et al. 2006, 2008, 2009). The ataxia telangiectasia gene codes for ATM, the kinase responsible for phosphorylation of a broad range of DNA repair factor including H2AX (Shiloh 2003). Preclinical mouse models of radiosensitivity testing allowed researchers to verify different DSB repair deficiencies after whole-body irradiation (DNA-PK, SCID, AT). Even slight impairments caused changes in DSB repair that were detected in both lymphocytes and tissues using the g-H2AX focus formation assay. Thus, future examinations of patient blood ex vivo could give precious information about individual repair capacity (Rube et al. 2008). Finally, the use of g-H2AX for radiation biodosimetry was also valuable to measure daily space radiation exposure in the International Space Station. The study showed that measured doses were similar between biological (g-H2AX) and physical (track and luminescent detectors) dosimetries (0.7 mSv/day vs. 0.5 mSv, respectively) (Ohnishi et al. 2009). 1.4.2.2 Chemotherapy In addition to its growing role in drug development in research laboratories, g-H2AX is increasingly used in clinical trials. Most cancer therapeutic treatments cause DSBs directly or indirectly (Bonner et al. 2008). Since g-H2AX is a robust DSB biomarker, measuring g-H2AX in cells may provide clinicians valuable information about individual patient responses. Numerous clinical trials have measured g-H2AX signals in patients (Bonner et al. 2008). Because collecting blood is relatively noninvasive, this tissue has been the most often used for g-H2AX detection (Lobrich et al. 2005; Rothkamm et al. 2007). However, in contrast to radiotherapy where DSBs are induced regardless of cell

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cycle, analysis of g-H2AX signals in the nondividing lymphocytes during chemotherapy could be challenging as most of the cancer drugs target dividing cells. The use of skin biopsies and plucked hairs, both containing dividing cells, can be used in addition to blood (Qvarnstrom et al. 2004; Fong et al. 2009). However, because of their genetic makeup and/or differences in microenvironments such as vascularization, normal tissues may respond more differently than the targeted tumors. Therefore, g-H2AX detection in tumor biopsies will still be required. In recent years, phase 1 clinical studies have started utilizing the g-H2AX assay. A study using clofarabine (a ribonucleotide reductase and DNA polymerases inhibitor) and cyclophosphamide (an alkylating agent effectively used for treatment of leukemias) for adults with refractory cancers showed increased g-H2AX levels in circulating leukemia cells of 12 of the 13 treated patients (Karp et al. 2007). Another trial confirmed the inhibition of the poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) by the drug olaparib by analyzing g-H2AX in plucked-eyebrow hairs (Fong et al. 2009). Still another study demonstrated the genotoxic activity of the minor groove binding agent SJG-136 by increased g-H2AX levels in both patient lymphocytes and tumor biopsies (Hochhauser et al. 2009). Finally, a trial testing the combination of tipifamid (a farnesyltransferase inhibitor) and etoposide (a topoisomerase II inhibitor) as orally bioavailable regimen for elderly adults diagnosed with myelogenous leukemia used a g-H2AX assay in AML marrow blasts (Karp et al. 2009). This study showed good patient responses to the drug combination demonstrated by H2AX phosphorylation correlating with increased subdiploid DNA content. Quantifying H2AX phosphorylation has also been used for clinical diagnostics. Increased levels of g-H2AX and shorter telomeres were found in colonocytes of patients with ulcerative colitis, a chronic inflammation disease linked to colorectal cancer (Risques et al. 2008). Potentially, g-H2AX assays could be used to prescreen individuals, determining the ones at risk for cancer that would result in preemptive treatments (Martin and Bonner 2006). Other diagnostic tools using g-H2AX revealed its usefulness in the diagnosis of metastatic renal cell carcinoma (Wasco and Pu 2008) and predicting the recurrence of bladder urothelial carcinoma (Cheung et al. 2009). 1.4.2.3 Environmental Toxins The formation of DSBs can be used as a sensor to determine the genotoxicity of compounds/chemicals, occupational activities, and other human behaviors. For example, g-H2AX has been used to determine the DNA-damaging effects of cigarette smoking (Albino et al. 2004; Toyooka and Ibuki 2009), nonthermal microwaves of cell phones (Markova et al. 2005; Belyaev et al. 2009), electromagnetic fields (Luo et al. 2006), dental resin composites (Urcan et al. 2009), crude oil (Ibuki et al. 2007), chromium VI (Ha et al. 2004), actinomycin D (Mischo et al. 2005), norethindrone (Gallmeier et al. 2005), NO-aspirin (Tanaka et al. 2006), carbon

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nanotubes (Zhu et al. 2007), isocyanates (Mishra et al. 2008), and the herbicide paraquat (Kim et al. 2004). Thus, the future use of g-H2AX in routine screens could be valuable to examine the genotoxicity of doubtful agents and/or human activities that, if detected in time, would help reduce preventable human diseases.

1.5 Conclusions H2AX is phosphorylated in response to DNA DSB formation, irrespective of whether those breaks are induced directly by environmental agents or indirectly as a result of programmed and nonprogrammed biological processes. In addition, the amplification of the process makes it applicable for use at physiological levels of DSBs, leading to possible clinical assays. While the phosphorylation of H2AX is understood at the level of the individual protein, how it contributes to foci formation is still unclear. Also, evidence is accumulating that foci have considerable complexity and undergo changes with cell cycle and possibly other states. Understanding the dynamics of foci formation as opposed to repair protein recruitment will help further elucidate how cells deal with DSB damage. Acknowledgements This work was supported by the Intramural Research Program of the National Cancer Institute, NIH.

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Terai C, Wasson DB, Carrera CJ et al (1991) Dependence of cell survival on DNA repair in human mononuclear phagocytes. J Immunol 147:4302–4306 Thiriet C, Hayes JJ (2005) Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair. Mol Cell 18:617–622. Toyooka T, Ibuki Y (2009) Cigarette sidestream smoke induces phosphorylated histone H2AX. Mutat Res 676:34–40 Unal E, Arbel-Eden A, Sattler U et al (2004) DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16:991–1002 Urcan E, Scherthan H, Styllou M et al (2009) Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites. Biomaterials 31:2010–4 van Attikum H, Gasser SM (2005) ATP-dependent chromatin remodeling and DNA double-strand break repair. Cell Cycle 4:1011–1014. Wang B, Hurov K, Hofmann K et al (2009) NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control. Genes Dev 23:729–739 Warters RL, Adamson PJ, Pond CD et al (2005) Melanoma cells express elevated levels of phosphorylated histone H2AX foci. J Invest Dermatol 124:807–817 Wasco MJ, Pu RT (2008) Utility of antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma. Appl Immunohistochem Mol Morphol 16:349–356 Whitaker SJ, Ung YC, McMillan TJ (1995) DNA double-strand break induction and rejoining as determinants of human tumour cell radiosensitivity. A pulsed-field gel electrophoresis study. Int J Radiat Biol 67:7–18 Wood JL, Singh N, Mer G et al (2007) MCPH1 functions in an H2AX-dependent but MDC1independent pathway in response to DNA damage. J Biol Chem 282:35416–35423 Wu RS, Tsai S, Bonner WM (1982) Patterns of histone variant synthesis can distinguish G0 from G1 cells. Cell 31:367–374 Wu RS, Tsai S, Bonner WM (1983) Changes in histone H3 composition and synthesis pattern during lymphocyte activation. Biochemistry 22:3868–3873 Xie A, Puget N, Shim I et al (2004) Control of sister chromatid recombination by histone H2AX. Mol Cell 16:1017–1025 Xie A, Scully R (2007) Hijacking the DNA damage response to enhance viral replication: gammaherpesvirus 68 orf36 phosphorylates histone H2AX. Mol Cell 27:178–179 Yang J, Yu Y, Hamrick HE et al (2003) ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 24:1571–1580 Yasui LS (2004) GammaH2AX foci induced by gamma rays and 125idU decay. Int J Radiat Biol 80:895–903 Yoon AJ, Shen J, Wu HC et al (2009) Expression of activated checkpoint kinase 2 and histone 2AX in exfoliative oral cells after exposure to ionizing radiation. Radiat Res 171:771–775 Yu T, MacPhail SH, Banath JP et al (2006) Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability. DNA Repair (Amst) 5:935–946 Zablocka A, Janusz M (2008) The two faces of reactive oxygen species. Postepy Hig Med Dosw (Online) 62:118–124 Zhao B, Benson EK, Qiao R et al (2009) Cellular senescence and organismal ageing in the absence of p21(CIP1/WAF1) in ku80(−/−) mice. EMBO Rep 10:71–78 Zhou H, Randers-Pehrson G, Suzuki M et al (2002) Genotoxic damage in non-irradiated cells: contribution from the bystander effect. Radiat Prot Dosimetry 99:227–232 Zhu L, Chang DW, Dai L et al (2007) DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells. Nano Lett 7:3592–3597

Chapter 2

DNA Damage Signaling Downstream of ATM Fred Bunz

Abstract ATM is the apical signaling molecule that triggers diverse cellular responses to double-strand DNA breaks. Directly and indirectly, ATM initiates a two-tiered cascade of protein kinase activation, composed of upstream phosphatidylinositol 3-kinase-like kinases, mediator proteins, and checkpoint kinases. Together, these proteins signal a broad network of downstream effectors that modulate virtually every aspect of cell growth and death. This review will focus on the signaling molecules required for the diverse ATM-dependent responses to DNA damage, with an emphasis on the extensively characterized pathways that suppress proliferation and promote DNA repair. Keywords DNA damage • ATM • ATR • Checkpoints • Signaling network

2.1 Introduction Damage to genomic DNA stimulates a profound and functionally diverse cellular response that affects fundamental cellular processes. As described in previous chapters, ATM plays a central role in initiating the collective responses to a particularly lethal form of DNA damage, the double-strand DNA break (DSB). Elegant biochemical experiments have demonstrated the fundamental mechanisms by which ATM kinase activity is activated at DSB sites (Bakkenist and Kastan 2004). The transduction of ATM signals arising from focal sites on damaged chromosomes to the many cellular compartments that mount responses to DSBs will be the focus of this review. F. Bunz (*) Associate Professor, Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, David H. Koch Cancer Research Building (CRB2), Room 453, 1550 Orleans Street, CRB II, Room 462, Baltimore, MD 21231, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_2, © Springer Science+Business Media, LLC 2011

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Fig. 2.1 Conserved DNA damage signaling pathways. The general organization of the two-tiered DNA damage signaling cascade was defined by genetic studies in budding and fission yeasts. Double-strand DNA breaks activate orthologs of ATM, while DNA replication intermediates trigger the activation of orthologs of ATR and its binding partner ATRIP. Mediator proteins facilitate the activation of the checkpoint kinases by the upstream PIKKs. In mammalian cells, a family of BRCT domain-containing proteins appears to provide the functions of the single mediator proteins in the yeasts. Downstream signals converge on cell cycle regulatory proteins including Cdc25 protein phosphatases. Mammalian cells contain additional regulators of cell cycle checkpoints, including p53. Both the PIKKs and the checkpoint kinases have many additional downstream substrates. See text for details

Studies of the DNA damage pathways in evolutionarily divergent yeasts have revealed a two-tiered kinase cascade that is conserved in human cells (Fig. 2.1). DNA lesions directly activate apical serine/threonine protein kinases that are structurally related to the phosphatidylinositol 3-kinase and are accordingly known as the (PI3-kinase-like kinase) PIKK family. Identified as the gene mutated in ataxiatelangiectasia patients in 1995, ATM is one of six human PIKKs (Lavin et al. 2005). A second PIKK known to be functionally conserved in human cells is the ataxia-telangiectasia and Rad3-related (ATR) kinase. In both humans and yeasts, upstream PIKK proteins activated by DNA damage phosphorylate downstream serine/threonine protein kinases known as checkpoint kinases (Bartek and Lukas 2003; McGowan and Russell 2004; Reinhardt and Yaffe 2009). Chk1 and Chk2, the human orthologs of the yeast checkpoint kinases, transduce DNA damage signals from lesions and stalled replication forks to spatially and functionally distinct compartments of the cell (Stracker et al. 2009). An emerging class of phosphoprotein-interacting proteins cumulatively play the role of yeast Rad9, the first identified checkpoint protein, and mediate the activation of the checkpoint kinases by the PIKKs (Mohammad and Yaffe 2009). By illuminating the relationship between PIKKs, mediator proteins and the checkpoint kinases, genetic analyses of yeast have provided the theoretical framework for understanding the functional organization of the DNA damage response signaling pathways in human cells.

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2.2 Ataxia-Telangiectasia and Rad3-Related ATR is a large serine/threonine protein kinase with significant homology to ATM and the other members of the PIKK family (Abraham 2001; Cimprich and Cortez 2008). Both ATM and ATR preferentially phosphorylate serine residues that are followed by glutamine (SQ sites), and therefore target overlapping sets of substrates. However, ATM and ATR respond to distinct stimuli and thus have nonredundant functions. ATM is primarily activated by DSBs. ATR responds to DSBs but is additionally activated by a wide range of DNA lesions and DNA structures caused by environmental and therapeutic agents that inhibit or impede DNA replication, including ultraviolet (UV) radiation, DNA cross-linking agents and antimetabolites that interfere with nucleotide metabolism (Abraham 2001; Osborn et al. 2002). From a genetic perspective, the ATM and ATR signaling pathways exhibit important differences that suggest distinct roles in the cell. Both ATM and Chk2 are encoded by tumor suppressor genes that confer cancer predisposition – most prominently an increased risk of breast cancer – when inactivated by germline mutations. The complete loss of ATM signaling in patients with ataxia-telangiectasia or in ATM-knockout mice largely eliminates the response to DSBs and leads to extreme sensitivity to ionizing radiation (Shiloh 2006) but does not obviously impair unperturbed cell growth. In contrast, both ATR and Chk1 are essential genes that are required for cell viability and proliferation (Liu et al. 2000; Brown and Baltimore 2003). Only rare hypomorphic ATR alleles are tolerated in the germline; it is unclear if these predispose carriers to cancer (O’Driscoll et al. 2004). While ATR and CHK1 mutations have been found in small number of mismatch repair-deficient cancers, these genes do not have the requisite characteristics of established tumor suppressors. Inhibition of ATR activity in the presence of low levels of DNA replication stress causes DNA breaks at defined loci known as fragile sites (Casper et al. 2002). Detected at the cytogenetic scale, fragile sites are thought to represent structurally distinct regions in the genome where DNA replication forks tend to stall. The requirement of ATR for normal cell growth, its activation by DNA replication inhibitors and its role as a suppressor of fragile site expression all support a critical role for ATR in the stabilization of DNA replication forks (Fig. 2.2). Recent studies have determined the fundamental mechanism by which ATR is activated at stalled replication forks (Zou and Elledge 2003; Kumagai and Dunphy 2006; Cimprich and Cortez 2008; Mordes et al. 2008). The molecular machines that replicate genomic DNA include multiprotein DNA polymerase complexes that function to synthesize nascent strands and DNA helicase complexes that unwind the DNA template ahead of the advancing fork. Critical to the activation of ATR is the decoupling of polymerase and helicase activities. When a polymerase complex runs out of nucleotide substrate or if it encounters a DNA lesion, such as a strand break or an adduct, DNA synthesis can stall (Zegerman and Diffley 2009). In this case, the helicase complex can continue unwinding DNA, exposing a stretch of single-stranded DNA that would otherwise be rapidly replicated. The single-stranded DNA at the lagging strand of the stalled fork

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Fig. 2.2 Activation of ATR. (a) Advancing DNA replication forks are driven by the coordinated activities of multiprotein DNA polymerase (Pol) and DNA helicase complexes. (b) DNA polymerases that synthesize the leading and lagging strands may run short of nucleotides, or encounter a DNA lesion that impairs DNA synthesis. In such cases, the polymerase complexes will pause and become uncoupled from the helicase complexes, which continue unwinding DNA ahead of the stalled fork. Regions of single-stranded DNA are rapidly coated with the trimeric replication protein A complex (RPA). (c) ATR is recruited to the RPA-coated single-stranded DNA via association with ATRIP. The Rad9–Rad1–Hus1 (9-1-1) complex is loaded at the single strand–double strand DNA junction by the ATP-dependent Rad17 clamp loader. The TopBP1 protein associates with both the 9-1-1 complex and the ATR–ATRIP complex, which are all required for ATR activation. (d) Activated ATR phosphorylates numerous proteins assembled at the stalled fork (phosphates shown in yellow), which apparently serves to increase fork stability and eventually facilitate resumption of DNA synthesis. Thus activated, ATR also phosphorylates Chk1 and many other downstream substrates

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becomes rapidly coated with the heterotrimeric single-strand DNA-binding protein complex known as replication protein A (RPA) (Fanning et al. 2006). The junction between RPA-coated single-stranded DNA and adjacent double-stranded DNA creates a structure that is recognized by the protein complexes that function to restart stalled forks. The RPA-coated single-strand DNA attracts binding by the ATR-interacting protein (ATRIP), the binding partner of ATR (Cortez et al. 2001). A heterotrimeric ring-like protein complex composed of Rad9-Rad1-Hus, known as the 9-1-1 sliding clamp, is loaded onto the single-strand DNA/double-strand DNA junction by an ATP-dependent clamp loader derived from the replicative replication factor C (RFC) complex (Parrilla-Castellar et al. 2004). The 9-1-1 complex and ATR function together to recruit the topoisomerase-interacting protein TopBP1 (Lee et al. 2007). The binding of ATR–ATRIP to RPA-coated single-strand DNA and the recruitment of TopBP1 are all required for activation of ATR kinase activity at stalled, unwound replication forks (Kumagai and Dunphy 2006; Mordes et al. 2008). ATR is known to phosphorylate several proteins involved in DNA replication, including subunits of RFC and RPA, proteins in the minichromosome maintenance (MCM) complex required for replication initiation and fork progression, and several DNA polymerases (Matsuoka et al. 2007; Cimprich and Cortez 2008). The consequences of individual ATR-dependent phosphorylation events remain poorly understood, but the overall effect of ATR on the replication fork appears to be to stabilize the protein complexes of the replisome and thereby maintain fork integrity and velocity (Cimprich and Cortez 2008; Wilsker et al. 2008). The role of ATR– ATRIP as both a sensor of stalled replication forks and as a stabilizer of the multiprotein complexes at the fork would appear to represent, in essence, a feedback circuit that ensures the efficient replication of the genome.

2.3 The Checkpoint Kinases The human homologs of the yeast checkpoint kinases serve as transducers of the signals generated by upstream PIKKs (Bartek and Lukas 2003). PIKKs are large kinases that, once activated, appear to remain closely located with DNA breaks and stalled replication forks. The smaller, more mobile checkpoint kinases are the mechanism by which signals originating at chromatin are transmitted throughout the cell to spatially and functionally diverse effectors (Smits 2006; Stracker et al. 2009). The checkpoint kinases preferentially phosphorylate R-X-X-S/T sites flanked by hydrophobic residues, and like the PIKKs have overlapping sets of substrates. In addition to the well-described ATM–Chk2 and ATR–Chk1 pathways, an additional complex consisting of the stress-responsive kinases p38MAPK and MK2 have been found to share substrate preferences with Chk1 and Chk2, and to function in the activation of checkpoints (Reinhardt and Yaffe 2009). The crossspecificity between Chk1, Chk2, and MK2 substrates has made it difficult to evaluate the respective roles of these kinases in downstream pathways. Recent studies

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suggest that Chk1 plays a primary role in cell cycle arrest pathways (Jallepalli et al. 2003; Wilsker et al. 2008) and is the most relevant checkpoint kinase in terms of therapeutic responses (Xiao et al. 2006; Wilsker et al. 2008).

2.3.1 Chk1 The most extensively characterized of ATR’s many substrates is the checkpoint kinase Chk1. The phosphorylation and activation of Chk1 requires a binding partner called Claspin that mediates the ATR–Chk1 physical interaction (Kumagai and Dunphy 2000; Chini and Chen 2004; Liu et al. 2006). Associated with the clamp loading complex at active replicons, Claspin is phosphorylated when replication forks stall (Wang et al. 2006). The association of Claspin with Chk1 is mediated by repeated phosphopeptide motifs that are phosphorylated by Chk1 and perhaps by other kinases as well (Kumagai and Dunphy 2003; Chini and Chen 2006). Thus, the Chk1–Claspin interaction is promoted by stalled replication forks and by the basal activity of Chk1 itself. Brought into proximity of the Chk1–Claspin complex, ATR phosphorylates Chk1 at two serine residues (S317 and S345) in a regulatory domain located in the c-terminus (Zhao and Piwnica-Worms 2001). While the Chk1 regulatory domain is evolutionarily divergent, the serine residues required for human Chk1 activation are within short regions of homology that are conserved in yeast. Detailed analyses of the functions of the individual Chk1 phosphorylation sites have provided insights into the functions of Chk1 and the distinct roles played by Chk1 in unperturbed and damaged cells. Genetic model systems in mouse and human cells have demonstrated Chk1 to be essential for cellular viability, as is ATR (Liu et al. 2000). Phosphorylation of Chk1 on its S345 site appears to be intrinsic to the essential function of Chk1; CHK1 alleles with mutations (S to A) that disrupt the S345 site do not support cellular viability (Niida et al. 2007; Wilsker et al. 2008). In contrast, mutations of the S317 are tolerated. However, cells harboring S317 mutant Chk1 proteins lose the ability to mediate the G2/M checkpoint in response to DNA damage and exhibit defects in DNA replication, including decreased replication fork velocity and increased fork stalling in unperturbed cells (Wilsker et al. 2008). Studies of Chk1 mutants have therefore revealed that Chk1 plays an essential role during normal cell growth and also has a nonessential role in the DNA damage response. These distinct roles are genetically separable by mutation of a single ATR phosphorylation site at S317 (Niida et al. 2007; Wilsker et al. 2008). Phosphorylation of Chk1 plays a prominent role in its localization. In unstressed cells, a pool of Chk1 protein remains stably bound to chromatin. Phosphorylation of Chk1 on S317 and S345 by ATR results in the rapid dissociation of this bound fraction (Smits et al. 2006). Following DNA damage or replicative stress, the majority of Chk1 in the cell becomes rapidly phosphorylated, suggesting that both chromatin-bound and unbound fractions of Chk1 are ultimately modified by ATR. In the prevailing model, unphosphorylated Chk1 in effect circulates to sites of

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activated ATR (Smits 2006). Once phosphorylated by ATR at regions of RPA-coated single-stranded DNA, Chk1 is released from chromatin to phosphorylate its downstream substrates. Chk1 localization is also controlled by cellular pathways generally unrelated to DNA damage signaling. The AKT kinase, which is negatively regulated by the PTEN tumor suppressor, phosphorylates Chk1 on S280 (Puc et al. 2005). The phosphorylation of Chk1 at this site promotes its monoubiqutination and promotes its sequestration in the cytoplasm. It is believed that the enhanced phosphorylation of Chk1 by AKT contributes to the checkpoint deficiencies observed in PTENdeficient cancers (Puc and Parsons 2005; Puc et al. 2005). Chk1 controls the intra-S and the G2/M checkpoints, DNA damage-responsive pathways by which damaged cells transiently halt DNA replication and are prevented from entering mitosis, respectively (Stracker et al. 2009). Key components of the checkpoint pathways are the Cdc25 family of phosphatases. The three Cdc25 proteins Cdc25A, Cdc25B, and Cdc25C remove inhibitory phosphate moieties from the cyclin-dependent kinases (CDKs), and thereby promote cell cycle transitions (Boutros et al. 2006; Karlsson-Rosenthal and Millar 2006). The effect of Chk1 on the Cdc25 proteins is to inhibit their activity by triggering their degradation (in the case of Cdc25A) or causing their sequestration in the cytoplasm (Cdc25B and Cdc25C). By inhibiting the Cdc25 phosphatases required for CDK activity, activated Chk1 is required for the checkpoint pathways that block cell cycle transitions. Other functions of Chk1 include the activation of DNA repair by the Fanconi Anemia pathway. Required for the repair of interstrand DNA cross-links, the Fanconi Anemia genes encode a multisubunit protein complex that is initially activated by ATR (Gurtan and D’Andrea 2006). Chk1 phosphorylates the FancE subunit on two residues (T346 and S374) that promote its degradation (Wang et al. 2007). The Chk1 sites on FancE are required for cell survival after treatment with DNA cross-linking agents.

2.3.2 Chk2 As Chk1 is directly phosphorylated by ATR in response to RPA-coated singlestranded DNA, so is Chk2 is phosphorylated by ATM in response to DSBs (Bartek et al. 2001; Ahn et al. 2004; Stracker et al. 2009). The kinase domains of Chk2 and Chk1 are highly related, and pharmacological inhibitors of these kinases typically exhibit cross-specificity. Beyond their kinase domains, Chk2 and Chk1 are structurally and functionally distinct (McGowan 2002). In striking contrast with the ATR–Chk1 pathway, the ATM–Chk2 pathway is nonessential. The Chk2 gene can be homozygously disrupted in human and mouse cells (Hirao et al. 2000; Jallepalli et al. 2003), and therefore does not play a critical role in cell proliferation. Chk2 mutations occur at a significant frequency, but the role of Chk2 in cancer has been controversial. The original identification of Chk2

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mutations in patients with a variant of Li Fraumeni syndrome known as Li Fraumeni-like syndrome suggested that Chk2 mutations might phenocopy highly penetrant p53 mutations (Bell et al. 1999). In accordance with this hypothesis, studies of Chk2 activity in vitro demonstrated that Chk2 can phosphorylate p53 on a site (S20) involved in its activation (Hirao et al. 2000; Shieh et al. 2000). However, the most prevalent Chk2 alteration is a truncating mutation (designated 1100delC) that is found in some populations at a frequency as high as 1% (Sodha et al. 2000). The discordance between the high frequency of the CHK21100delC allele and the low incidence of Li Fraumeni-like syndrome suggests that Chk2 mutations are not functionally equivalent to p53 mutations (Sodha et al. 2002). Studies in human cells demonstrate that p53 can be activated in the absence of Chk2 (Jallepalli et al. 2003), while analyses of knockout mice suggest that Chk2 is required for some p53dependent functions in some cells types (Jack et al. 2002). It thus would appear that Chk2 can promote p53 activation in some tissues, perhaps in response to tissuespecific stimuli. Large-scale population-based studies have demonstrated that carriers of the CHK21100delC allele have three- to fivefold increased risk of developing breast cancer (Meijers-Heijboer et al. 2002; Weischer et al. 2008). CHK2 is therefore a tumor suppressor gene with incomplete penetrance, similar to ATM. Chk2 has an N-terminal cluster of ATM/ATR recognition sites known as the SCD, a Forkhead-associated domain (FHA) that is involved in phosphorylationdependent protein–protein interactions and a kinase catalytic domain toward the c-terminus (Ahn et al. 2004; Stracker et al. 2009). More than 25 distinct Chk2 phosphorylation sites have been identified. Activated ATM phosphorylates Chk2 on many of the recognition sites in the c-terminal SCD, including residue T68. Once phosphorylated, the SCD becomes a docking site for a second Chk2 molecule. The multimerization of Chk2 brings the activation domains of the kinase loops into close proximity, thus promoting the autophosphorylation of multiple residues in the catalytic site. The autophosphorylation of the N-terminal sites increases enzymatic activity. This two-stage activation mechanism is common to the homologs of Chk2 in yeast (Oliver et al. 2007). The substrate specificity of Chk2 closely resembles that of Chk1 (Stracker et al. 2009). Accordingly, Chk2 has been shown to phosphorylate key cell cycle regulators such as p53 and the Cdc25 protein phosphatases. The phenotypes of Chk2 knockout human cells and mice are generally mild and do not phenocopy mutations in ATM. Chk2 knockout cells retain their ability to upregulate p53 and activate checkpoints following DNA damage, and apoptotic pathways appear to be defective in only a subset of tissues (Jack et al. 2002; Jallepalli et al. 2003).

2.3.3 p38MAPK/MK2 The mitogen-activated protein kinase-activated protein kinase-2 (MK2) is potently stimulated by the various activators of the p38 family, which response to diverse forms of cell stress (Roux and Blenis 2004). Loss of function of MK2 in knockout

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mice leads most apparently to deficiencies in immune responses. A role for the p38MAPK/MK2 in checkpoint control was suggested by the finding that MK2 can function downstream of ATR and phosphorylate the same sites on Cdc25 proteins as Chk1 and Chk2, following exposure to UV radiation (Manke et al. 2005). Cdc25B and Cdc25C are sequestered in the cytoplasm following MK2-dependent phosphorylation.

2.4 Cooperation Between ATM and ATR ATM and ATR are both activated by DSBs (Abraham 2001; Osborn et al. 2002). The activation of ATM occurs within minutes of a DSB, in cells that are in any phase of the cell cycle. In contrast, activation of ATR by DSBs is delayed and restricted to cells that are in S and G2 (Jazayeri et al. 2006). Cells from ataxiatelangiectasia patients deficient in ATM function exhibit markedly impaired phosphorylation of downstream substrates and diminished DSB responses, despite the presence of wild-type ATR. Such observations underscore the primary importance of ATM in the DSB response. Recent studies have demonstrated that ATM is in fact required for activation of ATR by DSBs (Adams et al. 2006; Cuadrado et al. 2006; Jazayeri et al. 2006; Myers and Cortez 2006). Unlike other downstream components of the DSB response, ATR is not known to be an ATM substrate. Rather, ATR is activated indirectly as a result of ATM-initiated processing of DSBs that involves the components of the MRN complex. The DNA end processing by the MRN complex in effect causes DSBs that efficiently activate ATM to be converted to regions of RPA-coated single-stranded DNA that efficiently activate ATR. This DSB conversion is CDK-dependent, which causes ATR activation to be cell cycle phase-specific (Jazayeri et al. 2006). In the absence of ATM, the conversion of DSBs to RPA-coated single-stranded DNA is significantly slower and less robust, and the activation of ATR is similarly decreased. Following its indirect activation by ATM, ATR plays a critical role in the overall DSB response. Most notably, cells engineered to be completely or even partially deficient in ATR function exhibit clear defects in the G2/M checkpoint triggered by ionizing radiation (Hurley et al. 2007). Additionally, ATR-deficient cells exposed to ionizing radiation fail to enter S-phase and complete DNA replication, suggesting a failure to stabilize early replication complexes in the presence of DSBs (Hurley et al. 2007). ATM has been observed to be detectably activated by agents that primarily inhibit DNA replication (Dodson and Tibbetts 2006; Stiff et al. 2006); this crossactivation has been attributed to phosphorylation by ATR of the ATM S1981 autophosphorylation site (Stiff et al. 2006). The ATR-dependent phosphorylation of this site is independent of the MRN complex, suggesting that ATM activation by this pathway is mechanistically distinct from its autoactivation after ionizing radiation. Cumulatively, these data show that the cooperation between ATM and ATR appears

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to work in both directions. While ATM and ATR control parallel pathways, these apical sensor kinases clearly work together to respond to structurally diverse DNA lesions (Hurley and Bunz 2007). Is there crosstalk between the ATM and ATR pathways? The shared substrate specificity of ATM and ATR presents the possibility that downstream substrates could be activated by either kinase. Activation of the ATM substrate Chk2 has been shown to result from treatment with DNA replication stressors known to activate ATR (Stiff et al. 2006). Chk1, known to be strongly phosphorylated by ATR, is phosphorylated in an ATM-dependent manner after exposure to ionizing radiation (Gatei et al. 2003). Each of these observations could be interpreted as evidence of crosstalk. However, recent insights into the cooperation between ATM and ATR have cast these observations in a new light. It is important to consider that DNA lesions are not static structures, but rather are rapidly processed and metabolized (Jazayeri et al. 2006). DSBs can be converted to regions of single-stranded DNA by the processing of broken ends that is ATM-dependent and MRN-mediated. DSB can alternatively arise from stalled replication forks that expose single-stranded DNA to the effects of nucleases. It now appears that relationships that on the surface appear to be crosstalk between upstream kinases are in fact the result of interconversion between DSBs and RPA-coated single-strand DNA. It is now generally believed that the respective activation of Chk1 and Chk2 by ATR and ATM is highly specific.

2.5 Mediators of the DNA Damage Response: BRCT-Containing Proteins Downstream of ATM and ATR are a complex family of proteins that share a common, highly conserved motif first identified in the breast cancer susceptibility gene 1 (BRCA1). The BRCA1 c-terminal (BRCT) motif is a phosphoprotein-binding domain involved in protein–protein interactions and oligomerization (Yu et al. 2003). Proteins containing tandem BRCT domains at their C-termini function to facilitate the interaction between signaling (PIKK) and transduction (checkpoint kinase) molecules, and thus act as mediators of the signaling cascade activated by DNA damage or DNA replication stress (Mohammad and Yaffe 2009). Among the BRCT-containing proteins recently found to function in DNA damage signaling are BRCA1, 53BP1, MDC1/NFBD1, and MCPH1/BRIT1. Together, these proteins represent the functional homologs of the first checkpoint protein ever described, budding yeast Rad9 (which is unrelated to the human Rad9 component of the 9-1-1 complex). Importantly, while yeast Rad9 functions to unidirectionally mediate signals from the PIKKs to the checkpoint kinases, several of the human homologs appear to additionally affect the upstream activation of ATM (Mochan et al. 2003; Aglipay et al. 2006; Wilson and Stern 2008). Both the diversity of mediators and the bidirectionality of the signaling pathways suggest a higher order of complexity in mammalian cells as compared with yeasts.

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2.5.1 BRCA1 Many of the genes involved in the DNA damage repair pathways function as tumor suppressors. Prominent among these is BRCA1, which mutated in about one-half of inherited breast and ovarian cancers. Many of the cancer predisposing mutations in BRCA1 affect the BRCT domain, suggesting that the role of BRCA1 in the DNA damage response is intrinsic to its role in tumor suppression. BRCA1 is phosphorylated after DNA damage on multiple sites by ATM (Cortez et al. 1999) and Chk2 (Zhang et al. 2004) and relocates rapidly to sites of damage and stalled DNA replication forks (Okada and Ouchi 2003; Ouchi 2006). Cells containing BRCA1 mutant proteins display defective intra-S and G2/M checkpoints and hypersensitivity to ionizing radiation (Deng 2006). The individual phosphorylation sites are differentially required for the G2/M and the intra-S checkpoints, demonstrating the independent regulation of these pathways and the overall complexity of the relationship between BRCA1 and checkpoints (Xu et al. 2001). The phosphorylation of BRCA1 by Chk2, also encoded by a tumor suppressor gene involved in breast cancer, is required for efficient repair of DSBs (Lee et al. 2000; Zhang et al. 2004).

2.5.2 53BP1 53BP1 is a component of the DNA damage response that was originally identified by virtue of its physical interaction with the tumor suppressor protein p53 (Iwabuchi et al. 1994). Genetic analyses have demonstrated the importance of 53BP1 for the stabilization of p53, the phosphorylation of Chk2 and the activation of intra-S and G2/M checkpoints after DNA damage (DiTullio et al. 2002; Wang et al. 2002; Ward et al. 2003). After exposure to ionizing radiation, 53BP1 relocalizes rapidly to nuclear foci that also contain ATM, which correspond to sites of DSBs. Cells deficient in ATM exhibit defects in the relocalization of 53BP1, indicating that 53BP1 functions in ATM-dependent pathways (DiTullio et al. 2002). 53BP1-knockout mice are cancer prone, radiosensitive, and develop similar cancers as ATM-knockout mice (Ward et al. 2004). Interestingly, loss of one or both copies of 53BP1 in p53-null mice significantly accelerated the rate of cancer development, demonstrating that 53BP1 and p53 proteins function together to suppress tumors (Ward et al. 2005). While a significant body of data suggests that 53BP1 mediates the phosphorylation of ATM substrates, the exact function of 53BP1 remains unclear. 53BP1 contains tandem BRCT motifs, but these are not required for its oligomerization or for downstream DNA repair functions (Ward et al. 2006). Localization of 53BP1 to damage sites has been shown to require a tandem Tudor domain, a methyl-proteinbinding motif required for the initial recruitment of 53BP1 to chromatin (Huyen et al. 2004). 53BP1 contains 15 identified ATM/ATR phosphorylation sites (Matsuoka et al. 2007). These sites are phosphorylated after ionizing radiation as well as UV radiation, suggesting that ATR as well as ATM can play an upstream role in its activation (Jowsey et al. 2007).

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2.5.3 MDC1/NFBD1 Mediator of DNA damage checkpoint protein 1 (MDC1/NFBD1) is a BRCT-domain-containing protein that is phosphorylated in response to DNA damage (Stucki and Jackson 2004). After its phosphorylation, MDC1/NFBD1 relocalizes to sites of DNA lesions and functions to facilitate the recruitment and accumulation of cell cycle checkpoint and DNA repair factors to sites of DNA damage (Lukas et al. 2004; Xu et al. 2008), a characteristic shared with the other BRCTcontaining proteins. Originally identified in a random screen of large cDNAs, MDC1/NFBD1 was predicted to function in the DNA damage response on the basis of its domain structure (Stucki and Jackson 2004). Depletion of MDC1/NFBD1 causes defects in checkpoint activation and apoptosis and sensitizes cells to DNAdamaging agents (Goldberg et al. 2003; Stewart et al. 2003).

2.5.4 MCPH1/BRIT1 MCPH1/BRIT1 was identified independently as a disease gene that encodes microcephalin and as a BRCT domain-containing inhibitor of the catalytic component of telomerase (hTERT). Like the more extensively characterized BRCT-domain protein 53BP1, MCPH1/BRIT1 regulates the intra-S and G2/M checkpoints, and localizes to gH2AX foci after irradiation (Xu et al. 2004; Lin et al. 2005; Alderton et al. 2006). Depletion of MCPH1/BRIT1 leads to decreased expression of BRCA1 and Chk1, and causes radiosensitivity. The clinical similarity of primary microcephaly, caused by MCPH1/BRIT1 mutations, and ATR–Seckel syndrome, a recessive disease caused by hypomorphic mutations in ATR (O’Driscoll et al. 2004), suggest the importance of an ATR–BRCA1–Chk1 signaling pathway in brain development (Lin et al. 2005; Alderton et al. 2006). MCPH1/BRIT1 contains three BRCT domains that facilitate its interactions with the chromatin-remodeling complex SWI/SNF. By recruiting this complex to the sites of DNA lesions, MCPH1/BRIT1 promotes chromatin relaxation which is thought to facilitate the access to the lesions by DNA repair proteins (Peng et al. 2009).

2.6 Activation of p53 by Upstream Kinases Among the first human proteins found to contribute to the DNA damage response was the tumor suppressor p53. The p53 gene is among the most widely mutated of cancer genes, and contributes to the development of approximately one-half of all cancers. Upon its activation by upstream signaling pathways, p53 contributes significantly to numerous growth inhibitory pathways, including cell cycle checkpoints, apoptosis and senescence (Vogelstein et al. 2000). The p53 protein is modified by a broad array of posttranslational modifications, many of which are believed to

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contribute to its activation (Horn and Vousden 2007; Kruse and Gu 2008). The best characterized among these are the phosphorylation moieties that are added by the kinases of the DNA damage signaling network (Appella and Anderson 2001). P53 normally has a very short half-life, due to its association with Hdm2, a ubiquitin E3-ligase that targets p53 for proteasomal degradation (Kubbutat et al. 1997). In unperturbed cells, p53 is therefore maintained at low levels. After DNA damage, p53 is phosphorylated on multiple sites. These phosphorylation events are concurrent with the dissociation of p53 from Hdm2 and its stabilization. Thus activated by DNA damage, p53 transactivates the transcription of a large number of genes that contribute to the diverse outcomes of the DNA damage response (Vogelstein et al. 2000). While p53 is known to be strongly activated by DNA damage and replication stress, the exact mechanism of its activation remains unclear (Kruse and Gu 2009). ATM-null cells from patients with ataxia-telangiectasia exhibit a markedly diminished p53 response to ionizing radiation, that is both less robust and temporally delayed compared with cells with wild-type ATM (Canman et al. 1994; Kastan and Lim 2000). ATM, ATR, Chk1, and Chk2 have all been reported to directly phosphorylate p53 on several N-terminal sites (Canman et al. 1998; Tibbetts et al. 1999; Chehab et al. 2000; Hirao et al. 2000), most prominently S15 and S20, that appear to play a role in protein stabilization in vitro. It has been unclear whether the direct phosphorylation of p53 by any one of the upstream kinases is primarily important, or to what extent the p53 response is due to indirect effects of DNA damage signaling. In recent years, studies of knock-in mouse models have called the role of the N-terminal p53 phosphorylation sites into question. Mutations of the mouse equivalents of human S15 and S20 (S18 and S23 in mouse) notably fail to eliminate the responsiveness of p53 levels to DNA damage and other stimuli (Wu et al. 2002; Saito et al. 2003; MacPherson et al. 2004; Sluss et al. 2004), although double mutants in both of these residues do exhibit significant apoptotic defects in some tissues (Chao et al. 2006). These results suggest that p53 stabilization may be a combination of direct and indirect effects of the DNA damage signaling pathways, including the stimulation of other types of posttranslational modifications (Kruse and Gu 2009).

2.7 Diverse Substrates of the Human PIKKs The two-tiered structure of the yeast DNA damage and replication stress pathways (Fig. 2.1) has guided the identification of homologous proteins that function in the human DNA damage response. The checkpoint and mediator proteins that play critical roles in the growth arrest and survival following DNA damage remain the most highly characterized substrates of ATM and ATR. Recently, unbiased proteomic approaches have significantly broadened our view of the extent and scope of the DNA damage responses. Over 700 proteins are robustly phosphorylated at more than 900 sites by the combined activation of ATM and ATR in response to the

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DSBs caused by ionizing radiation (Matsuoka et al. 2007). Similarly, UV radiation, a potent inhibitor of DNA replication, was found to induce the phosphorylation of nearly 500 proteins (Stokes et al. 2007; Stokes and Comb 2008). Within these two unexpectedly large sets of proteins are many that are commonly activated by both stimuli and therefore probably represent the combined effects of ATM and ATR activation. A significant number of substrates appear to be exclusively dependent on either ATM or ATR (Stokes and Comb 2008). A common theme among the downstream targets of ATM/ATR is the phosphorylation of multiple targets within individual pathways, suggesting many potential nodes of regulation (Matsuoka et al. 2007). For example, many substrates participate in the regulation of the successive stages of DNA replication. Prevalent among the ATM/ATR target proteins are those that are involved in each step of DNA replication, including origin recognition (ORC proteins), replication complex assembly (MCM proteins), clamp loading (RFC1 and RFC3), and DNA synthesis (DNA polymerase epsilon, GINS). Identification of these substrates promises mechanistic insights into the genetically observed roles of ATM and ATR in controlling DNA replication after DNA damage. Other functional modules impacted by the combined functions of ATM and ATR are the Fanconi anemia pathway and the nucleotide excision repair pathway, and combined pathways required for DNA repair by homologous recombination (Matsuoka et al. 2007). The functional diversity of the ATM/ATR phosphoproteome is striking. Many of the recently identified ATM/ATR substrates implicate pathways with no prior relationship to DNA damage signaling. For example, multiple proteins in the PTEN/ AKT pathway, which responds to growth stimuli such as insulin, were found to be robust ATM/ATR substrates, including AKT, its adaptors, regulators, and downstream effectors (Matsuoka et al. 2007). The elucidation of these pathways, and the ways they are functionally integrated will provide experimental challenges for years to come.

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Chapter 3

Checkpoint Control Following Radiation Exposure Markus Lobrich, Aaron A. Goodarzi, Tom Stiff, and Penny A. Jeggo

Abstract The DNA damage response involves processes of DNA repair and signal transduction pathways. DNA double-strand breaks (DSBs) are the most biologically significant lesion induced by ionizing radiation (IR). DNA nonhomologous end-joining and homologous recombination represent the major DSB repair pathways and ataxia telangiectasia mutated (ATM) lies at the heart of the DSB signaling response. However, ATM and Rad3 related (ATR) can also be activated in S and G2 phase following IR exposure. A major end point of damage response signaling is the activation of cell cycle checkpoint arrest. In addition to the initiation of checkpoint arrest, recent studies have demonstrated that the signaling pathway monitors the progress of DSB repair to ensure timely checkpoint release, a process called the maintenance of checkpoint arrest. In this chapter, we overview DNA damage-induced cell cycle checkpoint arrest following exposure to IR. We discuss how cell cycle stage impacts upon the roles of ATM and ATR, how they influence the initiation and maintenance of checkpoint signaling, and the interface between checkpoint arrest and DSB repair. We evaluate current insight into the sensitivity of the processes and the impact of higher order chromatin structure on damage response signaling. Keywords DNA double strand breaks • Cell cycle checkpoint arrest • Radiationinduced damage responses • G1/S checkpoint arrest • G2/M checkpoint arrest

P.A. Jeggo (*) Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RQ, UK e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_3, © Springer Science+Business Media, LLC 2011

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3.1 Introduction The DNA damage response (DDR) encompasses processes of DNA repair and two overlapping signal transduction pathways. A range of distinct DNA repair pathways recognize different classes of DNA damage, some of which have inbuilt additional specificity, such as the distinct glycolyases that function in base excision repair. Collectively, these processes allow the recognition and repair of a broad range of DNA lesions. In contrast, the signal transduction response is activated by one of two broad classes of lesion, a DNA double-strand break (DSB) or a single-stranded (ss) region of DNA. Two phosphoinositol 3-kinase-like kinases (PIKKs) lie at the center of the signaling response; ataxia telangiectasia mutated (ATM) regulates the response to DSBs and the related ATM and Rad3-related protein (ATR) is activated by ss DNA regions. The major DNA repair processes [e.g., nucleotide excision repair (NER), single-strand break (SSB), and DSB repair] function largely independently of the signal transduction pathways. However, there is increasing evidence of interplay between DNA repair pathways and ATM/ATR signaling. An additional and arguably more significant aspect of the signaling response is the regulation of cell cycle checkpoint arrest and, in higher organisms, the process of apoptosis. In this review, we focus on the processes of cell cycle checkpoint arrest and their regulation by damage response signaling. Our major focus lies on the response to DNA DSBs. Since DSBs are a biologically significant lesion induced by ionizing radiation (IR), our analysis will primarily consider IR-induced checkpoint arrest. Three damage response checkpoints have been described: G1/S, G2/M, and intra-S-phase arrest. We will review the current knowledge of the processes underlying these checkpoints and specifically consider their efficacy in enhancing survival and limiting genomic instability.

3.2 PIKK Activation and Signaling The first step in signaling DNA damage to the cell cycle checkpoint machinery is the activation of one or both of the PIKKs. IR induces base damage, SSBs, DSBs and a lower number of DNA inter- and intrastrand cross-links. Base damage can be processed into SSBs, and the ratio of SSB:DSB damage induced by IR is 20:1 (Nikjoo et al. 2002). A critical aspect in considering the mechanisms underlying cell cycle checkpoint arrest is the distinct structures that lead to PIKK activation. First, we will overview the current knowledge of the steps activating ATM and ATR and then consider PIKK activation following IR exposure.

3.2.1 ATM Activation The mechanism leading to ATM activation is, perhaps, better understood than that resulting in ATR activation. Several excellent recent reviews have discussed the

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steps involved in ATM activation and the assembly of ionizing radiation-induced foci (IRIF) and only an outline of the process will be provided here (Lavin 2008; Panier and Durocher 2009; van Attikum and Gasser 2009). Increasing evidence suggests that the Mre11-Rad50-NBS1 (MRN) complex represents the initial DNA DSB sensor (Carson et al. 2003; Uziel et al. 2003). Importantly, the extreme C terminus of NBS1 encompasses an evolutionary conserved motif that promotes interaction with ATM, providing a route by which ATM is initially recruited to DSBs (Falck et al. 2005). In undamaged cells, ATM exists as an inactive homodimer and its DSB recruitment results in autophosphorylation and monomerisation, a process leading to sustained activation (Bakkenist and Kastan 2003). An early step following ATM activation is phosphorylation of the C terminus of H2AX, the variant form of the histone H2A, creating phosphorylated H2AX (phospho-S139 H2AX), termed gH2AX (Rogakou et al. 1998). Although H2AX phosphorylation initiates in the region directly flanking the DSB, phosphorylation occurs at increasing distances from the break site with time so that the region containing phosphorylated H2AX can cover mega DNA base pairs at persisting DSBs (Rogakou et al. 1999). Thus, an extended region of H2AX flanking the DSB becomes phosphorylated, a critical factor leading to visible IRIF. gH2AX binds to the tandem BRCT domains of the mediator protein, MDC1 (Mediator of DNA damage checkpoint 1), providing a critical first step in mediator protein recruitment (Stucki and Jackson 2004). The N terminus of MDC1 encompasses a recently identified SDT (Ser–Asp–Thr) repeat motif in close proximity to the tandem BRCT domain. The SDT motifs are phosphorylated by CK2 both endogenously and following IR exposure and mediate an interaction with the FHA domain of NBS1, providing a route to tether MRN and ATM at the DSB site (Chapman and Jackson 2008; Melander et al. 2008; Spycher et al. 2008). A highly significant finding is that MDC1 facilitates the recruitment of two ubiquitin E3-ligases, RNF8 (RING finger 8) and RNF168 (RING finger 168), to the DSB site, a pivotal result exposing the importance of ubiquitylation as a post-translational modification in the DDR (Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007; Doil et al. 2009; Stewart et al. 2009). Ubiquitylation requires a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3). The FHA domain of RNF8 interacts with an ATM phosphorylation motif in the N terminus of MDC1. RNF8 also interacts with UBC13, an E2 ubiquitin-conjugating enzyme and catalyzes K63-linked ubiquitylation (Plans et al. 2006). The second E3 ligase, RNF168, was identified as the gene defective in a human disorder termed RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) Syndrome (Stewart et al. 2007, 2009; Doil et al. 2009). RNF168 recruitment requires the RNF8 RING finger motif suggesting that its recruitment itself requires ubiquitylation at DSBs. In very recent work, HERC2 has been identified as a further protein that is required for ubiquitylation at DSBs (Bekker-Jensen et al. 2010). HERC2 encompasses HECT domains and is an ATM substrate. ATM-dependent phosphorylation of HERC2 mediates its interaction with the FHA domain of RNF8, which facilitates assembly of UBC13 with RNF8, thereby promoting K63-linked ubiquitylation. The main target of these ubiquitin ligases is H2A/H2AX. An important end point of H2A

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ubiquitylation is recruitment of another mediator protein, 53BP1 (Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007). The recruitment of BRCA1 to the DSB also depends on the RNF8-RNF168 ubiquitin ligases although the precise mechanism and function of this loop of the signaling process is unclear. BRCA1 is itself an ubiquitin ligase and it has been suggested that it further enhances the concentration of ubiquitin moieties at the DSB. Recent studies have provided evidence that 53BP1 interacts with Rad50 via 53BP1’s BRCT domain, providing an additional step promoting MRN hyper-accumulation at the DSB (Lee et al. 2009). This step also appears to be critical to promote ATM chromatin retention and the formation of p-1981-ATM foci (Noon et al. 2010). Importantly, although the phosphorylation of H2AX and the recruitment of the mediator proteins are important steps leading to p-1981-ATM foci formation, they are not essential for ATM activation and have only a modest impact on ATM substrate phosphorylation, although the precise impact is substrate specific. Collectively, these findings demonstrate that ATM regulates an orchestrated and sophisticated assembly of proteins at the DSB, which results in modifications of histones in the DSB vicinity. Increasing evidence suggests that changes to the higher order chromatin superstructure, including factors influencing heterochromatinisation, promotes DSB repair within regions of heterochromatin (Goodarzi et al. 2008; Noon et al. 2010).

3.2.2 ATR Activation ATR activation and signaling has also previously been reviewed in detail and will only be briefly discussed here (Cimprich and Cortez 2008). In contrast to ATM, ATR is essential since it is activated during every S phase. ATR is activated by ss regions of DNA, following their coating with the ss DNA-binding protein, RPA (Zou and Elledge 2003). Localization of ATR to ss DNA also requires ATR interacting protein (ATRIP), in part because ATRIP directly binds to RPA (Ball et al. 2007). However, additionally ATR and ATRIP closely interact and co-regulate each other’s stability (Cortez et al. 2001). Although ATR localization to RPA-coated ss DNA may require only ATRIP, sustained ATR activation also requires the colocalization of Rad17 and the so-called 9-1-1 complex, involving Rad9-Rad1-Hus1. The 9-1-1 complex represents a heterotrimeric ring-shaped molecule akin to PCNA. Loading of the 9-1-1 complex requires the recruitment of the damage-specific clamp loader, Rad17, to the RPA-coated ss DNA (Zou et al. 2003). Critically, the recruitment of the 9-1-1 complex promotes the recruitment of TOPBP1, a multiBRCT domain protein, which appears to be a critical step in ATR activation (Furuya et al. 2004; Kumagai et al. 2006; Delacroix et al. 2007; Lee et al. 2007). Interestingly, the recruitment and assembly of the Rad17/9-1-1 complex to RPA-coated ss DNA occurs largely independently of ATR-ATRIP recruitment, a feature which is distinct to the more step-wise orchestrated recruitment of proteins at a DSB. A critical issue in evaluating ATR signaling is the length of ss DNA required for ATR activation. Consistent with the findings discussed above that RPA binding to

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ss DNA regions is required for ATR activation, neither base damage nor ss nicks appear to activate ATR. ss DNA of sufficient length to bind RPA occurs predominantly at stalled or collapsed replication forks, and indeed this is a situation where ATR signaling plays its major role. ss DNA regions can also occur during the processing of certain DNA lesions, such as a pyrimidine dimer, although it is unclear whether ATR signaling is activated by the ss regions generated during NER or whether it requires exonucleolytic processing to generate longer and perhaps more accessible ss DNA regions (Nakada et al. 2004; Marini et al. 2006). ss DNA of sufficient length can also be generated in G2 phase at a resected DSB, which will be discussed in further detail below.

3.2.3 Activation of ATM vs. ATR Following IR Exposure Although base damage and SSBs are abundant lesions induced by IR, they are rapidly repaired and, importantly, do not directly activate either ATM or ATR signaling. In G0/G1 phase, SSBs do not undergo extensive resection and the available evidence suggests that ATR is not activated even at prolonged times after IR exposure (Stiff et al. 2004). Thus, following IR exposure, the signaling response and G1/S checkpoint arrest is predominantly ATM dependent in G0/G1 phase cells (Kastan et al. 1992; Lavin et al. 1994). SSBs are also rapidly repaired in G2 phase and it appears that, as in G1 phase, they do not activate damage signaling response (Shibata, unpublished findings). However, in G2 phase, resection can occur at least at a subset of DSBs resulting in ATR activation (Jazayeri et al. 2006). Importantly, resection at DSBs in G2 phase is ATM dependent (Jazayeri et al. 2006). Thus, in G2 and in G1 phase cell cycle checkpoint arrest after IR is ATM dependent although ATR may additionally contribute to checkpoint signaling in G2 phase as resection ensues (see below for further discussion of the interplay between ATM and ATR in G2/M checkpoint arrest) (Lavin et al. 1994; Shibata et al. 2010). In S phase, the situation is different since ATR can be activated as a consequence of replication fork stalling. Given the 20:1 ratio of SSBs:DSBs, replication fork stalling at SSBs can be significant, although the relevance of SSBs for damage response signaling is diminished by their rapid rate of repair. The prevailing evidence suggests that DSBs generated in S phase can directly activate ATM, as they do in G1 or G2 phase, and that, additionally, signaling can be complemented by ATR activation at stalled or collapsed replication forks (Byun et al. 2005). In addition to ATM-dependent activation of ATR following resection in S or G2 phase, there is also evidence that the generation of one-ended DSBs at collapsed replication forks can lead to ATM activation (Mirzoeva and Petrini 2003; Helleday et al. 2007). Further it has been proposed that ATR can phosphorylate ATM leading to ATR-dependent ATM activation (Stiff et al. 2006). Thus, the interplay between ATM and ATR signaling is complex and critically depends upon cell cycle phase. In summary, in G1 and G2 phase, almost all damage response signaling after IR exposure arises from DSBs and is ATM dependent at early times (Fig. 3.1). In S phase, ATR can additionally contribute due to its direct

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Fig. 3.1 Overview of roles of ATM and ATR in cell cycle checkpoint arrest processes. (1) DSBs in G1 phase directly activate ATM leading to Chk2/p53 activation and G1/S checkpoint arrest. This is a sensitive process. (2) In S phase, replication fork collision with SSBs or DSBs can lead to ATR and Chk1 activation and inhibition of origin firing. ATM-Chk2 can also be directly activated by DSBs in S phase. (3) In G2 phase, DSBs directly activate ATM-Chk2 causing G2/M phase arrest. Additionally, ATM activation promotes DSB resection leading to ATR-Chk1 activation. G2/M phase arrest has a threshold sensitivity of 10–20 DSBs. S phase arrest slows progression through S phase but cells can eventually enter G2 phase with damage. ATR also contributes to G2/M phase arrest of irradiated G1 or S phase cells. (a) Describes the progression through the cell cycle and pathways activated and (b) Describes the sensitivity of the checkpoint response

activation at stalled/collapsed replication forks. ATM-dependent resection can lead to ATR activation in S and G2 phase cells.

3.2.4 Signaling from ATM/ATR to the Transducer Kinases ATM and ATR signal to the checkpoint machinery via one of two transducer kinases, Chk1 and Chk2 (Matsuoka et al. 1998, 2000; see Bartek and Lukas 2003,

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for a review). A major function of these kinases is to relay the presence of DNA damage or the lack of genome integrity to the proteins that control cell cycle progression, namely the cyclin-dependent kinases (Cdks), and is achieved via inhibition/activation of phosphatases or kinases that regulate Cdk phosphorylation and activity. More recently, however, there is evidence that the transducer kinases may also regulate aspects of DNA repair (Sorensen et al. 2005). Perhaps surprisingly, although ATM and ATR share many substrates, their signaling to the transducer kinases appears to be specific, with ATM primarily phosphorylating and activating Chk2 while ATR signals in a similar way via Chk1 (Chaturvedi et al. 1999; Liu et al. 2000; Jazayeri et al. 2006). Chk2 is constitutively expressed but becomes activated uniquely in the presence of DNA damage (Lukas et al. 2001). In contrast, Chk1 is labile and its expression is increased in S/G2 phase (Lukas et al. 2001). Like ATR, Chk1 is activated during normal S phase progression. The activation of both Chk1 and Chk2 is regulated by PIKK-dependent phosphorylation. Chk2 is phosphorylated at a unique site in its regulatory N-terminal region, T68, predominantly by ATM. This triggers dimerisation and autophosphorylation in the C-terminal kinase domain (Matsuoka et al. 2000; Ahn et al. 2002; Xu et al. 2002b). Although p-T68-Chk2 can be observed in foci after DNA damage, live cell imaging analysis has shown that p-Chk2 is rapidly released from the damage site consistent with the notion that it functions as a signal transducer (Bartek and Lukas 2003). Chk1 is phosphorylated by ATR at several SQ sites in its C terminus including S317 and S345. Further details of the pathways leading from transducer kinase activation to cell cycle arrest will be discussed for the individual checkpoints below.

3.3 Cell Cycle Checkpoint Activation, Maintenance, and Adaptation The process of cell cycle checkpoint arrest can be divided into a number of stages which, at least to some extent, may be genetically and mechanistically distinct. The exploitation of IR is particularly useful for considering these distinct stages since the damage is rapidly induced allowing the processes of checkpoint initiation and maintenance to be distinguished. The presence of phosphorylated Chk1 or Chk2 (p-Chk1 or p-Chk2) can be observed at very early times (5–30 min) following IR treatment, although full checkpoint arrest takes more time to become evident. Nonetheless, in most cell lines, G2/M checkpoint arrest assessed by a dramatic reduction in mitotic cells is evident by 1 h post-IR. This process represents checkpoint activation. Subsequently, as DSB repair ensues a point is reached when the molecular signals relaying checkpoint arrest are no longer maintained and entry into mitosis recommences. Following exposure to higher IR doses, the process of initial checkpoint activation can be distinguished from the maintenance of checkpoint arrest. At lower doses, this is difficult to distinguish since the duration of checkpoint arrest may be short. It is also difficult to verify whether checkpoint initiation occurs at very low doses if the arrest is of short duration. From our own

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work, we have found that the presence of pChk1 or pChk2 provides a good correlation with checkpoint arrest and that addition of a Chk1/2 inhibitor (e.g., 600 nM UCN-01, which inactivates both checkpoint kinases) abolishes both the initiation and maintenance of checkpoint arrest (examined by adding UCN-01 after initiation has been established) (Shibata et al. manuscript submitted). Published findings have shown that the duration of checkpoint arrest depends upon dose and DSB repair capacity (Deckbar et al. 2007). Collectively, these findings provide strong evidence that the checkpoint machinery monitors the status of DSB repair to ensure timely checkpoint release. This process will be discussed further in the section considering G2/M checkpoint arrest. An additional factor in considering the maintenance of checkpoint arrest is the progression of cells through the cell cycle, which will also be considered below. In evaluating the distinct steps leading to checkpoint initiation and maintenance, the phenomenon of checkpoint escape should be clarified. Here, we define this to represent cells, which most likely due to their position in the cell cycle, fail to undergo checkpoint arrest – that is, they escape arrest. We distinguish this phenomenon from cells which initiate arrest but are subsequently released earlier than anticipated, which we describe as premature release. Cells that “escape” checkpoint arrest may be significant in the analysis of mitotic breakage when the time of sample collection is within the period of checkpoint arrest. Thus, although the majority of cells remain checkpoint arrested, a few “escape” arrest and enter mitosis. Since the results of most cytogenetic analysis are expressed as chromosome breaks per mitotic cell, the analysis can be highly dependent upon the small number of mitotic cells which have “escaped” checkpoint arrest rather than the majority of the population that has undergone checkpoint arrest (Lobrich and Jeggo 2007). Finally, checkpoint adaptation is a distinct process which was originally described in yeast and, more recently, in mammalian cells (Toczyski et al. 1997; Pellicioli et al. 2001; Yoo et al. 2004; Syljuasen et al. 2006). This represents a regulated process, whereby the checkpoint is released in cells that have endured prolonged arrest despite multiple persisting DSBs. Rather than remain arrested, cells appear to make a last ditch attempt to progress through the cell cycle albeit with the potential for increased chromosome breakage.

3.4 Mechanism Underlying DNA Damage-Induced G2/M Checkpoint Arrest The G2/M checkpoint has, arguably, been the best studied of the checkpoints, in part because the process is relatively well conserved between yeast and mammalian cells. Further, unlike the G1/S checkpoint, the process can be readily studied in immortalized or tumor cell lines. The G2/M checkpoint prevents the mitotic progression of cells which incurred DNA damage in G2 phase or which progressed into G2 phase with DNA damage. Progression from G2 into mitosis is regulated primarily by Cyclin B1/Cdk1 kinase. Cdk1 activity is itself regulated by inhibitory Tyr15 and Thr14 phosphorylation by the Wee1 and Myt1 kinases, respectively (Booher

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et al. 1997). Activation of Cdk1 requires dephosphorylation of Thr14 and Tyr15 by the Cdc25 phosphatases. Three Cdc25 phosphatases, A, B, and C, have been described in mammalian cells and their interplay is still poorly understood. Cdc25A and C are important as mitotic regulators with Cdc25B triggering the initial process. A critical step in damage-induced checkpoint arrest is the inactivation of the Cdc25 phosphatases, which is regulated by the Chk1/Chk2 kinases (Matsuoka et al. 1998). Cdc25A and C are phosphorylation targets of the Chk1/2 kinases. Cdc25A is phosphorylated by the transducer kinases at multiple sites and the regulation of its activity by phosphorylation is complex. Phosphorylation can directly inhibit Cdc25A activity and its proteasome-dependent degradation (Mailand et al. 2000; Molinari et al. 2000; Falck et al. 2001). However, Cdc25A is also phosphorylated by cyclin B-Cdk1 in mitosis, which results in its stabilization rather than degradation, a step which appears to consolidate mitotic entry (Mailand et al. 2002).

3.4.1 The Initiation of G2/M Checkpoint Arrest Early checkpoint arrest, representing the sharp reduction in mitotic entry within 1–2 h of IR exposure, must arise in irradiated G2 phase cells since primary and transformed cells take ~5 h to progress from S into G2 phase. As discussed above, IR exposure of G2 phase cells initially only activates ATM, since IR directly induces DSBs and SSBs but not regions of ss DNA, the lesion activating ATR. In G2 phase, DSB repair can occur by DNA nonhomologous end-joining (NHEJ) or by homologous recombination (HR). Although a widely accepted dogma is that HR represents the major DSB repair pathway in G2 phase while NHEJ predominates in G1 phase, recent studies have shown that ~80% of DSBs are repaired with fast kinetics by NHEJ in G2 phase while HR functions to repair those DSBs (approximately 20% of IR-induced DSBs) which are repaired with slow kinetics (Beucher et al. 2009). Making the assumption that the DSBs rejoined by NHEJ do not undergo resection but activate the ATM-Chk2 arm of the signaling response, the finding argues that ATM-Chk2 signaling must play a major role in the initiation of checkpoint arrest. Resection at the slowly repaired DSBs in G2 phase is ATM dependent but leads to ATR activation at the ss DNA regions generated (Jazayeri et al. 2006). Thus, signaling at resected DSBs occurs via ATM-ATRChk1. Recent in vitro studies have suggested that as resection ensues at DSBs, there is a switch from ATM to ATR signaling since ATM activity becomes diminished as the length of the ss DNA tail increases (Rhind 2009; Shiotani and Zou 2009). It is important to appreciate, however, that although the completion of DSB repair by HR appears to represent a slow process, resection occurs relatively rapidly after IR exposure. Indeed, using RPA foci formation to assess resection, we have observed that maximal numbers of RPA (and Rad51) foci can be observed by 30 min post-IR (earlier times are difficult to evaluate using this method of assessing resection) (Shibata, manuscript submitted). Consistent with this notion, pChk1 levels in G2 phase cells are also at a maximum level at 30–60 min post-IR

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(Shibata et al. 2010). Thus, for cells in G2 at the time of irradiation, we have observed that both ATM-Chk2 and ATM-ATR-Chk1 contribute to the initiation of checkpoint arrest (Shibata et al. 2010). In summary, early checkpoint arrest in G2 phase cells is ATM dependent, but Chk1/Chk2 have overlapping, redundant functions (Fig. 3.2). Indeed, the lack of dependency of G2/M checkpoint initiation upon Chk2 has been reported previously (Hirao et al. 2002). It might be expected, however, that ATM-Chk2 have the most significant impact since the majority of DSBs are repaired without resection by NHEJ. Such a dependency might be detectable at lower doses when the number of DSBs undergoing resection becomes small (and potentially less than one per cell), if Chk1 is only activated at 20% of IR-induced DSBs. Additionally, the significance of Chk1 vs. Chk2 in the initiation of G2/M checkpoint arrest may vary between cell types and may be distinct in tumor cells. One factor influencing this may be the rapidity with which DSBs undergo resection. Interestingly, checkpoint arrest in DT40 chicken cells is Chk1 dependent after 4 Gy, a finding consistent with the suggestion that more DSBs undergo resection and repair by HR in G2 in DT40 compared to mammalian cells (Sonoda et al. 2006; Rainey et al. 2008).

Fig. 3.2 Initiation and maintenance of G2/M checkpoint arrest in irradiated G2 phase cells. Irradiated G2 phase cells initially activate ATM and Chk2 from directly induced DSBs. However, ATM-dependent resection at DSBS can occur at early times after IR exposure in G2 cells leading to ATR-Chk1 activation. Both processes can contribute to the initiation of checkpoint arrest. Recent studies have shown that homologous recombination repairs the DSBs that are located at heterochromatin and are repaired with slow kinetics. Thus, resected DSBs represent a greater fraction of unrepaired DSBs at later times post-IR and ATR-Chk1 signaling makes a greater contribution to the maintenance of checkpoint arrest

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3.4.2 The Maintenance of G2/M Checkpoint Arrest Studies have demonstrated that the duration of checkpoint arrest is dependent upon dose and DSB repair capacity with DSB repair defective cells undergoing prolonged arrest compared to repair proficient cells (Wang et al. 2002b; Sturgeon et al. 2006; Deckbar et al. 2007). This demonstrates that G2/M checkpoint arrest is not activated for a defined period of time but instead supports a model by which the checkpoint machinery responds to the status of DSB repair. Thus, it is likely that DSBs are continuously sensed and Chk1/2 continuously activated. The maintenance of checkpoint arrest involves two distinct classes of cells. First, checkpoint arrest must be maintained in irradiated G2 phase cells. Since, the prevention of mitotic entry can be sustained for prolonged times after higher doses or in cells lacking DSB repair capacity, the checkpoint signal must be capable of being maintained in G2 phase cells for prolonged times. Additionally, at longer times after IR exposure (>5 h), irradiated and potentially damaged S phase cells progress into G2 phase. Such cells can also undergo G2/M arrest for prolonged periods (Krempler et al. 2007; Fernet et al. 2009). Interestingly, these findings suggest that the intraS-phase checkpoint arrest process does not efficiently prevent irradiated S phase cells progressing into G2 phase and that the G2/M checkpoint plays a significant role in preventing progression of such cells. However, the efficacy of the S phase checkpoint has not been carefully evaluated and will not be discussed in detail in this review. As discussed above, ATR can be activated in an ATM-independent manner in S phase cells after IR exposure as a consequence of replication fork arrest. Consistent with this notion, G2/M checkpoint arrest at later times after IR occurs by a molecularly distinct process to the early G2/M arrest being ATR-Chk1 dependent and ATM independent (Beamish et al. 1994; Xu et al. 2002a; Wang et al. 2003; Fernet et al. 2009). In recent studies, we have employed aphidicolin, an inhibitor of the replicative polymerase, to prevent S phase cells progressing into G2 phase postirradiation to allow an analysis of the maintenance of checkpoint arrest and DSB repair in irradiated G2 phase cells (Beucher et al. 2009; Shibata, manuscript submitted). Since HR repairs the slow DSB repair component, it is likely that, at later times postirradiation, the persisting DSBs will be those that have undergone resection, and hence ATR-Chk1 activation will be important for the maintenance of checkpoint arrest. Consistent with this notion, we observed that loss of either ATR or Chk1 results in premature mitotic entry of irradiated G2 phase cells (Shibata et al. 2010). However, additionally, ATM-Chk2 signaling contributes to the maintenance of checkpoint arrest, although less significantly compared with the role of Chk1 (Fig. 3.2). Thus, it appears likely that as DSB repair ensues and the checkpoint signal nears a threshold level required to maintain checkpoint arrest, both Chk1 and Chk2 provide some contribution. This starkly contrasts to the redundant and overlapping functions in initiating checkpoint arrest likely due to either activity alone being sufficient to reach a threshold signal (after 3 Gy IR).

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3.4.3 Sensitivity of the G2/M Checkpoint An important question in evaluating the efficacy of the G2/M checkpoint is its ability to respond to low levels of DNA damage. A simple and possibly the anticipated prediction would be that G2/M checkpoint arrest would be triggered by a single DSB. This is a reasonable expectation given that the checkpoint arrest has evolved to prevent genomic instability arising from the progression of cells with DSBs through mitosis. However, several studies have now provided evidence that the G2/M checkpoint is not sensitive to a single DSB but rather has a defined threshold of 10–20 DSBs (Deckbar et al. 2007; Fernet et al. 2009). In one approach, we evaluated the duration of checkpoint arrest with the status of DSB repair, using gH2AX to monitor the progress of DSB repair. We observed that cells enter mitosis after 3 Gy when approximately 15–20 gH2AX foci remain. Although the duration of arrest changes in repair proficient vs. deficient cells, the number of gH2AX foci remaining at the point of mitotic entry was similar, providing suggestive evidence that there is a defined sensitivity threshold (Deckbar et al. 2007). One limitation of this approach is that there may be a lag between completion of DSB repair and loss of gH2AX foci. Indeed, this is a possibility suggested by the finding that although the kinetics of DSB repair monitored by pulsed field gel electrophoresis (PFGE) is similar to that monitored by gH2AX foci enumeration, the repair lags 1–2 h when assessed by gH2AX foci enumeration compared to the use of PFGE. However, even 2 h after commencing mitotic entry, gH2AX foci remain detectable in the remaining G2 phase cells. Further supporting the notion that mitotic entry occurs prior to the completion of DSB repair, we also observed that mitotic cells derived from G2 phase cells that were released from G2/M arrest harbor 1–2 chromosome breaks (Deckbar et al. 2007). We also assessed chromosome breakage in G2 phase cells using calyculin A to induce premature chromosome condensation (PCC) of G2 phase cells. At the time of mitotic entry, we observed ~3 PCC chromosome breaks per G2 phase cell. Thus, mitotic entry occurs when ~15 gH2AX foci and 3 PCC breaks remain in G2 phase cells and gives rise to 1–2 chromosome breaks in the cells that enter mitosis after checkpoint arrest. Given that gH2AX foci detect nearly all DSBs, while a smaller subset of DSBs may be manifest as PCC breaks and an even smaller subset are visualized as chromosome breaks, this ratio appears reasonable. Importantly, since the majority of the G2 phase population undergoes G2/M arrest, our findings show that the major contribution to chromosome breakage arises in cells that undergo checkpoint arrest (Fig. 3.3). This contribution is more significant than the higher number of chromosome breaks observed in cells that “escape” checkpoint arrest, since the latter represents just a tiny percentage of the irradiated G2 population (Lobrich and Jeggo 2007). Collectively, these results provide strong evidence that the G2/M checkpoint has a defined threshold that allows for a low level of chromosome breakage in cells that are released from arrest. The ATMdependent G2/M checkpoint is therefore inefficient and lacks sensitivity. Interestingly, a recent study has suggested that cells that traverse S phase after irradiation have a lower threshold sensitivity compared to cells that were in G2 at the time of irradiation (Fernet et al. 2009).

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Fig. 3.3 Estimation of the kinetics for total chromosome breakage considering the level of chromosome breaks per mitotic cell and the number of cells in mitosis. The number of mitotic cells assessed by phosphoH3 analysis at various times post 1 Gy IR (upper left panel) was multiplied with the number of chromosome breaks per mitotic cell (upper right panel) providing an estimation of the total number of mitotic breaks (total chromosome breakage). Artemis cells harbor more chromosome breaks per mitotic cell than wt cells at any time point. Strikingly, however, both Artemis and wt cells harbor a similar level of chromosome breaks per mitotic cell at times when the checkpoint is released (which is delayed in Artemis cells). Hence, the total number of chromosome breaks is only marginally higher in Artemis than in wt cells although they arise with delayed kinetics in Artemis cells. This demonstrates the efficiency of checkpoint arrest in limiting the impact of a repair defect, i.e., the co-operative interplay between checkpoint and repair function. Because of the lack of checkpoint arrest, A-T cells display elevated numbers of chromosome breaks early after irradiation which then decreases because of repair and depletion of the G2 population. The total number of chromosome breaks in a population of irradiated G2 A-T cells is several-fold higher than in normal and Artemis cells (see Deckbar et al. 2007 for details of this analysis)

A consideration of these issues raises the interesting question of how signaling is maintained as cells initiate the process of HR. The available evidence suggests that NHEJ and ATM signaling can function together at DSBs – thus NHEJ is largely unaffected by the absence of ATM and similarly, ATM signaling is activated

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normally at DSBs in the absence of NHEJ proteins (although arrest is maintained for a prolonged period due to persisting unrepaired DSBs). This suggests that ATM can be recruited and remain active at non-resected DSBs despite ongoing NHEJ. Following resection at a DSB, the ss DNA generated is rapidly bound by RPA, initiating the recruitment and activation of ATR and Chk1 as described above. However, this process is rapidly followed by the replacement of RPA-coated ss DNA with Rad51. This raises the important question of how signaling to Chk1 is maintained at DSBs undergoing HR. Interestingly, studies in yeast, where most DSB repair is carried out by HR, show that Crb2, the yeast homologue of 53BP1, is required for the maintenance of checkpoint arrest raising the possibility that the mediator proteins help maintain damage response signaling as the process of HR proceeds (Nakamura et al. 2005). Additionally, HR proteins themselves have also been suggested to help sustain checkpoint arrest (Badie et al. 2009). A further possibility is that the lack of efficient ATM and ATR signaling to DSBs during later stages of HR contributes to the insensitivity of G2/M checkpoint maintenance. The discussion above reflects the sensitivity of the G2/M checkpoint in maintaining arrest once initiated. A related, and arguably more important, question is the number of DSBs required to activate the G2/M checkpoint. Substantial evidence has now shown that for most primary cell lines, the G2/M checkpoint arrest is not efficiently activated following exposure to doses below 0.5 Gy (Marples et al. 2004; Deckbar et al. 2007; Fernet et al. 2009). Although it is difficult to distinguish transient checkpoint activation from the complete lack of checkpoint activation, we have not observed any significant perturbation in primary human fibroblasts exposed to 0.25 Gy X or g-rays. Assuming that 1 Gy X-rays induce ~25 DSBs in G1 phase (i.e., 50 DSBs in G2 phase cells), this suggests that the introduction of 12–15 DSBs does not activate G2/M phase checkpoint arrest – a number of DSBs remarkably similar to the threshold at which checkpoint arrest is not maintained (Kegel et al. 2007). Interestingly, the phenomenon of low dose radiation hypersensitivity has also been attributed to a failure of low dose radiation exposure to activate G2/M checkpoint arrest (Marples et al. 2004; Fernet et al. 2009).

3.4.4 Role of Damage Response Mediator Proteins in G2/M Checkpoint Arrest Perhaps surprisingly, although the DNA damage mediator proteins have frequently been described as “checkpoint” proteins, and, indeed MDC1 derives its name from this phenotype, loss of mediator protein function confers only a modest defect in checkpoint arrest, only being observed following exposure to low radiation doses (Fernandez-Capetillo et al. 2002; Wang et al. 2002a; Stewart et al. 2003; Lou et al. 2006). It has, therefore, been argued that the mediator proteins, H2AX, MDC1, and 53BP1, function primarily to amplify ATM signaling with their role only being detectable at low doses when amplification is required to generate a sufficiently strong signal to activate the checkpoint arrest machinery (Fernandez-Capetillo et al.

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2002). A possible model based on current understanding of DDR signaling is that this is achieved by their ability to enhance the retention of ATM at the DSB (Noon et al. 2010). In this context, it is also significant that recent in vitro studies suggest that 53BP1 enhances ATM activation when the MRN concentration is low (Lee et al. 2009). Strikingly, our recent studies suggest that the mediator proteins are important for maintaining checkpoint arrest following exposure to high radiation doses (Shibata et al. 2010). Significantly, this demonstrates a potential value of their “amplification” role after exposure to high doses of IR, a role which appears to be significant for the maintenance of genomic stability (see below). Interestingly, ATM and the mediator proteins have recently been shown to be required for the slow component of DSB repair, which represents the repair of DSBs located at regions of heterochromatin (Goodarzi et al. 2008; Noon et al. 2010). Further, their role involves the concentrated, localized phosphorylation of KAP-1, a heterochromatic building factor (Noon et al. 2010). Thus it is possible that KAP-1 phosphorylation is also important for signaling from DSBs located at regions of heterochromatin and that this represents the “amplification” step. Interestingly, previous studies have suggested that chromatin compaction limits the strength of the DNA damage signaling response and that an enhanced response is observed when histone H1 levels are diminished (Murga et al. 2007). Indirect evidence suggests that heterochromatin may also create a barrier to ATM signal expansion, which is, at least partially, relieved by ATM-dependent phosphorylation of Kap1 (Jeggo, unpublished findings). This may be important in maintaining checkpoint arrest since heterochromatic DSBs are repaired slowly.

3.5 G1/S Arrest In mammalian cells, there is strong evidence for a process of G1/S phase arrest that is dependent upon p53 (Kastan et al. 1992). This process, which does not appear to be present in yeast, appears to be particularly important in multicellular organisms in preventing the proliferation of damaged cells and, thereby, represents an important step in cancer avoidance (Kastan and Bartek 2004). Additionally, there is evidence for another process that prevents G1/S progression that functions via ATM-Chk2. We will first describe p53-dependent G1/S arrest, then discuss evidence for a second process more akin to the mechanism underlying G2/M checkpoint arrest.

3.5.1 p53 Dependent G1/S Arrest p53 is subject to complex regulation, which occurs at both the transcriptional and post-translational level. p53 stability is regulated predominantly by the ubiquitin ligase, MDM2, which binds to the N-terminal region of p53, ubiquitylates the C terminus and targets it for proteasome-dependent degradation, thereby ensuring low levels of p53 in undamaged cells (Khosravi et al. 1999). ATM, ATR, and the transducer kinase, Chk2, can phosphorylate both MDM2 and p53 (Kastan et al. 1992;

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Hirao et al. 2000; Bartek and Lukas 2001b). Phosphorylation of p53 or MDM2 diminishes MDM2 binding to p53, leading to decreased p53 ubiquitylation and consequently increased p53 stability. These processes additionally impact upon p53 transcriptional activity. p53 is a transcriptional regulator of proteins that harbor p53 binding motifs. The Cdk inhibitor, p21waf1/Cip1, is the most significant p53 regulated gene in the context of G1/S checkpoint arrest (Harper et al. 1993; Sherr and Roberts 1999; Bartek and Lukas 2001b). However, p53 also regulates MDM2, providing a feedback, autoregulatory mechanism. Thus, following DNA damage, p53 is rapidly activated by post-translational modifications, leading to enhanced levels of p21, which occurs more slowly since it requires transcriptional activation. Entry into S phase is regulated by phosphorylation of Rb protein, which in turn is controlled by cyclindependent kinases (Sherr 2000). p21, a Cdk2 inhibitor, prevents Cdk2 phosphory lation of Rb and S phase entry (Sherr and Roberts 1999; Bartek and Lukas 2001b). Previous studies have suggested that the G1/S checkpoint is highly sensitive and is activated by a single DSB (Di Leonardo et al. 1994; Huang et al. 1996; Linke et al. 1997). In a recent study, we examined the sensitivity of the G1/S checkpoint and found that it was transiently activated by doses less than 100 mGy, suggesting that it is, indeed, highly sensitive (Deckbar et al. 2010). We observed that irradiated G2 cells that were released from G2/M checkpoint arrest were subsequently arrested at the G1/S checkpoint, suggesting that the G1/S checkpoint provides a barrier to counteract the inefficiency of the G2/M checkpoint. However, we revealed a significant limitation of the G1/S checkpoint, namely that full arrest did not occur for >4 h post-IR. We attribute this feature to the time required to fully activate p21 since transcriptional activation is a slower process than post-translational modification. It should also be mentioned that although p53 is a substrate for both ATM and ATR, after IR exposure G1/S checkpoint arrest is ATM-dependent, most likely because ATR is not activated in G0/G1 cells as discussed above (Lavin 2008).

3.5.2 A Second Process Inhibiting S Phase Entry After Radiation Exposure Previous studies have shown that, in a similar manner to the G2/M checkpoint, the Cdc25 phosphatases can regulate CyclinE-Cdk2 activity in G1 phase and that DNA damage results in the rapid destruction of Cdc25A via ubiquitylation and proteasome mediated degradation (Mailand et al. 2000). These findings, therefore, provide strong evidence for the existence of another process regulating G1/S entry (Bartek and Lukas 2001a). This process has been proposed to represent an early response, thereby providing a dual wave of G1/S regulation; an early Cdc25A-dependent process and a more slowly activated p53-dependent process (Bartek and Lukas 2001a). Our analysis of G1/S entry provided findings consistent with this (Fig. 3.4). Following IR exposure, we did not observe complete blockage to S phase entry until >4 h post-IR treatment but rather a slowing in the rate of S phase entry. Such slowing was ATM-Chk2 dependent and Chk1 independent demonstrating that it represents a regulated DNA damage response. Since p53 enhances S phase entry even in undamaged cells, it was unclear

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Fig. 3.4 Two processes delay entry from G1 into S phase following IR exposure. DSBs activated in G1 phase lead to ATM activation. ATM phosphorylation of Chk2 leads to Cdc25 activation and causes a delay but not complete arrest of entry into S phase. ATM also phosphorylates p53, which activates p21. This process causes a complete block in S phase entry. However, this process requires transcriptional activation and thus leads to a block in S phase entry that is only observed >4 h postirradiation. Thus, at early times post-IR, a delay in S phase entry is observed following by a defined blockage at slightly later times

whether this process is p53 dependent. Taken together with the previous analysis of the impact of IR exposure on Cdc25 stability, our findings suggest that this represents an initial process that regulates G1/S entry but does not confer complete blockage (Mailand et al. 2000). At >4 h after IR exposure, ATM activates p53-dependent G1/S checkpoint arrest via p21 activation. Although the p53-dependent process has a low threshold of sensitivity, i.e., it is activated by 1–3 DSBs, it is activated slowly allowing damage cells to enter S phase at early times after IR exposure.

3.5.3 Maintenance of G1/S Checkpoint Arrest Previous studies have suggested that p53-dependent checkpoint arrest permanently eliminates damaged cells rather than providing a transient delay to enhance the time for DSB repair (Di Leonardo et al. 1994; Wahl et al. 1997). However, in our recent study, we observed that cells can reenter S phase following full G1/S checkpoint activation (Deckbar et al. 2010). Indeed, given the fact that primary fibroblasts are predominantly in G0/G1 phase and that G1/S phase is activated in all cells even following low dose exposure, the ability of these cells to survive IR demonstrates that

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they must be released in a timely manner following checkpoint activation. Indeed, we observed that following exposure to low IR doses (<2 Gy), the duration of G1/S checkpoint arrest is dose dependent and that cells released from G1/S checkpoint arrest enter S phase at a rate similar to that of untreated cells (Deckbar, et at. 2010). However, following exposure to higher IR doses, the G1/S checkpoint was not efficiently maintained. Following exposure to doses >4 Gy, we observed that a low number of cells (~5%) can enter S phase at later times even though DSB repair is not yet completed. The rate of S phase entry is slower than observed in untreated cells, suggesting that it does not represent release of the entire G1 population but rather the failure of a few cells to remain arrested. Analysis of DNA DSB levels assessed by gH2AX foci enumeration and by PCC formation provided strong evidence that DSB repair is not completed when the cells enter S phase. Thus, we suggest that the G1/S checkpoint even if fully activated also has limitations that can affect its maintenance and hence its efficacy for preventing genomic instability (Fig. 3.5).

Fig. 3.5 The limitations of G1/S and G2/M checkpoint arrest. At early times post-IR, G1/S checkpoint arrest slows but does not abolish S phase entry allowing cells to enter S phase. By 6 h postirradiation, full arrest of S phase entry is observed following exposure to doses >1 Gy. However, G1/S arrest is not efficiently maintained and slow release of cells with DSBs can occur. Such cells can progress into G2 phase with unrepaired DSBs. The G2/M checkpoint arrest is initiated within 1 h of IR exposure. However, the G2/M checkpoint has a defined threshold of sensitivity and cells are released into mitosis when they harbor 10–20 DSBs. This can generate mitotic cells with DSBs. The hatched regions represent periods when cells can enter S phase or mitosis with DSBs

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3.6 Intra-S-Phase Checkpoint Arrest Studies in yeast and mammalian cells have also demonstrated that there is a process(es) that monitors replication fork integrity in S phase and functions to stabilize replication forks following their arrest (Branzei and Foiani 2009; Segurado and Tercero 2009). Additionally, DNA damage in S phase can lead to transient arrest of origin firing (Caspari and Carr 1999). These processes will only be discussed briefly. An extremely early characterized feature of cell lines derived from ataxia telangiectasia patients, a human disorder caused by mutations in ATM, was a phenomenon called radioresistant DNA synthesis, now known to be due to a failure to elicit S phase checkpoint arrest (Painter and Young 1980). Thus, while control cells show a rapid and pronounced decrease in DNA replication, normally monitored by thymidine incorporation, at 1–4 h postirradiation, A-T cells continue to traverse S phase at a rate similar to that observed in untreated cells. This is now known to represent ATM-dependent inhibition of Cdc25A phosphorylation that likely occurs via two independent pathways that involve NBS1 and Chk2 (Falck et al. 2001; Falck et al. 2002). This process, at least in part, involves inhibition of Cdk2-dependent loading of Cdc45 onto replication origins (Falck et al. 2002). At later times (>4 h) post-IR, S phase arrest is ATM-independent but ATR-Chk1 dependent, a process considered to represent the inhibition of fork elongation and/ or origin firing via an ATR-dependent mechanism most likely from ss regions of DNA generated at stalled replication forks (Zhou et al. 2002). Although not studied in detail, it is likely that the intra-S-phase checkpoint also has limitations. This is perhaps most convincingly demonstrated by the observation that S phase cells can progress to G2 phase following high dose exposure (e.g. 6 Gy), even in cells with impaired DSB repair (Krempler et al. 2007).

3.7 Significance of Cell Cycle Checkpoint Arrest The DDR functions to achieve two distinct goals, enhancing survival and maintaining genomic stability. Although these two end points are related they are distinct. Indeed, the maintenance of genomic stability may necessitate decreasing survival to achieve the elimination of damaged cells and enhancing survival by increasing genomic instability is an important step in the etiology of carcinogenesis. Checkpoint arrest likely enhances survival by allowing more time for DSB repair. However, although repair can progress in the subsequent cell cycle stage, the accuracy of repair will be comprised. Thus, for example, progression through mitosis and cytokinesis in the presence of DSBs may result in loss of acentric fragments diminishing the opportunity to rejoin the correct DNA ends. Further, replication past a DSB enhances the opportunity for translocation events. The ability to repair DSBs makes the greatest impact on survival post-IR since unrepaired DSBs are particularly lethal lesions, and cells lacking DSB repair mechanisms, e.g., NHEJdeficient cell lines, are dramatically radiation sensitive. Although there are multiple

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routes for repairing DSBs including telomere fusion, end capping, and translocation, most of them utilize NHEJ proteins. Thus, mice and patients deficient in NHEJ proteins show dramatic radiation sensitivity but only modestly increased carcinogenesis. This is perhaps best demonstrated by SCID mice which show dramatic radiation sensitivity but little elevated radiation-induced tumorigenesis (Bosma and Carroll 1991). In short, diminished DSB repair dramatically enhances radiation sensitivity with only modestly enhanced instability. In contrast, checkpoint arrest most likely exerts a significant impact on genomic instability compared to survival. The phenomenon of low dose radiation hypersensitivity, however, represents the enhanced sensitivity to low radiation doses. Recent evidence has consolidated the model that this phenomenon arises as a consequence of a failure to activate G2/M checkpoint arrest following low doses strongly suggesting that checkpoint arrest can enhance survival (Marples et al. 2004; Marples and Collis 2008; Fernet et al. 2009). The overlapping roles of Chk1 and Chk2 in checkpoint arrest and the fact that Chk1 is essential makes it difficult to assess the impact of a checkpoint defect. However, loss of p53, which abolishes the late G1/S checkpoint results in enhanced survival and genomic instability, consistent with the notion that checkpoint arrest might exert a greater impact on the maintenance of genomic stability. However, p53 also regulates apoptosis limiting defined conclusions. The loss of ATM confers loss of checkpoint arrest coupled with comprised DSB repair (Lobrich and Jeggo 2005). Artemis defective cells harbor the same DSB repair defect as ATM defective cells but are checkpoint proficient. We previously compared chromosome breakage arising from irradiated G2 phase cells in A-T vs. Artemis null cells (Deckbar et al. 2007). Strikingly, we observed that although Artemis null cells showed only modestly enhanced chromosome breakage in mitosis, demonstrating the efficacy of the G2/M checkpoint in preventing the progression of damaged cells, loss of ATM conferred a high level of chromosome breakage. Use of UCN01, a checkpoint inhibitor, to abolish checkpoint arrest, conferred a small defect similar to that observed in Artemis-defective cells. Thus, we suggest that loss of either checkpoint arrest or a DSB repair defect enhances chromosome breakage but loss of both is more than additive (Deckbar et al. 2007; Lobrich and Jeggo 2007). This likely explains the dramatically increased cancer predisposition and genomic instability observed in A-T.

3.8 Conclusions The activation of cell cycle checkpoint arrest is a critical end point of DNA damage response signaling that plays an important role in limiting genomic instability and also contributes to enhancing survival following radiation exposure. Two distinct processes that arrest (or slow) entry from G1 into S phase have been described (Fig. 3.4). Intra-S-phase arrest slows progression through S phase and G2/M arrest prevents entry into mitosis. Radiation exposure induces DSBs, SSBs, and base damage, but only DSBs directly activate damage response signaling via ATM.

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However, ATR can be activated following replication fork stalling or collapse in S phase or following resection in S/G2 phase. This provides a complex signaling response that differs with cell cycle phase (Fig. 3.1). Cell cycle checkpoint arrest is a sensitive process yet has defined limitations (Fig. 3.5). An evaluation of these limitations is important for considering the impact of radiation on genomic stability and survival. Further understanding of the processes can enable them to be exploited to enhance the therapeutic potential of radiotherapy.

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Chapter 4

Chromatin Responses to DNA Damage Karina Falbo and Xuetong Shen

Abstract The preservation of genome stability depends upon tightly regulated mechanisms that continuously search the genome for irregularities that, if left ignored, could be dangerous to both the genomic integrity and cell survival. These mechanisms are mediated by a multiplicity of proteins and factors that physically approach the DNA to either search for the damage or to fix it. Therefore, in eukaryotic cells, where the DNA molecule is tightly packed into nucleosomes forming a highly compacted structure, the chromatin, access of these factors to the DNA molecule represents an additional step, as well as an opportunity for extra regulatory mechanisms. Although a plethora of information has been accumulated over the past years on the factors and mechanisms involved in the response to DNA damage, very little is known about the role of the chromatin structure itself on the DNA damage response (DDR). Interestingly, several remodeling complexes have recently been described to be involved in the DNA damage response. In particular, the INO80 remodeling complex seems to be involved in several aspects of the DDR response. Thus, this chapter will describe the novel roles of the INO80 remodeling complex in DNA damage tolerance, double strand break repair (DSB) and telomere maintenance. Keywords INO80 • Chromatin remodeling

4.1 Chromatin Remodeling is an Integral Component of the DNA Damage Response The therapeutic activity of ionizing radiation on tumors is primarily based on the cell cytotoxicity derived from the inhibitory effects of radiation on vital cellular processes such as DNA replication and transcription. Radiation also induces the activation of

X. Shen (*) Department of Carcinogenesis, The University of Texas, MD Anderson Cancer Center, MDA SP/RD, Unit 116, 1808 Park Road 1-C, Box 389, Smithville, TX 78957, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_4, © Springer Science+Business Media, LLC 2011

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the so-called DNA damage response, a surveillance mechanism responsible for the preservation of genetic stability. Interestingly, therapeutic inactivation of this response seems to represent a window of opportunity to enhance the effects of radiation therapy (Ljungman 2009). As part of the DDR, several proteins have been identified and studied in the past few years leading to a clear understanding of the different steps involved in these repair processes (Powell and Bindra 2009). However, since the DNA is not naked but wrapped around histone proteins forming a highly compacted structure, the chromatin, it became evident that remodeling of the chromatin might be an essential component of the DDR (Morrison and Shen 2006, 2009). Furthermore, recent investigations have shed light on the importance of chromatin structure during the DDR opening new questions on whether chromatin remodeling complexes could represent a new player in the field of radiation oncology.

4.2 The Chromatin Environment In order to fit the long DNA molecule into the nucleus, eukaryotic cells evolved to create a highly compacted structure, the chromatin. In the chromatin, the DNA molecule is wrapped around proteins called histones forming the basic chromatin unit, the nucleosome. Nucleosomes interact with each other to further fold the DNA molecule to create a higher level of compaction, the so-called 30 nm fiber (Staynov 2008). Thus, as a consequence of this complexity, the chromatin creates impediments and constrains to nuclear processes that require access to DNA, such as DNA replication or the DDR. Histones, a group of highly evolutionarily conserved proteins, are the main mediators of DNA folding into the chromatin structure, since they can interact with each other, with the DNA and with other chromosomal proteins. Each nucleosome core is formed by two molecules of each of four different histone proteins: H2A, H2B, H3, and H4, with histones H2A and H2B forming a stable dimer (H2A/H2B) and histones H3 and H4 forming a stable tetramer (H3/H4)2 (Vernet et al. 1990). The H3/H4 histone tetramer has a pivotal role in nucleosome organization, while the H2A/H2B dimer is mainly responsible for the interaction between the DNA and the nucleosome core. Finally, the histone fold domains of the core histones are responsible for organizing the DNA around the histone core, primarily through electrostatic interactions (Luger et al. 1997). Histones are actively involved in the regulation of the chromatin environment, through their ability to be evicted, exchanged or displaced along the DNA (Huertas et al. 2009). In addition, histones are posttranslationally modified by phosphorylation, ubiquitylation, methylation, acetylation, etc., at both the N-terminal tail as well as at the globular domain, modifications that are essential for many nuclear processes. Interestingly, while the role of histone mobilization, exchange, or eviction in the DDR is essentially an unexplored area, the involvement of posttranslational modifications is, instead, in active investigation (Altaf et al. 2007). Specifically, in the yeast Saccharomyces cerevisiae, histone H2AX is phosphorylated at Ser129 in

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the region around a double strand break (Rogakou et al. 1998). The phosphorylated form of H2AX, gH2AX, occurs in cis on the DNA and extends in just minutes to around 50–100 kb from the DSB in both directions after irradiation. Moreover, the recruitment of factors involved in the early steps of DSB repair depends on H2AX phosphorylation, supporting a pivotal role for this posttranslational modification in the chromatin structure and in the response to DNA damage. Finally, although very little is known about the role of chromatin structure in the DNA damage response, several lines of evidence indicate that nucleosome mobilization, exchange, and eviction mediated by chromatin remodeling complexes constitute a central component of this response. Therefore, this chapter will focus on the rapidly expanding research related to the role of chromatin structure and chromatin remodeling complexes in DNA damage-related nuclear processes.

4.2.1 Chromatin Remodeling Complexes and the Dynamic Nature of Chromatin The dynamic nature of the chromatin is possible thanks to the concerted activities of several chromatin modifier factors. In fact, these factors are responsible for the regulation of essential nuclear processes such as: covalent modification of histone tails, histone exchange, DNA methylation and nucleosome mobilization. Here, we will focus on a subgroup of chromatin modifiers that utilize the energy provided by ATP to mobilize nucleosomes or exchange histones, the ATP-dependent remodeling complexes. More specifically, we will focus our discussion on the INO80 remodeling complex, since the last few years have witnessed an accelerated rate on the discovery of novel functions for this complex, including the DNA damage tolerance pathways during replication and DSB repair.

4.2.2 The INO80 Remodeling Complex The INO80 remodeling complex is an evolutionarily conserved complex that bears both an ATPase activity and a 3¢–5¢ helicase activity. Even though the mechanism is still unclear, INO80 can mobilize nucleosomes using the energy provided by ATP (Shen et al. 2000). In fact, the first function attributed to INO80 was during transcription, where INO80 binds to the promoter regions of certain activated genes and mobilizes the surrounding nucleosomes, using energy provided by ATP, to allow access to the DNA by transcription factors (Shen et al. 2003b). Furthermore, in the last few years, several novel transcriptional-independent functions were discovered, including regulation of DSB repair, DNA damage tolerance response during DNA replication, and telomere maintenance (Fig. 4.1). Thus, the main discussion topics of this section will be the INO80 structural characteristics that relate to these novel research areas.

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a

b

DSB repair

Other Arp8 subunits

ATP

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DNA replication

Other Arp8 subunits ATP

Ino80

Nhp10 P

PCNA

H2AX Nucleosome eviction Strand invasion Homologous Recombination

c

Telomere maintenance Other subunits

ATP

Ino80

Ies3 Est1

Rad18 Rad51

Template switch Damage avoidance

Promotion of recombinational telomere maintenance?

Fig. 4.1 INO80 is involved in several nuclear functions. (a) INO80 activities at DSBs. After MMS treatment, INO80 binds to double strands breaks and mediates homologous recombination by interaction with the phosphorylated form of histone H2AX. This interaction depends on H2AX phosphorylation and the INO80’s subunit Nhp10. At the DSBs site INO80 evicts or displaces nucleosomes facilitating the proper recruitment of HR factors, such as Rad51. (b) INO80 activities during DNA replication. When replication forks face an obstruction, such as an MMS adduct, INO80 binds to the DNA to help process stalled replication forks. INO80 allows proper recruitment of DNA damage tolerance factors leading to fork resolution though template switching and the completion of DNA replication without DNA damage generation. (c) INO80 activities at telomeres. INO80 is involved in the regulation of telomere structure and function. Ies3 subunit interacts with the Est1 telomeric protein, suggesting a direct role for INO80 at telomeres

Structurally, INO80 is composed of 15 subunits: Ino80, Arp8, Rvb1, Nhp10, Rvb2, Arp4, Arp5, Actin, Ies1, Ies2, Ies4, Anc1/Taf14, Ies5, and Ies6. Interestingly, while some of these subunits are well characterized, some others still remain to be investigated. However, due to the extensive list of subunits and space limitations, we will consider only the subunits that are relevant to our discussion (Bao and Shen 2007). Probably the most important subunit is Ino80 that confers the ATPase activity. Ino80 contains the GXGKT motif at the ATPase domain, a highly conserved nucleotide-binding motif that also contains a lysine residue known to interact with an ATP phosphate, and whose alteration renders the INO80 complex nonfunctional. The INO80 complex also contains several Arp subunits with diverse functions. Arp5 and Arp8 are INO80 exclusive subunits, and both Arp4 and actin incorporate into the complex depend on Arp8. In addition, Arp5 and Arp8 are important for the chromatin remodeling activity of the complex, while Arp4 is the only subunit that binds ATP (Shen et al. 2003a). Furthermore, Arp8 and Arp4 are known to interact in vitro and in vivo with histones H3 and H4, possibly to perform chaperone activities between histones and the DNA. Another important subunit is Nhp10, an HMG-1 like protein that could potentially bind to structured DNA or nucleosomes. Nhp10 is not important for INO80-mediated nucleosomes mobilization, but it is relevant for the recruitment of Ies3 into the complex (Bao and Shen 2007). More importantly, we will discuss later in this chapter, a key interaction between Nhp10 and gH2AX during DSB mediated by INO80. Finally, the molecular functions of Ies1, 3, 4, and 5 are still unclear; however, we will discuss below recent findings that provide interesting clues about the putative roles of some of these subunits in important nuclear functions. Therefore, the

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diversity and complexity of activities performed by the different INO80 subunits support the view that the high degree of conservation reflects a pivotal, essential, and versatile role of this complex in vital nuclear processes.

4.3 INO80 is Directly Involved in the DNA Damage Response The most important effect of ionizing radiation is the generation of DNA double strand breaks, lesions that are repaired by either homologous recombination (HR) or nonhomologous end joining (NHEJ). Thus, cells that are sensitized by inhibition of DSB repair pathways could represent an excellent target for selective therapeutic radiation (Friedberg et al. 2006; Shrivastav et al. 2008). As such, numerous inhibitors have been developed to target DNA-dependent protein kinase (DNA-PK), a key player in the NHEJ pathway. However, due to the highly compacted nature of the chromatin, providing DDR proteins access to the DNA requires additional factors, such as chromatin remodeling complexes. Surprisingly, very little is known about the role of chromatin structure in the DDR response. Indeed, three chromatin remodeling complexes have recently been shown to be involved in DDR in the yeast S. cerevisiae: SWI/SNF, RSC, and INO80 (Osley and Shen 2006), opening a novel research area for the exploration of new therapeutic options. The INO80 remodeling complex was originally suspected to be involved in the DDR response due to the sensitivity of the ino80, arp8, and arp5 mutants to agents that cause DSB, such as ionizing radiation (IR) and methyl methanesulfonate (MMS) (Shen et al. 2000, 2003a; Morrison et al. 2004; van Attikum et al. 2004; Tsukuda et al. 2005). Also, it was well established that INO80 binds to the promoter regions of a multiplicity of genes to direct nucleosome mobilization. As such, one possibility was that the sensitivity of the mutants to DNA-damaging agents could be a consequence of INO80 remodeling activity at promoter regions of genes involved in DDR. Nonetheless, as it was later discovered, it was also possible that INO80 activity at DDR was direct instead of transcription-related.

4.3.1 The INO80 Remodeling Complex Binds to Double Strand Breaks After DSB formation, DNA integrity can be restored by two major mechanisms: an error prone process, where the DNA broken ends are directly ligated through a nonhomologous end-joining mechanism, or an homologous recombination process that involves copying information from a sister chromatid or homolog. Due to the highly compacted nature of the chromatin, both processes require chromatin changes to pave the way for the DDR machinery to access the DSB site. Interesting insights into the putative direct role of the INO80 remodeling complex during the DDR response came from investigations by Morrison et al. and van Attikum et al. with the discovery that the INO80 remodeling complex is directly involved in the DDR response.

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This original discovery was performed using a well-studied genetic system developed by J. Harber. In the yeast S. cerevisiae, a single DSB can be created at the MAT locus by induction of a specific HO endonuclease. This single DSB cannot be efficiently repaired due to the lack of recombination donors, thus, allowing the characterization of recruited DNA repair factors to the DSB using chromatin immunoprecipation (Moore and Haber 1996; Haber 2000). In this model system, chromatin immunoprecipitation (ChIP) analysis of flag-tagged versions of Ino80, Arp8, and Arp5 shows that these subunits are recruited to a DNA region that extends up to 9 kb from the HO-induced DSB break. In addition, the kinetics of recruitment to DSB sites is similar to that of several proteins involved in DSB repair, suggesting a coordinated function of INO80 with other repair factors (Morrison et al. 2004; van Attikum et al. 2004).

4.3.2 INO80 Binding to Double Strand Breaks Depends on Histone H2AX Phosphorylation One of the first responses to DNA damage in yeast is the recruitment of the phosphorylated form of histone H2AX (gH2AX) to DSBs (Downs et al. 2000; Redon et al. 2003). gH2AX is detected in the region surrounding the DSB within 15 min after induction of an HO cut (Shroff et al. 2004). Interestingly, the observation that INO80 associates with histones under physiological conditions, together with the finding that INO80 binds to DSBs, led Morrison and Van Attikum to envision a mechanism in which INO80 recruitment to DSBs is, somehow, mediated by phosphorylation of H2AX. In fact, the phosphorylated form of H2AX is enriched with the INO80 complex in cell cultures treated with MMS to induce DSBs. Using an antibody that specifically recognizes gH2AX, INO80 can be detected in association with gH2AX when the complex is purified under mild salt conditions. Moreover, INO80 recruitment to DSBs is significantly reduced in a strain bearing a mutation at the H2AX serine residue that is phosphorylated, indicating that INO80 recruitment to DSBs is mediated by H2AX phosphorylation. Therefore, after DSB induction, INO80 binds preferentially to the phosphorylated form of H2AX present at the DSB and this interaction depends on H2AX phosphorylation (Fig. 4.1). Interestingly, several INO80 subunits are dispensable for INO80 recruitment to DSBs, including the Arps. However, only Nhp10, an exclusive INO80 subunit, is necessary for the INO80–gH2AX interaction. Thus, since Nhp10 deletion does not significantly affect INO80 remodeling activity (Shen et al. 2003b), it can be reasoned that the Nhp10 subunit confers upon INO80 the exclusive capability to bind to gH2AX independently of its remodeling activity. Finally, the direct role of INO80 at DSBs is strongly supported by data collected by comparing the global transcription profile of the wild-type and the ino80 strains. As such, a survey of the global transcription profile of the ino80 mutant reflects no major changes in the expression profile of genes associated with DNA damagerelated pathways, including cell cycle arrest, checkpoint activation, homologous

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recombination and nonhomologous end joining (Mizuguchi et al. 2004), reinforcing the idea that INO80 activities at HO sites are not related to the complex ability to remodel the chromatin at promoters (Shen et al. 2000), but rather to a direct activity of INO80 at the HO cut.

4.3.3 INO80 is Involved in Homologous Recombination-Mediated DSB Repair INO80’s presence at DSBs and its interaction with gH2AX led to the early speculation that the complex could be necessary for NHEJ and/or HR at DSBs. In yeast, the Mre11–Rad50–Xrs2 complex is recruited to the DNA immediately after DSB formation to mediate DNA resection that leads to ssDNA formation (Lee et al. 1998; Nakada et al. 2004), an early step in HR. Interestingly, this original speculation was supported by the observation that in S. cerevisiae, 5¢–3¢ resection of HO-induced DNA ends is reduced in the arp8 mutant, when assessed by a quantitative PCR-based amplification assay (Morrison et al. 2004). However, there are currently two opposing views based on the investigations of two different labs. In one study, both the arp8 and nhp10 mutants were shown to be defective in strand resection when assessed by an RT-PCR method called QAOS (van Attikum et al. 2004). Using this method, ssDNA can be detected within 1 hr at a site located 1.6 kb of the HO cut in a wild-type strain, but the signal is significantly reduced in the arp8 and nhp10 mutants, which leads to the conclusion that chromatin remodeling driven by INO80 favors DNA end processing. Moreover, recruitment of Mre11, a factor involved in DNA resection, is significantly reduced in the arp8 mutant at HO sites, indicating that INO80 remodeling activity at HO sites facilitates Mre11 recruitment that leads to proper DNA resection. In a second independent study, using Southern blot to detect strand resection at the MAT locus, and a recruitment assay to measure the association and spreading of RPA with single-stranded MAT DNA, Tsukuda et al. described that end-strand resection occurs normally in the arp8 mutant even though the recruitment of Rad51 and Rad52, two proteins involved in stand invasion, is delayed in the same mutant (Tsukuda et al. 2005). Therefore, this data is in agreement with a theory in which the INO80 complex binds to HO sites and allows the recruitment of early HR factors, possibly by regulating DNA resection at the DSB. Further research is required to clarify these disparate views.

4.3.4 INO80 is Involved in the Early and Late Steps of Homologous Recombination INO80 has been described to be indispensable for HR repair in several organisms. In Arabidopsis, the frequency of HR is reduced by 15% in a mutant that does not

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express INO80. Interestingly, reports in yeast seem to indicate that INO80 has no significant effect on the frequency of HR-mediated DSB repair when assessed in a haploid HO MAT system that lacks the donor sequences (van Attikum et al. 2004; Tsukuda et al. 2005). Thus, the disparity in the data described above, as to whether or not INO80 affects the outcome of HR-mediated DSB repair, prompted the use of a different genetic system to further analyze DSB repair in a step-by-step approach to address this issue. Using a diploid HO MAT system where the donor sequence is present, Tsukuda et al. showed that the total allelic HR frequency is reduced approximately fivefold with all HR classes reduced to a similar extent in mutants that lack INO80. In this allelic system, homologous recombination between chromosomes is stimulated by a single HO DSB generated in an ura3 allele, allowing the analysis of conversion tract lengths, G2 crossovers, DSB-dependent chromosome loss, break-induced replication (BIR), and DSB-induced cell death. Remarkably, this study showed that Arp8 is necessary during early and late steps of HR (Tsukuda et al. 2009). In fact, during early HR, Arp8 accelerates the strand invasion step, despite the fact that it does not affect DNA resection. Indeed, in an HO MAT system where the donor sequences are available, strand invasion is dramatically impaired in the arp8 mutant when assessed using a single-end invasion assay (SEI), a PCR-based assay that allows quantification of the initial strand invasion/repair synthesis reaction after a DBS. Furthermore, dissection of the late HR steps using the ura3 system reveals important differences. First, the frequency of conversion tracts, a hallmark of defective HR, is increased in the arp8 mutants. Second, the frequency of marker conversion as a function of distance from the DSB, another parameter related to tract length, is altered in the arp8 mutants. In wild-type cells, markers 5¢ of the DSB convert more frequently than equidistant 3¢ markers, but in the arp8 mutant several 3¢ markers convert at significantly higher frequencies that in wild type. Thus, since the Arp8 subunit is indispensable for INO80 ATPase activity, it can be inferred from the analysis of HR in diploids that chromatin remodeling mediated by the INO80 complex regulates both early stand strand invasion and the later steps that control gene conversion, tract length, and continuity (Tsukuda et al. 2009).

4.3.5 INO80 Can Evict Histones at DSBs An important question on the role of chromatin remodeling during DSBs refers to the putative mechanism involved, and in this regard, several lines of evidence indicate that ATP-driven remodeling activity is involved in INO80 activities at DSBs. As described above, arp8 mutants that lack the INO80 ATPase activity are defective in several HR steps. Also, a point mutation that specifically abolishes the INO80 ATPase remodeling activity is sensitive to chemicals that induce DSBs. Nonetheless, robust data addressing whether or not the chromatin remodeling activity is important during DSB repair comes from the analysis of nucleosome and histone occupancy at DSBs showing that the INO80 complex is involved in nucleosome eviction at DSBs.

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The HM loci is tightly packaged into heterochromatin, thus, during MATa ating-type switching, the normally silenced HO site in the HMLa that is occluded m by a strategically positioned nucleosome is cleaved, indicating chromatin remodeling takes place at this locus. Similarly, during MATa switching, histones located at the HMRa region are displaced, indicating that the search for homology of the invading strand requires chromatin remodeling at the donor sequence. Accordingly, analysis of histone H2B occupancy by microccocal nuclease digestion in both MATa and HMRa shows that while H2A is rapidly evicted from Mata in both wild-type and arp8 strains, H2A presence at the HMRa is retained only in the arp8 mutant. In addition, transfer of a HR mediator, Rad51, from the recipient to the donor strand that is a key step in homology search, is less efficient in the arp8 mutant, indicating INO80 plays an important role in chromatin remodeling at the donor sequence, possibly by mediating histone eviction that facilitates strand invasion by the recipient template (Tsukuda et al. 2009). Similarly, ChIP analysis of histones using the haploid HO MAT system shows that INO80 is involved in general nucleosome eviction at an HO cut. Indeed, after 1 hr of HO induction gH2AX accumulates and spreads from the DSB at MAT, peaking at +9.6 kb from the DSB in wild-type as well as in arp8 and nhp10 mutant strains. However, after 2–4 hr of HO induction, gH2AX decreases dramatically in the wild-type but not in the arp8 and nhp10 mutants, indicating that gH2AX is removed from the chromatin at an HO DSB in an INO80-dependent manner. Moreover, ChIP analysis of core histones shows similar results, a marked reduction in the presence of core histones that correlates with the reduction in H2AX occupancy in the arp8 and nhp10, leading to the conclusion that INO80 is required for the general eviction of nucleosomes at HO sites after DSB generation (Tsukuda et al. 2009).

4.3.6 INO80 is Involved in the Recruitment of DNA Repair Factors to DSBs INO80-mediated nucleosome eviction, and consequent chromatin remodeling, allows the recruitment of several proteins involved in different steps of HR. In fact, the recruitment of DSB repair factors, such as Rad51 and Rad52 and Mre11 to DSB is significantly reduced in the arp8 mutant, when assessed in a MAT strain that lacks donor loci (Tsukuda et al. 2009). Although it is still not clear whether or not Rad51 recruitment affects DNA resection (see disparate results described before), the evidence supports the fact that Rad51 recruitment impacts the efficiency of the strand invasion step during HR-mediated repair at DSBs. Accordingly, when HM donor loci are present, Rad51 is transferred from recipient MAT ssDNA to the donor template to effect DNA strand exchange. Furthermore, ChIP analysis using a MAT system in which the donor locus is present shows that while Rad51 recruitment to the Mata locus is similar in wild-type and arp8 mutants, in the MATa locus Rad51 accumulates to higher levels in the arp8 mutant, indicating INO80-dependent

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chromatin remodeling is required for efficient Rad51 transfer from the invading strand to the donor HMRa strand (Tsukuda et al. 2009). In summary, a mechanism can be envisioned in which, by evicting or displacing nucleosomes at DSBs, INO80 allows proper recruitment of proteins involved in DSB repair. Specifically, strand invasion depends on the interaction of a ssDNARad51 nucleoprotein with a donor locus, interaction that is promoted by the INO80 chromatin remodeling activity. Therefore, INO80’s remodeling activity induces nucleosome eviction specifically at the donor sequences allowing or facilitating the transfer of Rad51 from the invading to the donor strand. Interestingly, since INO80 was never reported to have histone transfer activity (Shen et al. 2000), it is possible that INO80 originally disrupts the nucleosome structure to facilitate the subsequent nucleosome displacement by a chaperone, possibly Asf1, a global chaperon known to be involved in DSB repair (Tyler 2002; Adkins and Tyler 2004; Prado et al. 2004).

4.4 INO80 is Phosphorylated at the Ies4 Subunit An important question on the role of INO80 in DNA repair concerns the regulatory mechanisms that direct INO80 activities during the DDR. Insights into this issue came from the investigations published by Morrison et al., describing the presence of a posttranslational modification in the INO80 remodeling complex. Specifically, Morrison et al. found that the Ies4 subunit of the INO80 complex is phosphorylated after MMS treatment. Indeed, 2D gel analysis of MMS-treated wild-type cultures reveals five phosphorylated Ies4 spots that map to the Ies4 N-terminal peptide that contains two serines in Mec1/Tel1 kinase (S/T)Q consensus sites. In addition, Ies4 phosphorylation after MMS treatment is impaired in strains that lack either the Mec1 or Tel kinases, early effectors in the DNA damage-related checkpoint response. After DNA damage, Mec1/Tel1 phosphorylates several checkpoint proteins, many of which have a Mec1/Tel1 kinase (S/T)Q consensus site, leading to checkpoint activation (Fig. 4.2). Remarkably, purified INO80 can be phosphorylated at the Ies4 subunit in an in vitro kinase, assay, in a mix that contains precipitated Mec1 or Tel1, indicating that INO80 is, in fact, phosphorylated by Mec1 and Tel1 and thus involved in the checkpoint pathway (Morrison et al. 2007). Interestingly, while ies4 mutants that cannot be phosphorylated by Mec1/Tel1 are not sensitive to DNA-damaging agents such as MMS, a serine to glutamic acid mutation that mimics phosphorylation results in high sensitivity to MMS, suggesting Ies4 phosphorylation is relevant during DSB repair. However, analysis of several steps of HR using the HO MAT system that repair the DSB via an ectopic donor or single strand annealing shows that none of these phosphorylation mutants are defective in any of the steps analyzed, including strand invasion, ligation of repair ends, product formation, and cell survival, indicating that Ies4 phosphorylation by Mec1/Tel1 seems to be dispensable for HR-mediated DSB repair (Morrison et al. 2007).

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Fig. 4.2 INO80 activities at DSBs are influenced by Mec1/Tel1. When cells are treated with DNA-damaging agents, Mec1/Tel1 mediates either the phosphorylation of H2AX that leads to the recruitment of INO80, or the phosphorylation the Ies4. Following INO80 binding to a DSB, some subunits such as Nhp10, Arp8, and Arp5 are involved in repair mechanisms while Ies4 is involved in checkpoint activation

Therefore, since Mec1/Tel1 activation is not only involved in HR repair but also in the early steps of checkpoint activation, it is possible that Ies4 has an active role during checkpoint pathways. After DNA damage generation in yeast, ssDNA formation induces recruitment of RPA, a single strand DNA-binding complex that consequently recruits the Ddc2–Mec1 complex, leading to Rad53 phosphorylation and checkpoint activation (Harrison and Haber 2006). Interestingly, Rad53 phosphorylation and its activity are enhanced in the ies4 mutant cells that mimic a persistent phosphorylation, which leads to the consequent enhanced checkpoint response and pronounced cell cycle arrest. Surprisingly in the arp8 and nhp10 mutants, Rad53 phosphorylation and activity are significantly reduced after induction of an HO site, as compared with a wild-type strain. Moreover, this defect could be attributed to an impaired ability of the arp8 mutant to recruit Mec1, a Rad53 downstream effector. In fact, ChIP analysis using a HO MAT system lacking a donor locus shows that while Mec1 is present at HO sites within 1 h after DSB induction, this recruitment is significantly reduced in the arp8 and nhp10 mutants (Morrison et al. 2007). In summary, after DNA damage, the INO80 remodeling complex is posttranslationally phosphorylated by the Mec1/Tel1. Thus, while the lack of the Arp8 subunit impairs Mec1/Tel1 recruitment to DSB and the consequent Rad53 phosphorylation, failure to regulate Ies4 phosphorylation induces Rad53 hyperphosphorylation and prolonged cell cycle arrest, supporting the idea that different INO80 subunits could be responsible for different aspects of the DDR response. Finally, a future extension of this research could reveal a more detailed mechanism for INO80 posttranslational modifications and its role in checkpoint activation.

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4.5 INO80 is Important for Telomere Maintenance Telomeres, the end terminal structure of chromosomes, consist of an array of repetitive sequences that, due to their resemblance to DSBs, are important for the maintenance of genomic stability. In fact, to avoid recognition by the NHEJ machinery, telomeres are arranged in a higher order nucleoprotein structure that attaches to the nuclear matrix (Misri et al. 2008). Nonetheless, despite the extensive research accumulated on the protein component of telomeres, very little is known about the role of the chromatin structure at telomeres. The chromatin structure at telomeres is unique; it is known that in eukaryotes telomere DNA is organized in tightly packed nucleosomes separated by 10–20 bp of linker DNA (Pisano et al. 2008) and that the telomeric DNA is associated with several proteins important for telomere structure and maintenance. Interestingly, several lines of evidence indicate that most DNA damage response proteins are, somehow, involved in telomere maintenance. Indeed, in yeast, the MRN complex that plays a critical role in DNA DSB repair in eukaryotes, also binds to telomeres (Lombard and Guarente 2000; Nakamura et al. 2002; Takata et al. 2005). In human cells, the MRN complex, together with ATM (Tel1), is involved in the activation of the ATR kinase (Mec1) after IR exposure (Jazayeri et al. 2006). Moreover, a similar situation is observed in yeast, where Mre11, a MRN complex component, is implicated in telomere resection and Mec1 loading during late S phase (Larrivee et al. 2004), indicating that the telomeric function of Mre11 might reflect the role of Mre11 in the recognition and processing of DNA DSBs. Therefore, the involvement of chromatin remodeling complexes in DSBs leads to the possibility that a similar process is present at telomeres. In this regard, the discovery by Yu et al. of a physical interaction between a telomeric protein, Est1, and an INO80 subunit, Ies3, have shed light into the role of chromatin remodeling at telomeres (Yu et al. 2007). Using a two hybrid screening system Yu et al. found that the Ies3 subunit of the INO80 remodeling complex interacts directly with Est1, a highly conserved polypeptide essential for telomerase maintenance and protection in vivo. Est1 is involved in telomerase recruitment to chromosome ends as well as in activation functions after recruitment that are still not well understood (Virta-Pearlman et al. 1996; Pennock et al. 2001; Seto et al. 2002; Taggart et al. 2002). In addition, Yu et al. observed that some INO80 subunits could influence the length as well as the structure of telomeres (Yu et al. 2007). As such, the moderate but significant telomere elongation characteristic of the ies3, arp8, and nhp10 mutants, observed by analysis of telomere restriction fragments, together with the significant reduction of telomere position effect (TPE) in the ies3 mutant, suggest that the INO80 remodeling complex is involved in telomere structure and function as described below. In yeast that had lost telomere proteins, such as Est1, telomeres experience attrition that leads to growth defects. Interestingly, when these mutants are subcultured, after several passages, a subpopulation of survivor cells arises with growth rates that are similar to wild-type cell rates. Moreover, it is believed that these survivors use a recombination-mediated mechanism for telomere maintenance, since loss of several recombination proteins impairs the ability of this strain to form survivors (Lundblad

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and Szostak 1989; Lundblad and Blackburn 1993; Teng and Zakian 1999; ass-Eisler and Greider 2000). Interestingly, the est1 ies3 double mutant presents K this type of growth retardation. Moreover, est1 mutant subcultured cells recover after 3 days, similar to wild type, while the est1 ies3 double mutant fails to recover until day 7, suggesting that Ies3 is important for HR at telomeres. In addition, both strains accumulate long and heterogeneous terminal restriction fragments that are typical of telomere-related mutants. However, 2D gel analysis shows that only the double mutants accumulate unusual telomeric DNA structures, such as circular DNA, as well as high levels of G strand overhangs (Yu et al. 2007). Furthermore, altered structures at telomeres can also be repaired by nonhomologous end joining, whose main products are T–T fusions (van Gent et al. 2001). Interestingly, although PCR analysis of the ino80 and est1 mutants shows no significant presence of these aberrant fusions, a high level of T–T fusions is present in the double est1 ies3 mutant, suggesting a role for the ies3 protein in NHEJ at telomeres. Similar to the situation at DBSs, the question remains as to whether or not INO80 remodeling activity is necessary for INO80 telomere-related activities. Interestingly, the ino80 mutant that lacks the ATP-dependent remodeling activity (Yu et al. 2007) does not present any major telomere defects, suggesting that the ies3 mutant effect on telomeres could be independent of any remodeling activity. However, in the arp8 mutant that is also required for the chromatin remodeling activity of INO80, telomere length, as well as formation of survivor clones, are affected, suggesting that at least some INO80 activities at telomeres are remodeling-dependent. In support of this observation, chromatin immunoprecipitation of Ino80, Ies3, and Nhp10 indicates that these subunits bind to telomeric regions, supporting the view that INO80 could be remodeling the chromatin at telomeres. Furthermore, INO80 binds to telomeres independently of Est1, suggesting that Est1 direct interaction with Ies3 may not have a structural role, such as helping INO80 bind to telomeres, but instead a functional one, such as the activation of INO80 after recruitment (Yu et al. 2007). In summary, several INO80 subunits are involved in the regulation of telomere structure and function. Specifically, the Ies3 subunit directly interacts with Est1, a telomere protein, interaction that does not seem to be necessary for INO80 recruitment to telomeres (Yu et al. 2007). Moreover, the preferential localization of some INO80 subunits to telomeres suggests that INO80’s activities at telomeres might be direct, instead of transcription-related. Nonetheless, further investigations are required to shed light on several unaddressed issues, especially since current data seems to indicate that different subunits could have different roles at telomeres.

4.6 INO80 is Involved in the DNA Damage Tolerance Pathways 4.6.1 The DNA Damage Response During Replication To achieve self-perpetuation, the eukaryotic cell has to make an accurate copy of the entire genome. Thus, replication represents a main challenge to the cell, since the DNA molecule could be easily broken or altered during the process. Moreover,

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since the DNA is highly compacted into the chromatin, the DNA replication machinery depends on several chromatin remodeling factors to obtain access to the DNA molecule. As such, DNA replication has developed into a highly coordinated and regulated mechanism to ensure replication fidelity as well as the avoidance of replication-related damage (Falbo and Shen 2006). In fact, an important aspect of the DNA damage response is linked to the process of replicating the DNA, provided that replication forks might be able to process any dangerous DNA alterations that could compromise replication fork stability, and the correct completion of DNA replication. Fork instability leads to fork collapse and the generation of broken chromosomes, genome aberrations, and mutations, the most common hallmarks of genome instability. Thus, when replication forks encounter an obstruction, the S phase checkpoint is activated. S phase checkpoint activation halts DNA replication and induces the recruitment of several factors to stabilize the fork and to allow proper processing of the obstruction (Branzei and Foiani 2009). Interestingly, a stalled replication fork has two possible outcomes. On one hand, failure to stabilize obstructed replication forks leads to fork collapse and the generation of DSB that can be repaired by gene conversion or break-induced replication, two DNA damage repair processes characteristic of late S phase or G2. On the other hand, stalled replication forks can resume DNA replication by different mechanisms all of which are mediated by PCNA, a key molecule during unperturbed DNA replication. These so-called DNA damage tolerance mechanisms confer cell the ability to avoid DNA damage generation as a consequence of DNA replication itself. In fact, during DNA damage tolerance lesions can be bypassed by using specialized polymerases. Replication can also be restarted downstream of the lesion leaving a gap that is filled by translesion synthesis (TLS) polymerase, or it can undergo template switching, a mechanism that involves homologous recombination, where the newly synthesized DNA strand is utilized as a template for DNA synthesis across the gap (Branzei and Foiani 2009).

4.6.2 Chromatin Remodeling at the Onset of DNA Replication During DNA replication, the chromatin structure is radically affected, resulting in the rupture and establishment of histone–DNA interactions that are mostly mediated by chromatin remodeling complexes. Surprisingly, although the role of chromatin in transcription is well known from studies in the past two decades, the role of chromatin in DNA replication has not been widely investigated. In fact, from the limited research in the area we currently know that several chromatin remodeling complexes are involved in different steps during unperturbed DNA replication (Falbo and Shen 2006). However very little is known about the role of these complexes in the DNA damage tolerance response during DNA replication. Thus, this section will discuss the recent discoveries linking the INO80 remodeling complex to DNA replication and more specifically to the DNA damage avoidance pathways.

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4.6.3 A Direct Role for INO80 During DNA Replication Originally, accumulated evidence argued in favor of an indirect, transcriptionalmediated, role of chromatin remodeling during DNA replication. However, the observation that hydroxyurea (HU), a compound that blocks DNA replication, dramatically induces Ino80 expression during S phase, strongly supported the idea of a direct role of INO80 during DNA replication. Moreover, the ino80 mutant shows a prolonged cell cycle and is hypersensitive to DNA replication blocking agents, such as MMS and HU (Falbo et al. 2009). Therefore, these early observations suggested a defective response to stalled replication forks in mutants that lack INO80.

4.6.3.1 INO80 Binds to Origins of Replication During S Phase An important indicator of the direct role of INO80 during replication is the fact that INO80 binds to ARS during S phase. In fact, in the yeast S. cerevisiae, whole genome ChIP–chip analysis of cells tagged at the INO80 locus shows that INO80 binds to 45% of ARS in cells arrested in S phase with HU but only to 5% of cells arrested in G2 with nocodazole, indicating that INO80 binding to ARS is S phase specific. In addition, INO80 does not affect the transcription of genes known to be implicated in DNA replication, when assessed by transcription microarray analysis, supporting the idea that INO80’s role in S phase is direct instead of transcriptionrelated. Finally, INO80 does not seem to discriminate between early and late ARS, a characteristic shared by many factors involved in the S phase checkpoint. Instead, INO80 binds to early, as well as late ARS almost with the same probability genome wide (Falbo et al. 2009). Therefore, since HU induces the checkpoint response, and many checkpoint factors are recruited only to early ARS, INO80 binding distribution to ARS does not correspond with a checkpoint-related function, as it will be explain in the next section.

4.6.3.2 INO80 and the S Phase Checkpoint After HU treatment, replication forks stall, leading to S phase checkpoint activation that is mainly mediated by Rad53 phosphorylation. Checkpoint activation induces cell cycle delay, fork stabilization, and prevention of late firing origins activation. Thus, in the absence of Rad53 early replication forks collapse and late firing origins repression is lost (Branzei and Foiani 2007). Interestingly, the involvement of INO80 in the S phase checkpoint seems to be controversial, since analysis of Rad53 phosphorylation in the ino80 mutant by different research groups has shown dissimilar results. On one hand, Shimada et al. described a delayed Rad53 phosphorylation in the ino80 mutant after HU treatment. Moreover, this group also described that Rad53 inactivation is delayed in the

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ino80 mutant after release and recovery from HU arrest (Shimada et al. 2008). In addition, using 2D gel analysis to follow replication fork movement in cells released from HU treatment Shimada et al. observed a small, yet, significant delay in replication progression that could be due to the delayed effect on Rad53 phosphorylation. On the other hand, Falbo et al. found no significant differences in the Rad53 phosphorylation pattern between wild-type and ino80 mutants. More importantly, 2D gel analysis of replication intermediates that are generated only after fork’s collapse, a well-established method to test for replication fork collapse at specific ARS, indicates that unlike the rad53 mutant, the ino80 mutant shows no sign of fork collapse, such as the presence of a cone signal or the failure to repress late firing origins (Falbo et al. 2009). Therefore, available evidence indicates that INO80 does not seem to affect replication fork stabilization after HU treatment. As such, the hypersensitivity of the ino80 mutant to HU on plates, which is not connected to the defect in replication fork stabilization shown by 2D gel electrophoresis, is known to be a characteristic of mutants of genes that mediate DNA damage tolerance during replication. Therefore, as it will be discussed in the next section, INO80 has a pivotal role in the DNA damage tolerance pathways during replication. 4.6.3.3 INO80 and the DNA Damage Tolerance Pathways In S. cerevisiae, replication forks that encounter an MMS-induced adduct during S phase stall, and the synthesis of the leading and lagging strands becomes uncoupled. Thus, if the obstruction is in the template for the leading strand, a gap is formed, and the subsequent excision of this region creates a DSB by destruction of the replication fork leading to H2AX phosphorylation. Moreover, in yeast, bypass of this type of lesions is performed by the RAD18/RAD6-mediated DNA damage tolerance pathway and, similar to the ino80 mutant, mutants of genes involved in this pathway are hypersensitive to MMS (Hoege et al. 2002; Papouli et al. 2005; Watts 2006; Ulrich 2007). In support of a role for INO80 in DNA damage avoidance during replication, H2AX phosphorylation, a DSB marker, is significantly increased in the ino80 mutants when MMS treated, G1 synchronized ino80 mutant cells are released into the S phase (Falbo et al. 2009). Moreover, this phosphorylation depends on cells’ progression through the S phase, which strongly suggests that INO80 is necessary to avoid replication-related damage. Furthermore, analysis of S phase synchronized cells treated with MMS by two different techniques confirms these results. One of these techniques, pulse field gel electrophoresis (PFGE) allows differentiation of replicating DNA vs. non-replicating DNA, since replicating DNA is unable to enter the agarose gel, remaining as a single band on the top. Thus, PFGE analysis of the ino80 and arp8 mutants shows a significant delay in the mutants’ ability to reconstitute their chromosomes, indicating an impaired ability of the mutants to complete DNA replication after MMS treatment. The other technique, DNA combing analysis, that allows direct visualization and quantitation

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of replicating chromosomes, indicates that the length of the BrdU tracks, indicative of replication fork movement, is significantly reduced in the arp8 mutant after release from MMS (Falbo et al. 2009). Therefore, the altered chromosome mobility of arp8 cells in PFGE after MMS treatment is due to the persistence of unreplicated gaps that represents an impaired ability of the mutants to complete DNA replication. 4.6.3.4 INO80 Chromatin Remodeling Activity is Required for Efficient PCNA Ubiquitylation Mutants of genes involved in the RAD6/RAD18 pathway and the ino80 mutant have similar phenotypes, such as MMS hypersensitivity and an impaired ability to resume DNA replication after MMS treatment. In the budding yeast, the RAD6/ RAD18 damage tolerance pathway is dependent upon PCNA. After MMS treatment, PCNA is monoubiquitylated at its K164 residue by Rad6-Rad18, and subsequently, K164 is polyubiquitylated by the Mms2-Ubc13-Rad5 enzyme complex. Thus, PCNA mono and/or polyubiquitylation are the main cellular mechanisms to resolve replication fork obstructions that could potentially lead to DNA damage (Ulrich 2007). Remarkably, INO80’s impaired ability to resume replication fork movement after MMS treatment seems to be related to a defective PCNA ubiquitylation. In fact, pull down from trichloroacetic acid extracted proteins followed by western blot analysis using an anti-Ub antibody in the ino80 mutant, shows that in S phase synchronized MMS-treated cells, the ubiquitylated forms of PCNA are significantly lower in abundance as compared to the wild-type strain. Moreover, the same effect is observed in a point mutant (K737A) that specifically abolishes the ATPase activity of INO80, indicating that the INO80 ATPase activity is required for PCNA ubiquitylation. Furthermore, transcriptional microarray analysis of S phase synchronized MMS-treated ino80 mutants shows no significant regulation of genes involved in the DNA damage tolerance pathways, which supports a direct role of INO8O during this process (Falbo et al. 2009). 4.6.3.5 INO80 is Required for the Formation of Rad51-Dependent Recombination Intermediates Induced by MMS Treatment After MMS treatment, Rad51 is recruited to stalled forks to perform recombinationmediated activities conducing to fork resolution and completion of DNA replication. Rad51 activity at forks generates DNA hemicatenate-like structures called X-shaped structures. Moreover, Rad51-mediated accumulation of these structures depends on PCNA polyubiquitylation and Rad18 recruitment to the fork, indicating that Rad18 and Rad51 work in conjunction to promote lesion bypass by a template switching mechanism that uses the information in the newly synthesized chromatid (Branzei et al. 2008).

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Concordantly with the reduced PCNA ubiquitylation in the ino80 mutant, chromatin immunoprecipitation analysis of several ARS in S phase synchronized, MMS-treated cultures shows that Rad18 recruitment is significantly reduced in the ino80 mutant (Falbo et al. 2009). Thus, it is possible that INO80 binds to replication forks, and remodels the chromatin to allow proper recruitment of DNA damage tolerance factors like Rad18 and Rad51. Moreover, Rad51 recruitment to ARS is, in fact, significantly reduced in the ino80 mutant, when assessed by chromatin immunoprecipitation analysis. Thus, as explained previously, improper recruitment of Rad51 leads to impaired recombination and the consequent reduction in the generation of recombination intermediates, as it can be concluded by comparison of the arp8 mutant and wild-type cells using 2D gel electrophoresis analysis (Falbo et al. 2009). Therefore, INO80 is necessary to recruit Rad18 and Rad51 to origins of replication when forks face MMSinduced adducts. In conclusion, INO80 is a novel regulator of DNA damage tolerance during replication, through its ability to influence the recruitment of factors in both the RAD6 and RAD51 pathways. In S. cerevisiae, INO80 binds to origins of replication and allows the proper recruitment of proteins involved in replication fork resolution, probably through its ATPase-dependent remodeling activity. Thus, INO80 allows recruitment of Rad18 that leads to PCNA ubiquitylation, which is necessary for Rad51 recruitment. Rad51 recruitment leads to activation of recombinationmediated processes that possibly, as part of a template switching mechanism, will lead to fork resolution and DNA replication completion without the generation of DSBs, genomic aberrations, and genomic instability (Fig. 4.3).

Rad18 DNA adduct

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Fig. 4.3 INO80 is involved in the DNA damage tolerance pathway during DNA replication. When replication forks encounter obstructions caused by DNA damage the INO80 chromatin remodeling complex is recruited to blocked replication forks. Then, INO80 remodels the chromatin environment to facilitate the recruitment of factors from both the RAD6 and RAD51 pathways, such as Rad18 and Rad51. These initiating factors activate subsequent pathways to avoid DNA damage that could arise as a consequence of improper handling of stalled replication forks

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4.7 Conclusions and Perspectives The past few years have witnessed an accelerated phase in the discovery of chromatin regulation in eukaryotic organisms. From epigenetic modifications to nucleosome mobilization, it is now clear that chromatin structure and its respective modifiers are essential players in a multitude of DNA-related activities. Specifically, chromatin remodeling complexes, through their ability to remodel the structure of chromatin, act as DNA gatekeepers, granting or denying access to the molecule and, thus, regulating many nuclear functions. In particular, one remodeling complex, INO80, has been implicated in several unrelated novel nuclear activities, constituting a good example of the extreme versatility these complexes have achieved through evolution. INO80 is involved not only in transcription, but also in DNA damage-related activities, telomere maintenance and DNA replication, and an important clue to this multitasking skill is INO80’s structure and composition. Yet, it is known that each different subunit has its own specific function, but it is also clear that they work together to perform different tasks. A relevant example of INO80’s multitasking skills is the role of INO80 in DNA damage-related activities. INO80 is involved in both DSB repair and DNA damage avoidance during DNA replication, two processes that, despite their sharing of some mechanisms, are radically different in nature. In brief, the Nhp10 subunit is required during conventional DSB due to its ability to bind gH2AX, but it is not required for INO80’s role in DNA damage tolerance, since, for example, lack of Nhp10 does not affect fork recovery after MMS treatment as measured by PFGE. In addition, the presence of posttranslational modifications in some subunits adds a different level of complexity to the picture, related to the possible regulatory mechanisms that could be directing INO80 activities. Indeed, posttranslational modifications might increase INO80’s opportunities to participate in multiple and different processes, since these marks could constitute a code that, for example, would shift INO80 focus from one activity to another. Clearly, further research would certainly advance our knowledge of this complex as well as the intricate relations established among its subunits. Interestingly, the INO80 remodeling complex is highly conserved from yeast to humans, and in fact, an INO80 homolog was recently described in humans, leading to the question of whether the INO80 complex might have similar functions during DDR in humans (Cai et al. 2007). In support of this idea, it was described that deletion of the human homolog of INO80 increases a cell’s sensitivity to DNA-damaging agents. In addition, YYI, a transcription factor closely associated with INO80, has the ability to bind recombination intermediate structures (Wu et al. 2007). Therefore, further exploration of this new research area could bring innovative and invaluable tools to improve the knowledge and treatment of diseases such as cancer, where accurate DNA replication and proper activation of DNA damage avoidance pathways have an important impact on the disease progression.

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Moore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16:2164–2173 Morrison AJ, Highland J, Krogan NJ et al (2004) INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119:767–775 Morrison AJ, Kim JA, Person MD et al (2007) Mec1/Tel1 phosphorylation of the INO80 chromatin remodeling complex influences DNA damage checkpoint responses. Cell 130:499–511 Morrison AJ, Shen X (2006) Chromatin modifications in DNA repair. Results Probl Cell Differ 41:109–125 Morrison AJ, Shen X (2009) Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes. Nat Rev Mol Cell Biol 10:373–384 Nakada D, Hirano Y, Sugimoto K (2004) Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway. Mol Cell Biol 24:10016–10025 Nakamura TM, Moser BA, Russell P (2002) Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres. Genetics 161:1437–1452 Osley MA, Shen X (2006) Altering nucleosomes during DNA double-strand break repair in yeast. Trends Genet 22:671–677 Papouli E, Chen S, Davies AA et al (2005) Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol Cell 19:123–133 Pennock E, Buckley K, Lundblad V (2001) Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104:387–396 Pisano S, Galati A, Cacchione S (2008) Telomeric nucleosomes: forgotten players at chromosome ends. Cell Mol Life Sci 65:3553–3563 Powell SN, Bindra RS (2009) Targeting the DNA damage response for cancer therapy. DNA Repair (Amst) 8:1153–1165 Prado F, Cortes-Ledesma F, Aguilera A (2004) The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange. EMBO Rep 5:497–502 Redon C, Pilch DR, Rogakou EP et al (2003) Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage. EMBO Rep 4:678–684 Rogakou EP, Pilch DR, Orr AH et al (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273:5858–5868 Seto AG, Livengood AJ, Tzfati Y et al (2002) A bulged stem tethers Est1p to telomerase RNA in budding yeast. Genes Dev 16:2800–2812 Shen X, Mizuguchi G, Hamiche A et al (2000) A chromatin remodelling complex involved in transcription and DNA processing. Nature 406:541–544 Shen X, Ranallo R, Choi E et al (2003a) Involvement of actin-related proteins in ATP-dependent chromatin remodeling. Mol Cell 12:147–155 Shen X, Xiao H, Ranallo R et al (2003b) Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 299:112–114 Shimada K, Oma Y, Schleker T et al (2008) Ino80 chromatin remodeling complex promotes recovery of stalled replication forks. Curr Biol 18:566–575 Shrivastav M, De Haro LP, Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res 18:134–147 Shroff R, Arbel-Eden A, Pilch D et al (2004) Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr Biol 14:1703–1711 Staynov DZ (2008) The controversial 30 nm chromatin fibre. Bioessays 30:1003–1009 Taggart AK, Teng SC, Zakian VA (2002) Est1p as a cell cycle-regulated activator of telomerebound telomerase. Science 297:1023–1026 Takata H, Tanaka Y, Matsuura A (2005) Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication proteins in Saccharomyces cerevisiae. Mol Cell 17:573–583 Teng SC, Zakian VA (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol Cell Biol 19:8083–8093

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Chapter 5

Caenorhabditis elegans Radiation Responses Aymeric Bailly and Anton Gartner

Abstract Over the past 10 years a number of laboratories have started to focus on Caenorhabditis elegans radiation responses, taking advantage of a multi-cellular experimental model system that enables studying DNA damage responses at the organismal level. Here we provide a comprehensive review of C. elegans DNA damage responses, largely focusing on recombinational repair, DNA damage signalling and DNA damage-induced apoptosis in response to ionizing radiation. To better explain C. elegans DNA damage response phenotypes and DNA damage response pathway, we also provide an introduction to the C. elegans life cycle and indicate key experimental procedures. Keywords Germ cell • Nematode

5.1 Introduction The use of a model organism to dissect fundamental biological processes has been instrumental in the establishment of various fields in biology. The ionizing radiation (IR) response is no exception, and early studies focused on the effect of DNA damage on single cell organisms such as yeasts. Using the budding yeast Saccharomyces cerevisiae as a model system has been a powerful method to identify genes required for DNA damage repair. Most of the key factors in this process have been isolated by performing forward genetic screens for mutants sensitive to DNA-damaging agents (Weinert and Hartwell 1993). The majority of those genes are conserved in higher eukaryotic organisms, and some alleles lead to heritable cancer predisposition syndromes in the human population (Bartek et al. 2007). However, yeast cells lack an important aspect of the DNA damage response present in higher eukaryotic organisms – the removal of genetically compromised cells by programmed cell A. Gartner (*) Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_5, © Springer Science+Business Media, LLC 2011

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death (apoptosis). Furthermore, DNA damage response and repair pathways are more complex in higher organisms, and diverse cell types differentially respond to DNA damage. It is still technically challenging to perform forward genetic screens with vertebrate cell lines, and large-scale RNAi screening procedures do not permit the generation of stable mutations or hypomorphic alleles in essential genes. Therefore, the use of a model system that recapitulates the integral DNA damage response of mammalian cells but concurrently provides the ease of maintenance and manipulation of a small and simple organism is of great interest to the scientific community. Over the past years an increasing number of laboratories have engaged in establishing and exploiting the nematode worm Caenorhabditis elegans as an experimental system to study DNA damage response genes. Before going into the details of C. elegans DNA damage response pathways, it is important to summarize the basic biological features of this nematode, as well as to explain experimental approaches unique to the nematode system. A basic knowledge of worm physiology and life cycle is also necessary to comprehend the various strategies used by somatic and germ cell tissues to respond to DNA damage, as well as to understand the basic assays that are used to assess defects in DNA damage response pathways.

5.2 The C. elegans Life Cycle and Implications for Radiation Responses C. elegans is a relatively small animal and adults are approximately 1 mm long (Fig. 5.1a). Studies mostly focus on worm development and are aided by the invariant somatic cell lineage. All individual animals display the same cell division patterns to produce identical groups of differentiated cells, and the complete process of embryogenesis can be monitored by light microscopy. The pioneering work of Sulston and co-workers established the complete map of the C. elegans embryonic cell lineage (Sulston et al. 1983). Knowledge of the invariant lineage enables using the worm to ask basic biological questions, such as the molecular details of inductive developmental processes at the level of individual cells. Upon fertilization, the first cleavage divisions produce five somatic founder cells and one germline precursor cell (the P cell) (Sulston et al. 1983), and different adult tissues are produced according to a highly complex lineage pattern. During embryogenesis, which takes less than 12 h, 556 somatic cells are generated and two cells act as germ precursor cells (Sulston et al. 1983). Embryonic cell divisions occur very rapidly, especially during early development where some cell divisions occur in less than 15 min. Embryogenesis is followed by four larval stages, termed the L1 to L4 stages, before worms develop into fertile adults, 50–70 h after fertilization. During larval development, further cell divisions occur in somatic tissues, especially in the ventral cord and during development of the vulva. However, most cell proliferation happens in the C. elegans germline. The germline consists of only two germ cells at the L1 stage, which expand into two gonad arms each comprised of ~500 cells each in the adult worm (Sulston et al. 1983) (Fig. 5.1a). In the adult all somatic cells are post-mitotic and the germline is the only proliferative tissue. This dichotomy in proliferative capacity is important when

Fig. 5.1 (a) Adult Caenorhabditis elegans hermaphrodite (upper panel). Schematic representation of a C. elegans germline (lower panel). The distal end of the worm germline is comprised of mitotic cells. Germ cells enter meiosis at the “transition zone.” After passing though the transition zone most germ cells are in early and late pachytene; late pachytene germ cells can undergo apoptosis. (b) Apoptotic germ cells, highlighted by arrows appear as bottom-like structures under Nomarski optics. (c) CED-1::GFP accumulation around dying cells during the corpse engulfment highlights earlystage apoptotic cells. (d) Cell cycle arrest phenotype. Nuclei (arrow) and the surrounding cytoplasm are enlarged in irradiated cells due to continued cellular growth in the absence of cell division

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considering radiation responses in adult worms. It appears that germ cells and somatic cells are subject to divergent selective pressures. Somatic cells are optimized to contribute to the fitness for only one generation, whereas germ cells are optimized to maintain proliferative potential and to ensure the accurate transmission of the genetic material. It is thus not surprising that somatic tissues tolerate higher levels of DNA damage and use other DNA repair strategies compared to germ cells. Germlinespecific checkpoints and accurate recombinational repair are used to guard germ cells from acquiring deleterious mutations that could be passed on to the next generation. Germ cell proliferation occurs in a stem cell compartment, and germ cell divisions are regulated by the somatic distal tip cell (DTC) located at the tip of the mitotic region, which forms a stem cell niche that constantly restocks the germ cell popula tion (Kimble and White 1981) (see Fig. 5.1a). Within this mitotic germ cell compartment cell divisions occur much more slowly compared to embryonic divisions, and estimates for cell cycle timing range from 12 to 20 h, contrasting with cell division times of less than 15 min during early embryogenesis (Crittenden et al. 2006; Crittenden and Kimble 2008). Germ cell nuclei are actually not fully cellularized and are thus part of a syncytium, although for convenience they are referred to as cells both in this review and generally in the C. elegans germ cell field (Hirsh et al. 1976). The mitotic stem cell compartment (commonly referred to as the mitotic zone) is followed by the so-called transition zone where events related to the entry into meiotic prophase occur, such as the formation of DNA double-strand breaks (DSBs) and the initiation of meiotic chromosome pairing (Dernburg et al. 1998) (Fig. 5.1a). Proximal to the transition zone most cells are in meiotic pachytene; homologous chromosomes are tightly aligned to each other forming the synaptonemal complex. Germ cells subsequently complete meiosis and concomitantly undergo cellularization. C. elegans is a hermaphrodite, and thus both sperm cells and oocytes must be produced in the same animal. Worms achieve this by temporally switching the sex of the germ line (Hodgkin and Brenner 1977). The germline acquires a male fate during the L4 larval stage and all germ cells differentiate into amoeboid sperm cells, stored in an organ termed the spermatheca. Oocytes are produced when the germline switches to its female fate towards the end of the L4 larval stage. In adult worms, fertilization occurs when oocytes pass through the spermatheca, allowing oogenesis to occur at the proximal end of the gonad (Fig. 5.1a). Failure to undergo meiotic recombination does not lead to overt changes in germ cell identity or morphology, and meiotic chromosome pairing is not affected. Mutants defective in recombinational repair of DNA DSBs often also show defects in meiotic recombination, leading to defects in meiotic crossover formation and meiotic chromosome segregation, and resulting in embryonic lethality in the following generation.

5.3 The C. elegans as an Experimental System C. elegans is a unique and versatile experimental system, which is easy to propagate and amenable to long-term storage. Nematodes can be routinely propagated on bacteria on petri dishes, or grown in liquid culture to obtain large quantities of

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worms amenable for biochemical analysis, and are thus an inexpensive and scalable experimental system. Further, worms are tractable to long-term storage as frozen stocks can maintain their viability, which facilitates the development of worm strain collections. Shorter-term storage is also possible due to the ability of worms to differentiate into starvation-resistant dauer larvae, which enables them to survive up to several months under normal culture conditions. As C. elegans worms are predominantly hermaphrodites, self-fertilization results in the easy maintenance of isogenic populations. Genetic crosses in worms employ males, which arise spontaneously from hermaphrodite populations by sex chromosome non-disjunction. Furthermore, C. elegans was the first multi-cellular organism to be fully sequenced (1998) and also made headlines for uncovering the basic apoptosis pathway (Sulston and Horvitz 1977; Ellis and Horvitz 1986; Hengartner et al. 1992; Conradt and Horvitz 1998), for the discovery of RNAi (Fire et al. 1998) and for being the first animal where ectopic green fluorescent protein (GFP) fusion proteins were expressed (Chalfie et al. 1994). Over the past four decades since C. elegans was first introduced as a model system (Brenner 1974), the worm research community has grown and produced invaluable resources, such as a centralized strain depository, gene knock-out mutant collections, and genome-wide RNAi libraries. Indeed, C. elegans is unique as RNAi can be applied to the entire organism. This is conveniently done by expressing double-stranded RNA corresponding to both strands of a single worm gene in Escherichia coli, ingestion of which leads to specific gene inactivation (Kamath et al. 2001). Such RNAi feeding procedures can be performed in liquid cultures using a 96-well microtiter plate format, allowing such screens to be performed on a genome-wide scale (Kamath et al. 2003; Lee et al. 2003). Candidate genes identified using this procedure can then be validated by analyzing the corresponding genetic mutations. In addition to the revolution brought about by the advent of genome-wide RNAi screens, it is predicted that the C. elegans DNA damage response field will benefit tremendously from new technology driving the revival of classic genetic mutagenesis and screening approaches. While in the past the community was hampered by the bottleneck associated with tedious and time-consuming positional cloning approaches, next-generation sequencing, which already allows for re-sequencing multiple C. elegans strains in a single sequencing reaction, will revolutionize this approach (Hillier et al. 2008; Sarin et al. 2008; Shendure and Ji 2008).

5.4 Basic Phenotypes Associated with DSB Repair and DNA Damage-Signalling Defects Pioneering work conducted by Hartmann (Hartman and Herman 1982) first aimed to use C. elegans as a model system to isolate genes implicated in DNA damage. Nine radiation-sensitive genes, rad-1 to rad-9 were identified by their ability to protect worms against UV light during embryogenesis. Loss-of-function mutations in these genes had detrimental effects on worm viability under UV exposure. Some of these mutants were also sensitive to IR. Following these initial studies, it was

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almost two decades before work on those mutants was resumed and the first rad genes were mapped and positionally cloned (Ahmed et al. 2001) (see below). Even earlier, mutants were isolated that showed defects in meiotic chromosome segregation, a phenotype arising as a consequence of defects in meiotic chromosome pairing and recombination. Defective meiotic chromosomes segregation leads to embryonic lethality and an enhanced incidence of males due to sex chromosome non-disjunction. We now know that many of these “him” (high incidence of males) mutants are defective in meiotic recombination, and show an enhanced sensitivity to IR. The first recombinational repair gene isolated by mapping and positional cloning of a him mutant was him-6, the C. elegans orthologue of the mammalian Blooms syndrome gene (Wicky et al. 2004). C. elegans genetics has been instrumental in working out the conserved core apoptotic pathway. The early studies described above took advantage of the invariant development of the worm and the discovery that 131 cells die during the development of the worm by apoptosis (Sulston et al. 1983). Further studies on the germline revealed that an average of one to three corpses can be detected in each gonad arm at any given time. Taking the rapid engulfment of those corpses into account, it became apparent that every second germ cell is eliminated by so-called physiological germ cell apoptosis (Gumienny et al. 1999). However, preliminary attempts to study DNA damage-induced apoptosis failed, as IR-induced apoptosis could not be detected in either embryos or the somatic tissues of adult worms. Given that the germline is the only proliferative tissue of the adult worm and may therefore be relatively sensitive to damage, it was then asked whether DNA damage (caused by DNA DSB-inducing agents such as IR) was able to induce germ cell apoptosis. This was indeed found to be the case. A key study focusing on C. elegans DNA damage response phenotypes established that DNA damage activates apoptotic pathways in the germline, and revealed that germline apoptosis is induced in a dose-dependent manner (Gartner et al. 2000). Using this system, up to thirty dying cells can be counted 24 h after a dose of 120 Gy IR. These studies have exploited the unique opportunity provided by C. elegans to directly observe apoptotic cells in a living multi-cellular organism (Gumienny et al. 1999). Irradiated worms can be transferred individually onto a slide covered with agarose, anesthetized with levamisole, and analyzed using a microscope enabled for Nomarski optics (Gartner et al. 2004). Under these conditions, germline apoptotic cells appear as button-like structures (Fig. 5.1b). Alternatively, apoptotic cells can be visualized using live cell dyes such as acridine-orange, which visualizes the reduced intracellular pH associated with apoptotic dying cells. Finally, apoptotic cells can be visualized by the characteristic pattern of ectoptic CED-1::GFP localization around dying cells during the engulfment process (Fig. 5.1c) (Schumacher et al. 2005a). CED-1 is a transmembrane receptor related to the human SREC scavenger receptor and is expressed at the surface of engulfing cells that face the apoptotic corpses (Zhou et al. 2001). It is now evident that radiation-induced germ cell apoptosis relies on the same core apoptotic genes that are required for developmental and physiological germ cell apoptosis (Gartner et al. 2000). The CED-3 caspase and the CED-4 Apaf1-like

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molecules are both required to induce apoptosis, while the CED-9 Bcl-2 homologue prevents cells from undergoing apoptosis (Ellis and Horvitz 1986; Hengartner et al. 1992). In addition, the transcriptional induction of the C. elegans BH3 domain protein EGL-1, whose cell type-specific transcriptional induction is necessary for C. elegans somatic developmental apoptosis, is also required for DNA damageinduced germ cell apoptosis (Conradt and Horvitz 1998; Gartner et al. 2000) (Fig. 5.2). EGL-1 is not required for physiological germ cell apoptosis, which is not dependent on DNA damage (Gumienny et al. 1999). IR-induced EGL-1 transcription is mediated by C. elegans CEP-1, the sole worm primordial p53-like transcription factor (Derry et al. 2001; Schumacher et al. 2001). CEP-1 is the subject of extensive current studies and will be described in the last chapter of this review. Interestingly, germ cell apoptosis is restricted to a subset of germ cells, namely cells in the late pachytene stage, a stage of meiotic chromosome pairing where recombination has already occurred, and homologous chromosomes are tightly associated with each other within the synaptonymal complex. DNA damageinduced apoptosis does not occur in any other germ cell types such as the mitotic stem cell compartment, or during embryogenesis. Germ cell apoptosis is also induced by meiotic recombination failure, and either mutations or RNAi-mediated depletion of a number of recombination genes such as rad-51, and the worm homologues of the BRCA1 and BRCA2 genes (Gartner et al. 2000; Alpi et al. 2003; Martin et al. 2005).

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Fig. 5.2 Diagram depicting Caenorhabditis elegans germ cell apoptosis pathways in response to ionizing irradiation

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Thus, it is likely that the primary function of DNA damage-induced apoptosis is to provide a fail-safe mechanism to ensure the elimination of germ cells with recombination defects. Indeed, germ cell apoptosis appears to have a more general role in monitoring apoptosis progression, given that an independent checkpoint leading to the elimination of meiotic germ cells with meiotic chromosome pairing defects has been defined (Bhalla and Dernburg 2005). The importance of germ cell apoptosis induction in cells with meiotic chromosome pairing and a recombination defect becomes evident when partially pairing and recombination-defective mutants are combined with apoptosis-defective mutants. Under those conditions, an enhanced level of lethality is observed when analyzing the viability of embryos that result from such defective germ cells (Rinaldo et al. 2002; Bhalla and Dernburg 2005; Adamo et al. 2008). A second damage-induced germline phenotype is a transient cell cycle arrest that occurs in mitotic germ cells following treatment with DNA-damaging agents such as IR (Gartner et al. 2000; Chin and Villeneuve 2001). Cell cycle arrest induced by DNA damage can be simply observed in living anesthetized worms under Nomarski optics without any treatment, extraction, or fixation steps. Arrested mitotic germ cells can be easily recognized since they appear larger than their unirradiated counterparts (Fig. 5.1d), although quantification of the size increase is best quantified by DAPI staining of dissected germlines. The cellular enlargement occurs because cells stop proliferation due to checkpoint activation but continue to grow. Radiation-induced mitotic cell cycle arrest predominately occurs in the G2 stage, as demonstrated by staining with an antibody that recognizes a conserved epitope corresponding to tyrosine-15 of mammalian CDK1 (Hachet et al. 2007; Moser et al. 2009). Phosphorylation of this tyrosine residue is required to keep CDK-1 inactive before S-phase entry, and is antagonized by the CDC25 phosphatase which activates the CDK-1 kinase, allowing progression into mitosis (Russell and Nurse 1986). Elucidation of a DNA damage checkpoint-dependent phenotype initiated a search for mutants defective in either germline DNA damage-induced apoptosis or cell cycle arrest. The first such screen for apoptotic mutants was undertaken by merely scanning through radiation-sensitive mutants previously identified by the Herman laboratory. The sensitivity of these mutants was first assayed by the L4 survival assay, which was designed to measure the radiation sensitivity of meiotic pachytene cells. For this assay, worms in the fourth larvae stage (L4) are irradiated, allowed to mature into adults, and the survival rate of embryos laid by the adult worms on the following day is assessed after approximately 48 h. As cells progress from the pachytene stage to fertilized embryos in approximately 1 day, this assay measures the extent of repair of meiotic cells residing in the pachytene stage. Typically, 50% of the progeny of wild-type worms will die while the other half will develop into fertile adults for 60 Gy of irradiation. DSB-defective repair mutants, however, fail to develop into adults under those conditions. A slightly modified assay measures the radiation sensitivity of proliferative mitotic germ cells. In this assay, worms at the L1 larval stage are irradiated. These can be conveniently isolated from a developmentally asynchronous liquid culture by filtration through an

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11-mm filter. Early L1 stage worms only have two germ cells. These need to proliferate in the following four larval stages to generate the pool of approximately 500 cells contained in each of the two arms of the adult gonad. Thus, L1 larval stage germ cells are very sensitive to IR, which at a high dose leads to sterility and the lack of viable progeny in the next generation.

5.5 C. elegans DNA Damage Response Signalling The first mutations associated with defects in radiation-induced apoptosis turned out to impair all DNA damage responses, and were later mapped to the mrt-2, clk-2, and hus-1 loci (Ahmed and Hodgkin 2000; Ahmed et al. 2001; Hofmann et al. 2002). mrt-2 and hus-1 also function in telomere replication (see below), and both have homologues in mammals and yeasts. The proteins corresponding to mrt-2 and hus-1 form part of the conserved 9-1-1 PCNA-like DNA-sliding complex and MRT-2 is related to fission yeast and human RAD1. The DNA damage-specific clamp-loader, comprising RAD17 and the four smallest RFC subunits, recruits this complex to the dsDNA–ssDNA transition of resected DSBs (Kostrub et al. 1998; Green et al. 2000; Venclovas and Thelen 2000; Bermudez et al. 2003; Majka and Burgers 2003). The worm homologues of the mammalian ATM and ATR kinases, which act as upstream DNA damage sensors and are mutated in human genome instability syndromes, have also been implicated in DNA damage checkpoint signalling. Worm ATR (referred to as ATL-1) and CHK1 are needed for checkpoint responses to IR, UV and replication stress. Both are also essential for germ cell proliferation, possibly by being required for faithful DNA replication (Kalogeropoulos et al. 2004; Garcia-Muse and Boulton 2005). As expected, ATL-1 accumulates in DNA damage foci upon irradiation or DNA replication stress. ATM appears to play a more minor role in the C. elegans DNA damage checkpoint response, as atm-1 mutants are only partially defective in responding to IR, and progeny survival is reduced only upon high doses of IR (Stergiou et al. 2007). Mutants of the C. elegans Werner Syndrome helicase wrn-1 share the partial cell cycle arrest phenotype of atm-1 mutants in response to IR (Lee et al. 2010). It is possible that WRN-1 acts upstream of ATM-1 because ATM-1 nuclear accumulation and RPA-1 focus formation in response to IR are defective in wrn-1 mutants (Lee et al. 2010). The first conserved DNA damage checkpoint gene uncovered by C. elegans genetics was clk-2. The mn159 allele was found in an initial screen for mutations conferring increased lethality to the progeny of irradiated germ cells and was originally named rad-5 (mn159), while clk-2 (qm37) was found in a screen for mutations with slow development and an extended life span (Lakowski and Hekimi 1996; Ahmed et al. 2001). However, clk-2 (qm37) lifespan extension is weak, and may derive indirectly from its slow growth phenotype. Both clk-2 mutations are temperaturesensitive, resulting in embryonic lethality associated with checkpoint defects in response to IR, UV and DNA replication stress at the restrictive temperature (Moser et al. 2009). CLK-2 is the orthologue of S. cerevisiae Tel2, which has been

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implicated in regulation of yeast telomere length, and which binds telomeric DNA in vitro (Lim et al. 2001). In contrast, C. elegans clk-2 does not seem to affect telomere length. Several studies indicated that CLK2/TEL2 is functionally conserved between yeasts and humans. The fission yeast homologue has been implicated in S-phase regulation and DNA damage checkpoint responses (Shikata et al. 2007). Human CLK2/TEL2 is required for both the replication checkpoint and DNA cross-link repair pathways, where it affects the DNA damage-induced mono-ubiquitination of the FancD2 repair protein. CLK2/TEL2 appears to promote S-phase checkpoint activation by preventing CHK1 degradation (Collis et al. 2007). Intriguingly, human and fission yeast CLK2/TEL2 was recently shown to bind to all PI3 kinases, which include TOR2, ATM and ATR (Takai et al. 2007). It was argued that CLK2/ TEL2 might function by stabilizing all PI3 kinases, as their protein levels were dramatically reduced upon CLK2/TEL2 depletion. However, the notion that CLK2/ TEL2 might act in the DNA damage response pathway via the regulation of PI3 kinases was contended in recent studies using the budding yeast model (Anderson and Blackburn 2008; Anderson et al. 2008). Furthermore, ATR/ATL-1 and CLK-2 depletion lead to opposite phenotypes during C. elegans embryogenesis (Moser et al. 2009). In addition, while both C. elegans CLK-2 and ATR/ATL-1 are required for germ cell proliferation, germ cell cycle arrest phenotypes are distinct. clk-2 mutant germ cells arrest in a G2-like stage with condensed chromosomes while atl-1 mutation results in excessive genome instability. CLK2/TEL2 does not contain an obvious catalytic domain, but it is now clear that CLK2/TEL2 belongs to the ARM repeat superfamily of structurally related proteins. Tandem ARM repeats form a super-helical fold capable of forming a surface for protein–protein interactions. ARM repeat proteins are structurally related to proteins containing tandem HEAT motifs. The demonstrated interactions between TEL2/CLK2 and the HEAT repeat containing PI3 kinases suggest that TEL2/CLK2 might act as an adaptor protein that may impinge on multiple signalling pathways (Takai et al. 2007). Another conserved gene that was first implicated in DNA damage checkpoint signalling using C. elegans is gen-1 (Bailly et al. 2010). GEN1 belongs to the XPG family of endonucleases (Clarkson 2003) and is the worm orthologue of the recently purified GEN1 Holliday junction-resolving enzyme (Ip et al. 2008). Holliday junction-resolving enzymes are required for the resolution of Holliday junctions, four-way DNA structures that are crucial intermediates of homologous recombination (HR) and which have to be resolved for separation of the two DNA double strands, in order to complete recombination (Lilley and White 2001). A gen-1 mutant was isolated in a screen for radiation-sensitive mutants also defective in both checkpoint-mediated apoptosis and cell cycle arrest in response to IR. Positional cloning revealed that the mutant affected the worm homologue of the human GEN1-resolving enzyme. C. elegans GEN-1 facilitates repair of DNA DSBs but surprisingly is not essential for meiotic recombination. The role of the meiotic resolving enzyme is apparently taken over by the SLX-1/SLX-4 nuclease complex in worms (Saito et al. 2009). The mammalian homologues of this nuclease complex have been shown to exhibit Holliday junction resolvase activity in vitro (Fekairi et al. 2009;

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Munoz et al. 2009; Svendsen et al. 2009), and the phenotypes of fly and worm SLX-4 mutants are consistent with a defect in meiotic Holliday junction resolution (Yildiz et al. 2002; Andersen et al. 2009). Mutational analysis of C. elegans GEN-1 reveals that the DNA damage-signalling function of GEN-1 is separable from its role in DNA repair, and epistasis analysis indicates that GEN-1 defines a DNA damagesignalling pathway acting in parallel to the canonical pathway mediated by CHK-1 phosphorylation and induction of CEP-1/p53-mediated apoptosis. Furthermore, GEN-1 acts redundantly with the aforementioned 9-1-1 complex to ensure genome stability in response to endogenous replication stress. It is intriguing to speculate that GEN-1 might coordinate DNA damage-signalling and DNA DSB repair. It is possible that GEN-1 acts as a dual-function protein to resolve Holliday junctions, but also to signal and maintain cell cycle arrest until every Holliday junction is resolved (Bailly et al. 2010). It will be interesting to see if either a DNA repair function or a DNA damage-signalling function will be found for human GEN1.

5.6 C. elegans DSB Repair The major repair pathway used in the C. elegans germline to repair IR-induced DSB is the HR pathway. Many of the components of this pathway are used for DSB repair as well as for meiotic recombination. Indeed, many genes involved in recombinational repair are preferentially expressed in the germline. In contrast, nonhomologous end joining (NHEJ) appears to be the predominant repair pathway in somatic tissues (Clejan et al. 2006). Mutations in C. elegans NHEJ genes such as homologues of lig-4 or cku-70 exhibit developmental defects when embryos are irradiated, as well as fused somatic nuclei, indicative of a repair defect (Clejan et al. 2006). Nevertheless, reporter constructs that reconstitute functional LacZ by recombinational repair indicate that recombinational DSB repair contributes to DSB repair in somatic tissues during development (Pontier and Tijsterman 2009). The same study also indicates that the single-strand annealing pathway as well and alternative NHEJ pathway also contribute to DSB repair in somatic cells. HR is essential for meiotic recombination as well as for DSB repair in response to DSB-generating agents. During meiosis DSBs are generated endogenously by the activity of the conserved nuclease SPO-11 (Dernburg et al. 1998). C. elegans provides an excellent model to study meiotic chromosome pairing and recombination. As part of this review we, however, will focus on recombinational repair in response to IR. A crucial step in the initiation of HR is the processing of the DSB and the generation of a single-stranded DNA tail, which is the molecular substrate of the universal RAD-51 recombinase protein. This nucleolytic event, often referred to as resection, results presumably from the activity of nucleases and/or helicases, both of which might operate in concert (Mimitou and Symington 2009). In C. elegans the MRE-11 nuclease appears to be required for resection, as expected from previous work in budding yeast (Nairz and Klein 1997; Chin and Villeneuve 2001). Similarly, the CtIP nuclease was identified independently in human cells and

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worms (Penkner et al. 2007; Sartori et al. 2007). CtIP promotes DNA resection in DSB repair following IR exposure in mitotic human cell lines and is required for RAD51 loading (Sartori et al. 2007). Interestingly, the nematode orthologue of CtIP, called com-1, is required for DNA resection during meiotic DSB repair, a phenotype that is similar to the phenotype associated with mutations of the budding yeast homologue sae-2 (Penkner et al. 2007). As expected for a resection-defective phenotype, com-1 mutant worms are defective in RAD-51 loading, although the rate of DSBs generated by SPO-11 appears to be normal. Recombinational DSB repair requires the bona fide DNA strand exchange protein RAD-51, a member of the RecA family (Ogawa et al. 1993; Benson et al. 1994). RAD-51 oligomerizes on the resected ssDNA and catalyzes strand invasion into an intact DNA double-strand template. RAD-51 oligomerization can be detected by immunofluorescence as discrete foci, which are the most important markers for DNA DSBs in C. elegans (Alpi et al. 2003). Similar to the human situation, RAD-51 specifically interacts with BRC-2 (Martin et al. 2005), the orthologue of the human protein BRAC2 which has been implicated as a mediator of RAD51 loading (Galkin et al. 2005). Cytological evidence suggests that BRC-2, like its mammalian counterpart, is required for RAD-51 foci accumulation following exposure to IR (Martin et al. 2005). BRC-2, like RAD-51, is required for the repair of meiotic DSBs generated by SPO-11. BRCA1 was originally identified in the human population as being linked to a heritable form of breast and ovarian cancer, similar to BRCA2 (Futreal et al. 1994; Miki et al. 1994; Wooster et al. 1995). BRCA1-deficient cells are defective for HR, rendering them extremely sensitive to genotoxic treatment (Jasin 2002). In cells subjected to IR treatment, BRCA1 rapidly relocates to sites of DSB repair and forms discrete foci similar to other repair factors, supporting the idea of its direct role in repair (Scully et al. 1997a, b). BRCA1 is part of a heterodimeric complex with BARD1 (Wu et al. 1996). BRCA1 displays an E3 ubiquitin ligase activity in vitro when associated with the E2 enzyme UBC5 (Lorick et al. 1999). However, no substrate of BRCA1/BARD1 has been identified so far and its function in the repair process remains elusive. Nematode orthologues of BRCA1 and BARD1 have been identified (Boulton et al. 2004), and worms carrying either brc-1 or brd-1 mutations share the same phenotype, consistent with a joint function in vivo. These mutants display enhanced p53-dependent apoptosis, compromised survival and chromosome fragmentations following IR treatment, strongly suggesting a DNA repair defect (Boulton et al. 2004). BRC-1 and BRD-1 proteins form discrete structures called foci at sites of DNA damage and co-localize with RAD-51 (Polanowska et al. 2006). Interestingly, it appears that brc-1 might be specifically required for interchromatid repair as opposed to interchromosomal repair in meiotic germ cells (Adamo et al. 2008). Recent publications have provided important insights into novel mechanisms that prevent excessive recombination. The gene encoding for RTEL1 has been identified on the basis of the synthetic lethality associated with its deletion in conjunction with a loss-of-function mutation in the C. elegans Blooms helicase (Barber et al. 2008). Such double mutants confer an increased rate of meiotic recombination

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and elevated levels of RAD-51 foci. These genetic observations were supported by biochemical experiments, which directly demonstrated that RTEL disrupts RAD-51/ssDNA filaments. Indeed, worms carrying an rtel-1 deletion are hypersensitive to inter-strand cross-linking (ICL) agents such as nitrogen mustard. Interestingly, mutants of the human orthologue RTEL1 display a similar sensitivity towards ICL agents (Barber et al. 2008). The efficiency of using C. elegans to screen for synthetic lethal interactions has been recently highlighted by the identification of the HELQ-1 helicase as a factor required for the disassembly of RAD-51 filaments during meiotic DSB repair (Ward et al. 2010). Prior to this, the critical step of filament disassembly had remained poorly defined at the molecular level. helq-1 was isolated based on a synthetic lethal interaction with the only C. elegans rad-51 paralogue, rfs-1. RFS-1 has been shown to be required for ICL repair (Ward et al. 2007). Worms mutant for both rfs-1 and helq-1 show an accumulation of SPO-11-dependent RAD-51 foci (Ward et al. 2010). Purified RFS-1 and HELQ-1 both interact independently with RAD-51 in vitro and promote the RAD-51 disassembly from duplex, but not singlestranded DNA (Ward et al. 2010). These genetic and biochemical data strongly suggest that both RFS-1 and HELQ-1 function in concert to remove and process RAD-51 filaments during meiosis I. C. elegans genetics can be used to assess whether checkpoint-defective mutants lead to an increased mutation rate in the absence of DNA-damaging agents. For example, using a mutation in the unc-58 locus that leads to an “uncoordinated” phenotype (Hodgkin et al. 1979), it has been shown that the number of revertants in a growing worm population increases 8–15-fold when DSB repair is compromised by mutated checkpoint genes (Harris et al. 2006). Importantly, many of these reversions were small deletions, a finding that could be confirmed by mapping spontaneously arising mutations.

5.7 Divergence Between Vertebrates and Nematodes Although DSB repair pathways are highly conserved throughout eukaryotes, some critical genes present in vertebrates have not been identified in nematodes. This is notably the case for one of the first factors phosphorylated in response to DSB formation, the histone variant H2AX, also present in the budding yeast genome (Downs et al. 2000; Paull et al. 2000). In fact, none of the H2A variant genes present in the C. elegans genome encodes the conserved C-terminal tail phosphoserine that is targeted by the ATM kinase in response to DSB in vertebrates and yeasts (unpublished). Another critical factor of the vertebrate response to DSB is MDC1, which is also phosphorylated by ATM, and is absent in the worm genome. In vertebrates, MDC1 directly interacts with phosphorylated H2AX through its BRCT domains (Goldberg et al. 2003; Stewart et al. 2003) and subsequently recruits the RNF8 ubiquitin ligase (Mailand et al. 2007). This process leads to the polyubiquitination of histone H2A and the recruitment of further ubiquitination ligases to

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the vicinity of the DSB, and is believed to help maintain the structure of repair centres. It is intriguing to speculate why none of these factors (RNF8, H2AX, MDC1) have been found in the C. elegans genome. However, it is unlikely to be due to the low level of homology of these factors with putative C. elegans orthologues. We rather speculate that they have been lost during nematode evolution. Thus, the above repair factors might function to optimize recombination repair and DNA damage signalling without being integral parts of this process. Other DNA repair genes that could not be identified in C. elegans include a homologue of the NBS1 gene, which exists in a complex with the MRE11 nuclease and RAD50 in mammalian cells (Alani et al. 1989; Usui et al. 1998; Hopfner et al. 2002), and is implicated in recombinational repair, NHEJ and DNA damage signalling. Another noticeable absence is ATRIP, a protein needed to recruit ATR to sites of DNA damage. Both NBS1 and ATRIP are evolutionary very divergent and their homology to their respective budding yeast counterparts is barely detectable (Edwards et al. 1999; Paciotti et al. 2000; Rouse and Jackson 2000). It is thus likely that their orthologues have not been identified in nematodes due to a very low level of sequence identity.

5.8 Telomere Replication and Mortal Germline Mutations Studies in C. elegans have addressed the connection between DNA damage checkpoint pathways, germ cell apoptosis and DNA repair factors, and the maintenance of immortal germlines. Unbiased genetic screens led to the identification of mutants that are defective in germ cell immortality. These mrt (mortal germline) mutants proliferate normally for several generations, but produce reduced progeny numbers in later generations, before succumbing to sterility caused by defects in germ cell proliferation (Ahmed and Hodgkin 2000). Several such mutants have been linked to telomere replication defects (Ahmed and Hodgkin 2000; Boerckel et al. 2007). A telomere replication defect in C. elegans leads to a progressive shortening of chromosome ends and ultimately to sterility after 15–20 generations, due to anaphase bridges and mitotic catastrophe. In addition to the telomerase catalytic subunit TRT-1 (Meier et al. 2006), another group of genes, namely hus-1, mrt-2 and hpr-17 that encode for the aforementioned PCNA-like 9-1-1 complex (the RAD-17 clamp-loader), have been shown to be essential for telomere replication. These studies guided investigations in mammalian tissue culture cells that showed that the 9-1-1 complex is indeed required for telomerase loading (Francia et al. 2006). This finding and double mutant analysis with C. elegans telomerase and 9-1-1 mutants lead to the notion that all these genes act in the same telomere replication pathway as the worm telomerase. It was also argued that there might be a common process required for both recombinational DSB repair and telomere replication. This notion was recently challenged by the description of the MRT-1 nuclease, a C. elegans hybrid protein composed of a conserved SMC1 nuclease domain fused to an OB-DNA binding fold related to the second such fold of the POT1 telomere-binding

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protein (Meier et al. 2009). MRT-1 mutants, similar to mutants in the 9-1-1 complex, are DNA cross-link repair defective, but not IR-sensitive. In addition, a TRT-1 mutation leads to neither DSB repair nor DNA cross-link defects. However, given that MRT-1, the 9-1-1 complex and TRT-1 all act in the same telomere replication pathway, nucleolytic DNA processing akin to DNA cross-link repair is likely to be required for telomerease activity in vivo. In yeast and mammalian cells telomere end-protection proteins are known (Garvik et al. 1995; O’Sullivan and Karlseder 2010). These protect telomeres from being recognized and processed as DNA DSBs, and hence depletion of such genes leads to immediate checkpoint activation resulting in senescence and apoptotic cell death (Verdun and Karlseder 2006, 2007). However, no such genes have been detected in C. elegans. Only late-stage telomere replication mutants show chromosome fusions and evidence for activation of the DNA damage checkpoint pathway (data not shown). Several further DNA repair mutants are known to have a mortal germline defect, although none of these mutants have the progressive telomere shortening phenotype of mrt mutants. Nevertheless, RFS-1, the sole worm homologue of the mammalian RAD51 paralogue family, seems to be required for intact telomeres. rfs-1 mutants display fluctuating telomere length, and progression into germline mortality is much less uniform compared to telomerase mutants (Ward et al. 2007; Yanowitz 2008). Indeed, rfs-1 mutants were shown to be able to occasionally partially rescue (or delay) the onset of sterility of telomerase mutants, implying that RFS-1 might have a role in the recombination of subtelomeric sequences. In addition, mutants in DNA mismatch repair genes, as well as in the C. elegans him-6 helicase, can lead to a mortal germline phenotype (Degtyareva et al. 2002; Ahmed 2006). It is intriguing to speculate that the mortal germline phenotype of these DNA repair factors is due to the accumulation of deleterious mutations and/or chromosome fusions. Such a phenotype could resemble the phenotype of hypermorphic DNA double-strand repair mutants in mouse models, where actively proliferating tissues become progressively depleted (Morales et al. 2005).

5.9 The Regulation of DNA Damage-Induced Germ Cell Apoptosis Germ cells damaged by an exposure to genotoxic agents such as IR, UV light or N-ethyl-N-nitrosourea (ENU) are removed by programmed cell death mediated by the C. elegans p53 homologue, CEP-1 (C. elegans p53-like 1). Homology to the mammalian p53 family was initially found in the DNA-binding domain (Derry et al. 2001; Schumacher et al. 2001) and subsequent biochemical and bioinformatic analysis has also identified a SAM protein–protein interaction domain in CEP-1 (Ou et al. 2007). A domain structure that includes a SAM domain is reminiscent of the mammalian p63 and p73 superfamily members, which are indeed slightly more related to the CEP-1 DNA-binding domain than is p53. Surprisingly, both p63 and p73 have predominately developmental roles, in skin development and neurogenesis

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respectively, while cep-1 mutants do not have any overt developmental phenotype. Nevertheless, the exact phylogenetic relationship of CEP-1 to any of the three mammalian p53 members is not clear, given that gene radiation into the three p53 families occurred during vertebrate development. The recent discovery that an isoform of mammalian p63 is specifically required for the elimination of female meiotic germ cells indicates that germ cell apoptosis might be a primordial function of the p53 family, and that p63 might be evolutionarily ancient (Suh et al. 2006). CEP-1 appears to act as a transcription factor and is required for the transcriptional induction of the egl-1 (Hofmann et al. 2002) and ced-13 genes (Schumacher et al. 2005b), that encode BH3-only proteins (Fig. 5.2). These BH3-only proteins induce apoptosis in worms by binding to the Bcl-2 homologue, CED-9, which normally holds the Apaf-1 homologue CED-4 in an inactive state. EGL-1 binding to CED-9 releases CED-4, which can then activate the caspase CED-3. Loss of CEP-1 function through RNAi knockdown, gene deletion, or expression of a dominant-negative protein lacking the DNA-binding domain, results in viable worms without any overt developmental defect (Derry et al. 2001; Schumacher et al. 2001) (Fig. 5.2). In addition, CEP-1 does not affect IR-induced germ cell cycle arrest, and cep-1 mutants are only weakly radiosensitive in radiation survival assays. Besides egl-1 and ced-13, there are very few genes that are transcriptionally induced by IR, and the induction of most of these genes does not depend on CEP-1. Indeed, the transcriptional induction of genes in response to IR only seems to play a rather minor role in the damage response. None of the IR-induced genes encodes a DNA repair or a DNA damage checkpoint signalling protein. IR-induced genes rather seem to be involved in responding to oxidative stress, and overlap with the set of genes induced in response to infection with bacterial pathogens, oxidative stress and ageing (Greiss et al. 2008b). Several genes have been shown to negatively regulate CEP-1-dependent apop tosis. The ABL-1 and AKT kinases antagonize CEP-1-induced apoptosis following irradiation, and lead to elevated levels of CEP-1, but their mode of action is unknown (Deng et al. 2004; Quevedo et al. 2007). Loss of components of the SCFFSN-1 ubiquitin ligase and of the neddylation pathway also result in increased CEP-1 phosphorylation and protein levels, along with increased egl-1 transcription (Gao et al. 2008). However, it is not clear whether SCFFSN-1 regulates CEP-1 directly, as no direct interaction between FSN-1 and CEP-1 has been detected. The best-studied negative regulator of CEP-1 is GLD-1. GLD-1 is an mRNA-binding protein that inhibits the translation of multiple target mRNAs involved in germ cell differentiation, mainly through interaction with target sequences in their 3¢ UTRs. A hypomorphic allele of gld-1 was identified that, in contrast to gld-1 null alleles, shows no overt defects in germ cell differentiation but specifically leads to the hyper-activation of CEP-1-dependent apoptosis (Schumacher et al. 2005a). The corresponding mutant protein fails to recognize the cep-1 3¢ UTR target, which leads to increased CEP-1 expression, while other developmental targets of GLD-1 are regulated normally. In the absence of GLD-1 more CEP-1 is translated, resulting in increased apoptosis. It will be interesting to see if similar regulatory mechanisms are important for the regulation of the mammalian p53 family. In addition, the sole

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C. elegans ASSP proteins iASSP was shown to bind CEP-1, similar to mammalian p53 (Bergamaschi et al. 2003). Worm iASSP bound both CEP-1 and p53 in vitro, indicating that it might negatively regulate CEP-1 by direct interaction. Finally, the protein arginine methyltransferase PRMT-5 can bind to both CEP-1 and the coactivator CBP-1 (p300/CBP homologue) and methylates the latter (Yang et al. 2009). When PRMT-5 is absent, cep-1-induced apoptosis and egl-1 mRNA levels are increased following DNA damage, indicating that PRMT-5 normally acts to modulate CEP-1-dependent transcription of egl-1. Mutations in several other genes have been shown to specifically result in reduced apoptosis induction in response to IR, reminiscent of cep-1 mutants (Fig. 5.2). RNAi-mediated depletion of lag-3 the worm homologue of the mammalian MAML Notch transcriptional coactivator resulted in decreased apoptosis and decreased egl-1 and ced-13 induction following irradiation. LAG-3 may thus act as a CEP-1 coactivator similar to the function of MAML in p53 activation (Zhao et al. 2007), but these studies, however, still have to be confirmed by the analysis of a lag-3 deletion mutant. Unexpectedly, egl-1 and ced-13 are still normally induced in several further mutants that show defects in DNA damage-induced apoptosis. This phenotype indicates that the corresponding genes impinge on apoptosis induction in one or more genetic pathways that act either downstream or in parallel of the cep-1-dependent transcriptional induction of egl-1. The first such factors described are components of the C. elegans retinoblastoma complex (RB1) (Schertel and Conradt 2007) which also affect basal, DNA damage independentgerm cell apoptosis (Fig. 5.2). The worm homologue of the human SIRT1 histone deacetlylase SIR-2, which has been implicated in regulating transcriptional repression and ageing also affects IR-induced apoptosis (Greiss et al. 2008a). Interestingly, it was found that SIR-2 translocates from the nucleus to the cytoplasm in earlystage apoptotic germ cells, and evidence was provided that such translocation may be functionally linked to apoptosis induction (Fig. 5.2). Other such factors, including the ING-3 transcription factor (Luo et al. 2009) and components of the ceramide synthesis pathway, have been shown to be required for DNA damage-induced apoptosis (Deng et al. 2008) (Fig. 5.2). Interestingly, the apoptotic defect associated with these mutants is restored upon microinjection of long-chain natural ceramide, and ceramide concentrations in mitochondria are increased upon IR. The involvement of ceramide in mammalian apoptosis has been controversial, and this study indeed confirms that ceramide has a role in inducing apoptosis (Fig. 5.2). Finally, KRI-1, an orthologue of KRIT1/CCM1, which is mutated in the human neurovascular disease cerebral cavernous malformation, is required to activate DNA damagedependent cell death independently of cep-1/p53 (Ito et al. 2010). While it has been hypothesized that the major regulatory events controlling cell death occur by cellautonomous mechanisms, kri-1 and at least in part the retinoblastoma complex components appear to function in C. elegans somatic tissues to affect apoptosis induction in the C. elegans germline (Fig. 5.2). In summary, C. elegans encodes a primordial p53 pathway needed for DNA damage-induced apoptosis. Intriguingly, several other genes are also required in what appears to be parallel pathways to promote germ cell apoptosis in response to IR. At the moment, these factors cannot

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be placed into a simple genetic or biochemical pathway, and future studies will be required to mechanistically link these genes to apoptosis regulation. Furthermore, it is not clear whether those factors acting in parallel to CEP-1 are regulated by the ATM/ATR kinases. It will be interesting to see if the same genes have a role in apoptosis induction in vertebrate cells. Acknowledgements A CRUK career development award to Anton Gartner funded work in the Gartner lab. Aymeric Bailly was supported by a Wellcome Trust VIP award. We are grateful to Ashley Craig and Ulrike Gartner for proofreading.

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Part II Modulation of Radiation Responses: Opportunities for Therapeutic Exploitation

Chapter 6

Hypoxia and Modulation of Cellular Radiation Response Ester M. Hammond, Monica Olcina, and Amato J. Giaccia

Abstract Tumors possess many features that differentiate them from normal tissues which can potentially be targeted for therapy. One such difference is the presence of an inefficient vascular network, which is often unable to meet the tumor’s needs. The interaction of this faulty vascular system with the cells within the tumor leads to the development of a unique tumor microenvironment not found in normal tissues. This particular environment has a number of distinct features such as low oxygen concentrations, high interstitial fluid pressures and low pH. Previous studies have found that the presence of these microenvironmental conditions potentiates the development of certain adaptive responses by the tumor. These adaptive responses can have a number of adverse consequences, including increased resistance to chemo and radiotherapy. Nevertheless, since these microenvironmental characteristics are found in most solid tumor types, they have a profound effect on the tumor’s behavior and represent attractive therapeutic targets. Keywords Hypoxia • DNA damage response • DNA repair • Reoxygenation

6.1 Characteristics of the Tumor Microenvironment Normal tissues are perfused by a well-structured vasculature which ensures that the needs of the tissue are met. This vasculature comprises a network of blood vessels, which are capable of delivering oxygen and nutrients, as well as a comprehensive lymphatic system, that functions to eliminate waste products and excess fluids. Partial pressures of oxygen in normal tissues lies in the range of 10–80 mmHg (Brown and Giaccia 1998). Within tumors, however, the situation is very different. Blood vessels are chaotic, having a distorted structure that prevents the proper A.J. Giaccia (*) Department of Radiation Oncology, Center for Clinical Sciences Research, Stanford University, Stanford, CA 94303-5152, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_6, © Springer Science+Business Media, LLC 2011

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delivery of oxygen. Furthermore, tumor vessels usually have a number of structural abnormalities that make them more permeable than normal vessels. In addition, the organization of such vessels is often disturbed and chaotic, and many vessels are functionally incapable to adequately supply the tumor with oxygen and nutrients. The consequences of a malformed vasculature are areas of chronic hypoxia, where viable tumor cells exist in a state of low oxygen (typically 100–200 mm from the lumens of a blood vessel). Moreover, acute hypoxic areas may also occur where blood vessels temporarily collapse due to, for instance, a high interstitial fluid pressure (IFP) or hemodynamic changes. The majority of hypoxia research is carried out between 2% oxygen, which gives robust hypoxia inducible factor-1 (HIF-1) activation and 0.1% oxygen which induces a block in DNA synthesis because within a tumor, a hypoxic gradient exists, with tumor cells at physiologic oxygen levels near the vessels and regions of anoxia surrounding the necrotic core. Tumor cells can adapt to hypoxia in a number of ways (Vaupel and Harrison 2004). For example, hypoxia may lead to cell cycle arrest particularly at the G1/S phase border through p21 and p27 activation by HIF-1 (Green et al. 2001; Gardner et al. 2003). In addition, hypoxia may promote apoptosis by both a p53-dependent and a p53-independent manner (Alarcon et al. 1999, 2001; Soengas et al. 1999; Koumenis et al. 2001). Necrosis may also be induced in response to hypoxia if cells do not receive oxygen (Vaupel and Harrison 2004). In addition, hypoxia induces a number of proteomic changes, which will enable the tumor to survive and proliferate in a low oxygen environment. Interestingly, many of these same changes involved in adaptation to hypoxia also promote tumor progression and provide the tumor with an increased ability to invade and metastasize to distant sites. Many of these changes in gene expression are mediated by the HIF family of transcription factors. The HIF transcription factor family are heterodimers composed of an a-subunit (HIF-1a, HIF-2a, HIF-3a) and a b-subunit. In the presence of oxygen, HIF-1a undergoes a prolyl hydroxylase (PHD-mediated hydroxylation) and is degraded by the von Hippel Lindau (VHL) protein. As oxygen decreases, HIF-1a is stabilized due to a decrease in hydroxylation by the PHDs (Maxwell et al. 1999; Jaakkola et al. 2001; Kaelin and Ratcliffe 2008). HIF-1 acts as a transcriptional regulator for genes involved in angiogenesis, glycolysis, glucose transport, and increased oxygen delivery (Poon et al. 2009). Examples of critical HIF-regulated genes include vascular endothelial growth factor (VEGF, angiogenesis), GLUT1 (glycolysis) and carbonic anhydrase IX (CAIX, a regulator of pH). These and others have all been used as markers of hypoxia. Oncogene activation and tumor suppressor loss can also have an effect on the tumor’s response to hypoxia. Furthermore, clonal selection of those cells that have developed mutations in certain tumor suppressor gene pathways, which increase survival in the hypoxic environment, will also lead to increased tumor aggressiveness (Graeber et al. 1996). Thus, hypoxia will inevitably be an adverse prognostic factor that predicts for increased metastasis and treatment resistance (Brizel et al. 1996). Increased IFP is another unique characteristic of the tumor microenvironment. A number of factors lead to a high IFP. First, the gaps in the tumor vasculature contribute to the leakiness of the tumor, allowing fluids and proteins to escape.

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This creates a high pressure in the interstitium that cannot be corrected, since tumors possess an inefficient lymphatic system and reduced blood flow. This problem is exacerbated further since the extracellular matrix (ECM) is influenced by the action of platelet-derived growth factor (PDGF) and transforming growth factor (TGF) b receptors as well as b1 integrin and collagen (Cairns et al. 2006). As mentioned above, the hypoxic environment found in tumors perturbs normal gene and protein expression at a number of levels. Some of the targets induced by HIF-1 include enzymes that promote a switch to glycolysis. This switch will increase the amount of lactate present. However, while it was originally thought that the low pH found in tumors was solely due to the increased lactic acid produced as a result of this metabolic switch, it has now been shown that other factors may also play a role. The overall acidic pH in tumors seems to be influenced by both extracellular and intracellular pH. Gradients of pH exist within cells which require the action of ion channels such as the monocarboxylate H+ co-transporter (MCT), the vacuolar H+ATPase, the Na+/H+ exchanger and the Na+-dependent Cl/ bicarbonate exchanger. The expression of these membrane transporters is thought to be increased as part of the adaptive response to hypoxia, resulting in a decreased extracellular pH. Many elegant studies have demonstrated that the degree of hypoxia in human tumors negatively correlates with prognosis (Hockel et al. 1996; Nordsmark et al. 1996a, b). This has been attributed to the finding that the hypoxic regions of tumors are more resistant to killing by both radio- and chemotherapy. Chemotherapeutic drugs are delivered to tumors through the blood stream and are therefore reliant on the tumor vasculature for optimum delivery. As discussed above, the tumor vasculature is malformed and inefficient, therefore impeding the delivery of chemotherapeutic agents and antibodies to hypoxic regions. This is also compounded by the finding that some chemotherapies are less efficient in conditions of low oxygen. The reasons behind this include both reduced drug activity in hypoxia (e.g., bleomycin and etoposide) and reduced killing of cells which are not proliferating as found in severely hypoxic areas and decreased pH regions (e.g., alkylating agents) (Shannon et al. 2003; Minchinton and Tannock 2006). Many studies have described the effect of oxygen on radiosensitivity (Brown and Wilson 2004). Oxygen enhances radiosensitivity and conversely a lack of oxygen or hypoxia increases radiosensitivity. Hypoxic cells are two to three times more resistant to killing by ionizing radiation than cells at normal oxygen concentrations. This ratio is termed the oxygen enhancement ratio (OER) and is irrespective of cell type and inherent radiosensitivity but is dependent on radiation quality (O’Neill and Wardman 2009). The toxic effects induced by radiation are mediated through the generation of reactive oxygen species which, if in proximity to the DNA will cause damage. In the presence of oxygen this damage becomes “fixed” or permanent. This is, of course, a simplification of what actually occurs. It has been proposed that the presence of oxygen radicals on DNA bases leads to double-strand breaks (DSBs) as well as damaged bases and that this radical site can then be transferred to the sugar and result in an additional single-strand break. This is referred to as damage transfer by oxygen and results in an amplification of the initial damage.

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In order for this to occur, it has been estimated that oxygen must be present within a thousandth of second after the irradiation (Wardman 2009). This is also true for chemical radiosensitizers such as nitroimidazoles (Wardman 2007). The most toxic type of DNA damage is an unrepaired DSBs, which is lethal to a cell. More recently, hypoxia has been shown to negatively impact tumorigenesis by driving what is known as a mutator phenotype (Loeb 2001). Several studies recently reviewed by Bristow and Hill (2008) have demonstrated that mutation frequencies are increased both in vitro and in vivo in response to hypoxia (Reynolds et al. 1996; Sandhu et al. 2000; Li et al. 2001). More and more evidence is emerging that hypoxia has a significant impact on the DNA repair capabilities of a cell and that this directly impacts genomic instability (Bertrand et al. 2003).

6.2 Hypoxic Induction of a Unique DNA Damage Response In response to genotoxic agents, which induce DNA damage, a well-characterized signaling cascade is initiated by members of the PI3 Kinase family (Jackson and Bartek 2009). This includes; ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA protein kinase (DNA-PK) (Cimprich and Cortez 2008; Lavin 2008). Despite their accepted presence at the top of this signaling pathway, these kinases must first be activated or relocalized to the site of DNA damage. In order to achieve this, first the cell must register that it has received a damaging insult. In response to DSBs, the MRN complex, consisting of MRE11, Rad50, and NBS1, localizes to DNA ends and recruits ATM which is then activated through autophosphorylation (Carney et al. 1998; Costanzo et al. 2001; D’Amours and Jackson 2002; Lee and Paull 2004). Mutation of the ATM, MRE11, and NBS1 genes have all been demonstrated to result in radiosensitivity syndromes (Stewart et al. 1999; Lavin 2008). However, it has been shown experimentally that DNA damage signaling does occur in the absence of these sensor proteins, although with delayed kinetics (Uziel et al. 2003). In some cases, genotoxic stress results in a replication arrest. For example, an induced bulky lesion can block the DNA polymerases leading to stalled replication forks which are sensed by single-stranded DNA-binding proteins (Richard et al. 2008). The most characterized of these singlestrand binding proteins is replication protein A (RPA). Once bound to regions of ssDNA, RPA then recruits ATR and its binding partner ATRIP to sites of stalled replication, therefore bringing the kinase into proximity of the damaged DNA (Zou and Elledge 2003). Once localized accordingly and/or activated, the PI3 kinases phosphorylate numerous downstream targets to bring about cell cycle arrest, apoptosis, DNA repair, and senescence. More recently, both ATM and ATR have been described as being active in hypoxic conditions (Hammond et al. 2002; Gibson et al. 2005; Freiberg et al. 2006; Bencokova et al. 2009; Economopoulou et al. 2009). The finding that exposure of minimally transformed cells to hypoxia induces p53-dependent apoptosis provided one of the first pieces of evidence that hypoxia induces a DNA damage response

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(Graeber et al. 1996). Additional studies demonstrated that p53 was both p hosphorylated and active in hypoxic conditions but only under low oxygen tensions (<0.1%). Some groups have further proposed that p53 is induced by hypoxia and acidosis (Hammond et al. 2002; Pan et al. 2004). The kinase linking hypoxia and phosphorylated p53 was identified as ATR. As previously mentioned, ATR is activated in conditions of replication stress. A direct correlation was found between conditions of hypoxia which induced a replication arrest and p53 phosphorylation. In response to severe hypoxia, DNA synthesis is rapidly shut down in all cell types, although the kinetics vary (Hammond et al. 2003b). This was attributed to a lack of available nucleotides due to a decrease in ribonucleotide reductase activity (Reichard and Ehrenberg 1983; Pires et al. 2010). DNA fiber analysis of replicons demonstrated that in response to severe hypoxia, replication was blocked in both the initiation and elongation phases leading to an accumulation of single-stranded DNA (Fig. 6.1). This block of replication coincides with an accumulation of RPA foci and subsequent ATR-mediated signaling. Of the known ATR substrates, p53 and Chk1 have received the most attention in response to hypoxia (Brown and Baltimore 2003). Loss of either ATR or Chk1 sensitizes cells to hypoxia/reoxygenation indicating that they play a critical role in protecting stalled replication forks as has been described in response to other stresses (Heffernan et al. 2002; Bartek and Lukas 2003; Syljuasen et al. 2005; Maya-Mendoza et al. 2007). Loss of ATR during hypoxia was shown to lead to the accumulation of DNA damage specifically in the S-phase cell population (Hammond et al. 2004). The involvement of ATM in the hypoxic response was unexpected. Despite the wealth of evidence to support the presence of stalled replication forks in hypoxic conditions, actual DNA breaks have not been detected unless reoxygenation occurs (Hammond et al. 2002, 2003a; Pires et al. 2010). To date the majority of ATM signaling has been described in response to DSBs, although a number stresses, including hypoxia, prove that you do not necessarily need DNA damage. Other examples include, hypothermia, chloroquine, heat, and insulin (Hunt et al. 2007; Loehberg et al. 2007; Jeong et al. 2010; Pandita et al. 2009). In response to severe hypoxia, ATM is rapidly phosphorylated. This is at least in part a result of

Fig. 6.1 Hypoxia induces a replication arrest associated with a block of both initiation and elongation. RKO cells were exposed to hypoxia (0.02%) for a period of 6 h before DNA fibers were generated. For DNA fiber method (Petermann et al. 2006; Pires et al. 2010). In normoxic conditions actively replicating fibers can be seen, while in hypoxic there are only stalled replication structures

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Fig. 6.2 Hypoxia induces a distinct DNA damage response. DNA damage cannot be detected by Comet assay in response to hypoxia without reoxygenation but does lead to the accumulation of gH2AX foci. These foci colocalize with MDC1 as predicted from the literature but surprisingly 53BP1 foci are not formed. Phosphorylated MDC1 would be expected to recruit 53BP1 through RNF8 indicating that this signaling is disrupted in hypoxic conditions

autophosphorylation, although there is evidence that ATR may also play a role in this phosphorylation (Goodarzi et al. 2004; Pellegrini et al. 2006; Stiff et al. 2006). In support of the data indicating an absence of DSBs in hypoxia, the MRN complex is not required to activate ATM under low oxygen conditions, and ATM does not subsequently form nuclear foci (Hunt et al. 2007; Bencokova et al. 2009). Additional differences between the DNA damage response induced by DNA strand breaks and hypoxia include the finding that while gH2AX can be detected in discreet nuclear foci, 53BP1 fails to form such structures (Fig. 6.2). Thus, the gH2AX signal is at sites of chromosome abnormalities present at stalled replication forks. However, the lack of 53BP1 foci is attributable to a lack of actual DNA strand breaks and also a possible failure of MDC1 to recruit RNF8 (Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007; Minter-Dykhouse et al. 2008; Wang and Elledge 2007; Bencokova et al. 2009). Until recently hypoxia-induced DNA damage signaling was thought to occur only at low oxygen levels which are found surrounding the necrotic areas in tumors. However, it has been shown that ATR-dependent signaling to the histone gH2AX occurs at oxygen levels as high as 1% and that this plays an important role in angiogenesis (Coleman and Ratcliffe 2009; Economopoulou et al. 2009; Rankin et al. 2009). This recent study demonstrated that gH2AX was required to maintain proliferation of endothelial cells in hypoxic conditions (1% O2) and that loss of gH2AX suppressed tumor angiogenesis and tumor growth. Interestingly, this effect was only seen in tumor/pathological angiogenesis and not developmentally associated neovascularization. As discussed, in response to much more severe levels of hypoxia, replication stalls leading to regions of single-stranded DNA and ultimately ATR activation. It is less clear what is the signal that activates gH2AX in hypoxic (1% O2) endothelial cells, which are still replicating. The phosphorylation of

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gH2AX in these conditions was found to be ATR dependent and was attributed to damage associated with replication stress or the hypoxia-mediated repression of DNA repair, which will be discussed further. This study suggests that other factors involved in the DNA damage response, i.e., downstream of gH2AX, may also have a role to play in tumor-associated angiogenesis, therefore making the pathway an attractive therapeutic target. In contrast to endothelial cells, which seem protected by hypoxia-induction of the DNA damage response, the biological consequences of the induction of a DNA damage response in cells exposed to severe hypoxia is less clear. The most likely fate of these cells is necrosis unless they find an oxygen supply. However, as a result of the rapid changes in the tumor vasculature by therapy, reoxygenation events can and do occur (Brown 1979, 2002; Jain 2005; Kim et al. 2007; CardenasNavia et al. 2008). The majority of radiotherapy is delivered in fractions in order to take advantage of such reoxygenation events. Well-oxygenated tumor cells are more sensitive to radiation and are therefore preferentially killed over the hypoxic cells. The predicted consequence of fractionated radiotherapy would be an increase in the hypoxic fraction of the tumor, as these are the remaining cells. What actually occurs is that the hypoxic fraction stays constant as cells move from the hypoxic compartment to well-oxygenated regions of the tumor, i.e., they are reoxygenated. Reoxygenation has been demonstrated to induce significant levels of ROS-mediated DNA damage (Hammond et al. 2003a). This has led to the suggestion that one of the benefits to activating the DNA damage response during hypoxia is to prepare the cell for such an event. In support of this concept, ATM was shown to activate Chk2 in hypoxic conditions but with no obvious effect on the cell cycle. However, after reoxygenation a Chk2-dependent cell cycle arrest occurs in the G2 phase which would facilitate repair of reoxygenation-induced damage (Gibson et al. 2005; Freiberg et al. 2006). Loss or inhibition of key players in this response have been demonstrated to sensitize cells to hypoxia/reoxygenation (Hammond et al. 2004; Freiberg et al. 2006). It is plausible that this sensitization is entirely due to a lack of DNA damage signaling during reoxygenation. This conclusion is supported by the finding that loss of Chk1 during reoxygenation leads to an increased rate of origin firing during replication restart. The implications of this are that replication resumes before repair has taken place of reoxygenation-induced damage (Pires et al. 2010). The lack of efficient DNA repair in hypoxic cells also contributes significantly to the increased genomic instability associated with cycles of hypoxia and reoxygenation (Yuan et al. 2000; Bindra et al. 2007; Huang et al. 2007).

6.3 Hypoxic Inhibition of DNA Repair Pathways It is becoming increasingly clear that in response to hypoxia DNA repair is, in general, inhibited. There are now numerous examples of proteins, critical to different repair pathways, being repressed by a variety of mechanisms in response to hypoxia. These diminished repair capabilities under hypoxia directly contribute to

134 Table 6.1 A brief overview of the repair factors affected by hypoxia Gene/ Repair protein pathway Potential mechanism Rad23B NER HIF-1 induction of miR373 Rad52 HR HIF-1 induction of miR373 and miR210 Rad 54 HR RNA expression downregulated MLH1 MMR Downregulated at mRNA level MSH2 MMR Myc/Max network Ku70/80 NHEJ Downregulated expression in cervical carcinoma Ku70/80 NHEJ Upregulated (4 h at 1% O2) DNA-PKcs NHEJ mRNA changes but not protein levels DNA-PKcs NHEJ Upregulated along with DNA-PK activity Rad51 HR Independent of HIF-1 and cell cycle Changes in E2F mediated transcriptional transactivation and transrepression and translation Rad51 C HR Downregulated at the translational level BRCA1 HR Independent of HIF-1 and cell cycle Changes in E2F mediated transcriptional transactivation and transrepression BRCA2 HR Downregulated at the translational level HR shown to be decreased in hypoxia and cells sensitive to cross-linking agents XRCC3 NHEJ Downregulated at the translational level XRCC4 NHEJ Downregulated at mRNA level LIGASE IV NHEJ Downregulated at mRNA level MSH6 MMR HIF displacement of Myc to repress gene transcription in a p53-dependent manner NBS1 DSB HIF-1 dependent and requires phosphorylation of the PASB domain in HIF

E.M. Hammond et al.

Reference Crosby et al. (2009b) Crosby et al. (2009b) Meng et al. (2005) Mihaylova et al. (2003) Bindra et al. (2007) Lara et al. (2008) Um et al. (2004) Meng et al. (2005) Um et al. (2004) Bindra et al. (2004) Chan et al. (2008)

Chan et al. (2008) Bindra et al. (2005)

Chan et al. (2008)

Chan et al. (2008) Meng et al. (2005) Meng et al. (2005) Koshiji et al. (2005)

To et al. (2006)

NER nucleotide excision repair, HR homologous recombination, MMR mismatch repair, NHEJ non-homologous end joining, DSB double-strand break repair

an increase in genomic instability and carcinogenesis (Powell and Kachnic 2003; Stark et al. 2004; Collis et al. 2005; Bindra and Glazer 2007). A brief overview of the repair factors effected by hypoxia are listed in Table 6.1 and have been recently reviewed (Huang et al. 2007; Bristow and Hill 2008). One of the most striking observations when considering this data as a whole is the variety of mechanisms used to shut down repair. This is especially important when considering severely hypoxic conditions abrogate translation and transcription, raising the possibility that the levels of DNA repair proteins could decrease simply as a result of changes in protein/mRNA half-lives (Denko et al. 2003). Studies using polysome fractionation have been particularly helpful in addressing this point (Chan et al. 2008). Changes in the cell cycle also needs to be considered as many DNA repair genes are regulated in this manner, and therefore may appear altered due to an

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arrest in the cell cycle (Bindra et al. 2005). Most recently, micro RNAs have been implicated in hypoxia-mediated repression of DNA repair factors. Recent studies have shown that the HIF-regulated miRs-210 and 373 recognize Rad52 and Rad23B, respectively, and that expression of both miR-210 and miR-373 is associated with an adverse prognosis and metastatic potential (Crosby et al. 2009a). Some of the studies carried out have considered how long after reoxygenation it takes for the DNA repair proteins to return to normal levels. For Rad51, protein levels returned in 48 h, indicating that HR is not only compromised in hypoxia but also during reoxygenation (Bindra et al. 2004). This has significant implications for the repair of reoxygenation-induced DNA damage and the generation of genomic instability. The impact on radiotherapy of reduced DNA repair in hypoxic cells has been highlighted in a recent study. Chinese hamster cell lines either proficient or deficient in homologous recombination (HR) were irradiated under oxic or anoxic conditions and their OERs calculated. Cells deficient in HR in either oxic or anoxic conditions possessed a decreased OER (1.5–2.0) compared with the repair proficient cells, which had OERs of 2.6–3.0 (Sprong et al. 2006). The unanswered question is why, in response to hypoxia, is DNA repair inhibited? One possible explanation is that when the cell enters a limiting environment, such as hypoxia, nonessential pathways are inhibited in order to preserve energy and therefore increase survival. This hypothesis has also been used to explain why, in response to severe hypoxia, protein synthesis is repressed (Koritzinsky et al. 2006). Hypoxia does not induce DNA breaks, therefore making the pathways to repair DNA damage nonessential. We have recently demonstrated that cells exposed to severe hypoxia reach a point of no return where, even if oxygen conditions are restored they do not undergo replication restart. This loss of restart ability is attributed to disassembly of the replisome, i.e., the machinery required for replication (Pires et al. 2010). Under these oxygen conditions, a lack of DNA repair is unimportant as the cells are unable to resume replication and re-enter the cell cycle. However, the involvement of HIF in repressing DNA repair implies that repair capabilities are also inhibited at much more moderate levels of hypoxia where cells would certainly be replicating. Although we may never fully understand why repair is repressed in hypoxia, work has already begun on exploiting this facet of the tumor microenvironment for therapeutic gain.

6.4 Exploitation of the Hypoxic Environment One of the principle responses to hypoxia is a HIF-mediated increase in angiogenesis. This has lead to a huge scientific effort into the development of antiangiogenesis therapies. More recently, the concept of “context synthetic lethality” has been suggested. Synthetic lethality has been described and exploited by yeast geneticists for many years (Kaelin 2005). In brief, the concept is that while mutation in either pathway A or B may not affect cell viability, mutation in both pathways simultaneously leads to lethality. This has recently been effectively demonstrated in

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mammalian systems (Bryant et al. 2005; Farmer et al. 2005). These studies d emonstrated that breast cancer cells carrying mutations in the BRCA1 tumor suppressor were extremely sensitive to PARP inhibition. In the presence of PARP inhibitors single-strand breaks persist and form DSBs which are then unrepaired due to a lack of HR due to BRCA1 mutations. These studies have been extended to demonstrate that loss of many of the proteins involved in HR, not just BRCA1/2, sensitizes cells to PARP inhibition (McCabe et al. 2006). Interestingly, many of these same HR proteins are repressed by hypoxic conditions. The concept of context synthetic lethality is that the hypoxic microenvironment inhibits DNA repair pathways such as HR, therefore leaving the cell sensitive to inhibition of alternative pathways. Studies have demonstrated that a PARP inhibitor radiosensitizes malignant tumor cells in hypoxic conditions (Liu et al. 2008). Further work has shown that both acutely and chronically hypoxic cells repress HR and are sensitive to PARP inhibition (Hammond and Bristow Groups, unpublished). There are multiple oxygendependent mechanisms underlying this sensitivity. At oxygen tensions which permit replication the sensitivity is through the accumulation of damage in a HR-compromised background. While at more severe oxygen levels where replication is prohibited, the target is replication restart. This conclusion is supported by the finding that PARP regulates replication fork progression and that inhibition of PARP during reoxygenation increases replication restart rates (Hammond, unpublished data) (Sugimura et al. 2008). It has also been demonstrated that PARP inhibitors delay tumor growth and inhibit HIF-1 activity and gene expression (Martin-Oliva et al. 2006; QuilesPerez et al. 2010). These effects on HIF-1 may be attributable to the vasodilating effects of PARP inhibitors (Kovacs et al. 2004). An optimistic view is that the simultaneous targeting of the HR and single-strand break repair pathways will not be the only effective example of synthetic lethality. The hypoxic microenvironment has a significant effect on DNA repair, and it is hoped that this will result in sensitivity to inhibition of a number of pathways. It has already been demonstrated that cells defective in DNA repair show increased tumor radio and chemosensitivity when ATM, Chk1/2, NHEJ, and proteosome function are inhibited (Choudhury et al. 2006; Helleday et al. 2007; Martin et al. 2008). Significant effort is being spent in identifying further examples of context synthetic lethality.

6.5 Conclusion There are many approaches to exploiting the tumor microenvironment for therapeutic gain. These are at a variety of stages of development and show varying degrees of promise. Especially given the relative radioresistance and chemoresistance of hypoxic tumor cells, it is becoming clear that many of these approaches may not be effective as single agents and that instead we are going to have to combine them for the best effects. For example, while a PARP inhibitor may sensitize tissue culture cells to hypoxia, in all likelihood, it will be used in combination with radiotherapy to kill the aerobic cells. Despite numerous reports and years of study, the fact that

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cells in tumors do not behave as those kept in in vitro conditions is often overlooked. The concept of context synthetic effectively highlights how hypoxia-mediated changes in gene expression should be considered a new approach to exploit hypoxia and improve cancer therapy.

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Chapter 7

Inhibitors of DNA Repair and Response to Ionising Radiation Barbara Vischioni, Nils H. Nicolay, Ricky A. Sharma, and Thomas Helleday

Abstract Ionising radiation, and most chemotherapeutic agents currently used to treat cancer, target DNA to cause cytotoxicity. The cellular response to DNA damage is a complex set of intra-cellular processes involving multiple DNA repair pathways, leading either to cell death or to survival if the lesions are repaired or bypassed. Thus, the multiple and redundant pathways involved in the repair of DNA damage are important for therapeutic resistance. Within the past two decades the components of these DNA repair pathways have increasingly been investigated to generate new targeted drugs, which may be combined with conventional radiotherapy or DNA-damaging agents in the treatment of tumours. Furthermore, since many different types of human cancers arise from genetic or epigenetic mutations in proteins involved in DNA damage repair, over recent years targeted therapy approaches have been developed to utilise specific tumour-related DNA repair defects as monotherapy. This chapter gives an overview of the mechanisms involved in the repair of the lesions created by ionising radiation and provides details on the strategies that are currently under pre-clinical and clinical investigation to inhibit DNA repair pathways for potential therapeutic gain. Keywords DNA replication • Repair • Cancer • Therapy • Homologous recombination • Poly(ADP-ribose) polymerase

T. Helleday (*) Cancer Research UK-Medical Research Council, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, OX3 7DQ, UK and Department of Genetics Microbiology and Toxicology Stockholm University, Stockholm S-106 91, Sweden e-mail: [email protected]

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7.1 Introduction Ionising radiation (IR) and most chemotherapeutic agents currently used to treat cancer work by producing DNA damage, such as DNA single-strand breaks (SSB) and double-strand breaks (DSB), DNA adducts, DNA–DNA or DNA–protein crosslinks, or interference with important DNA-interacting proteins, e.g. topoisomerases. The most toxic lesion induced by radiation is the DSB. Cellular responses to DNA lesions are complex. Damaged cells attempt to repair the lesions and thereby activate several signal transduction pathways, leading either to cell death or to survival if the lesions are repaired or bypassed. The elucidation of DNA repair pathways and the definition of molecular partners for DNA repair proteins have revealed potentially “druggable” targets, suitable for the identification of new anti-cancer therapies or for the enhanced anti-tumour activity of DNA-damaging strategies. The efficacy of IR and anti-cancer drugs is highly influenced by cellular DNA repair capacity. Inhibitors of DNA repair increase the anti-tumour efficacy of radiation and DNA-damaging anti-cancer drugs in pre-clinical models. Changes in the levels of DNA repair proteins have been correlated with resistance to anti-cancer strategies. If the DNA repair machinery is defective, cells may become hypersensitive to damage. Somatic or inherited mutations in DNA repair genes have been described in tumours determining a selective loss of function that can cause reduced DNA repair capacity and increased genetic instability in tumour cells. A defect in one DNA repair pathway can be compensated by other pathways. Such compensating pathways can be identified in synthetic lethality screens and then specifically targeted for treatment of DNA repair-defective tumours. This chapter contains an overview of the main DNA repair pathways and their activation and interplay after radiation. In this chapter, we define strategies to target the DNA repair machinery and ways to potentially exploit different DNA repair defects for cancer therapy. Particular attention is given to polyadenosine diphosphateribose polymerase (PARP) inhibitors, not only as chemo- or radiopotentiating agents, but also as active agents in DNA repair-deficient cancers.

7.2 The Role of Different DNA Repair Pathways for IR-Induced DNA Lesions As each human cell incurs more than 50,000 DNA-damaging events in a day, an elaborate set of different DNA repair mechanisms has evolved to maintain the integrity of the cellular genome and to prevent genetic instability and carcinogenesis. Cells have developed several distinct DNA repair pathways that are activated upon DNA damage and are able to recognise and repair specific DNA lesions. These pathways share a certain level of redundancy, meaning that two or more pathways can interact to repair different types of DNA damage, or one pathway can compensate for deficiencies in another DNA repair pathway.

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The human body is constantly exposed to low doses of IR from natural sources and may also receive higher doses as used in diagnostic radiology (e.g. computed tomography scans) and radiation therapy of cancer. Depending on the energy content, IR can damage the DNA in various ways, either directly by creating strand breaks or by ionising water molecules to create hydroxyl radicals that can then cause DNA damage (Mahaney et al. 2009). Hence, various forms of DNA damage have been observed following IR treatment, such as damage to DNA bases or the sugar backbone. Often, IR induces complex or clustered strand breaks that are characterised by different forms of DNA damage in one region of the DNA (Goodhead 1994). If those lesions remain unrepaired, they may lead to cell death; their misrepair may result in mutations and genomic instability. Although base damage and SSBs are much more frequent than DSBs, these SSBs are rapidly and accurately repaired using the opposite, intact DNA strand. DSBs are considered the main toxic lesion by which IR kills cells and are produced either directly or as the result of two SSBs in close proximity on opposite strands. Therefore, many IR-induced DSBs exhibit overhangs of the 3¢ or 5¢ strands, as well as 3¢-phosphate groups that need processing before the breaks can be ligated (Mimitou and Symington 2009). Since IR-induced DSBs have a high potential for losing genetic information and causing cells to die, their immediate and efficient repair is crucial for genomic integrity and cell survival. Mammalian cells employ two major pathways to repair DNA DSBs, non-homologous end joining (NHEJ) and homologous recombination (HR) (Helleday et al. 2007). Since HR uses the sister chromatid or the homologous chromosome as a template for repair of the DSB, it is more accurate than NHEJ. However, HR generally takes place after DNA replication, so the majority of IR-induced DSBs are repaired by NHEJ (Branzei and Foiani 2008). “Clean” DSBs are in most of the cases repaired accurately by NHEJ (Helleday et al. 2007). However, since IR mostly leads to complex breaks containing strand ends with non-ligatable groups, NHEJ requires removal of those nucleotides from both sides of the break before realignment and ligation can take place. Processing of the strand end may lead to the loss of chromosomal material and is therefore potentially error-prone.

7.2.1 Non-homologous End Joining The NHEJ pathway employs several different proteins that participate in DSB recognition, strand end processing and ligation. Initially, a DSB is recognised by Ku, a heterodimeric protein consisting of Ku70 and Ku80 (Tomimatsu et al. 2007). Ku70 and Ku80 form a central DNA-binding domain that can bind to the DNA irrespective of the base sequence, encircling the DNA end. Additionally, interaction of Ku with the DSB ends may facilitate the alignment of the two strands (Feldmann et al. 2000). As the major DSB-sensing protein involved in NHEJ, Ku localises to sites of DSBs independently of other factors and is then required for the recruitment of different repair proteins to DSB sites. DNA binding enhances the interaction of

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Ku with several of those proteins in vitro, suggesting that binding to the DSB is required for the recruitment of the DSB repair proteins. Additionally, in vivo data have shown that the C terminal domain of Ku80 is able to interact with the DNAdependent protein kinase catalytic subunit (DNA-PKcs) and recruits the kinase to lesion sites, which is a key step in NHEJ (Schild-Poulter et al. 2001). DNA-PKcs is a member of the phosphoinositide 3-kinase-like family of protein kinases and forms a complex with Ku upon DNA binding. The role of DNA-PKcs in the repair of IR-induced DSBs has been well established, and cells lacking DNAPKcs exhibit a strongly radiosensitive phenotype (Meek et al. 2008). DNA-PKcs has weak enzymatic activity and is able to phosphorylate serine and threonine residues on various proteins. The enzymatic properties of DNA-PKcs are greatly enhanced by binding to Ku, and the DNA-PKcs-Ku complex is commonly termed DNA-PK. The phosphorylation activity of DNA-PK is essential for NHEJ. Inhibition of DNA-PK inhibits the repair of DSBs and thus sensitises cells to IR (Allen et al. 2003). Because of the enzymatic requirements of DNA-PK, the kinase has been investigated as a possible therapeutic target in combination with radiotherapy (RT). In vitro data have revealed several target proteins that may be phosphorylated by DNA-PK; however, in vivo data suggest that the main target for phosphorylation by DNA-PK is DNA-PKcs itself, and various phosphorylation sites have been identified that get phosphorylated after exposure to IR in vivo (Mahaney et al. 2009). In addition, the phosphoinositide 3-kinase-like kinases, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated- and Rad3-related (ATR), have been shown to phosphorylate DNA-PK in response to IR (Yajima et al. 2006; Chen et al. 2007). The role of DNA-PKcs autophosphorylation has only been elucidated in vitro, but studies suggest that upon phosphorylation, the DNA-PK complex disassembles and opens up the DSB ends to other proteins. Inhibition of the kinase activity of DNA-PK by small-molecule inhibitors or mutation of phosphorylation sites renders cells more radiosensitive than DNA-PKcs-null cells, which may be due to the blockage of strand ends for other NHEJ proteins (Allen et al. 2003). Once DSBs have been detected and tethered, successful religation requires processing of the DNA termini to remove other DNA lesions and non-ligatable groups. Since DNA DSBs are highly variable in nature and are usually part of complex lesions, several different enzymes or enzyme combinations are required for processing strand termini. Some proteins that have been linked to end processing include Artemis, poly-nucleotide kinase (PNK), and DNA polymerases l and m. Artemis is the main nuclease enzyme for the preparation of strand ends before ligation; it possesses both 5¢-3¢ exonuclease and endonuclease activity. Additionally, Artemis has been shown to remove 3¢-phosphoglycolate groups from break ends in vitro (Povirk et al. 2007). Artemis can interact with DNA-PKcs and can become highly phosphorylated both in vitro and in vivo. Cells lacking Artemis are very radiosensitive, and a deficiency of Artemis leads to a radiosensitive form of severe combined immunodeficiency (RS-SCID) (Rooney et al. 2002). Additionally, other factors have been attributed to end processing during NHEJ. PNK exhibits both 3¢-DNA phosphatase and 5¢-DNA kinase activity which both may play a role in end processing, and studies have shown PNK involvement in NHEJ (Koch et al. 2004).

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Additionally, lack of PNK increases cellular radiosensitivity and defects in DSB repair (Bernstein et al. 2008). In in vitro studies, it has been shown that, if IR-induced damage contains gaps that need to be filled prior to ligation, X-family DNA polymerases m and l are recruited to lesion sites by Ku and the X4L4 complex, consisting of DNA ligase IV and XRCC4. Both polymerases contain an N-terminal BRCA1 C terminal domain (BRCT) that is required for their function during end joining repair. Polymerases m and l exhibit differences in their template dependence, and polymerase m may be capable of filling gaps independently of a template. Lack of either polymerase does not significantly increase radiosensitivity, which implies that both enzymes are only involved in the end processing for a small sub-set of DSBs (Capp et al. 2006). Other studies have linked proteins such as APLF, WRN helicase and Tdp1 to end processing of DSBs before end joining (reviewed in Mahaney et al. 2009). Eventually, if strand ends are suitable for ligation, the protein complex X4L4, containing DNA ligase IV and XRCC4, carries out the last step of NHEJ. XRCC4 itself has no enzymatic activity and is believed to act as a molecular scaffold that recruits other factors involved in ligation to the break site. DNA ligase IV has been shown to interact with XRCC4 and is stabilised and activated upon binding. XRCC4 has also been shown to interact with Ku, PNK, APLF and DNA, and Ku is required for XRCC4 recruitment (Macrae et al. 2008). XRCC4 is phosphorylated by DNAPKcs in vitro, although phosphorylation seems not to be required for NHEJ. However, in vivo studies have shown SUMOylation of XRCC4, which contributes to the nuclear localisation of the protein (Yurchenko et al. 2006). An additional protein required for end joining is called Cernunnos, which exhibits a structure similar to XRCC4, although its role has not been fully elucidated yet (Yano et al. 2009).

7.2.2 Homologous Recombination Several forms of homology-directed repair have been described, among them HR, single-strand annealing (SSA) and break-induced replication (BIR). Whereas SSA and BIR are potentially mutagenic, HR is the pathway used for conservative repair of DSBs. Analogous to NHEJ, the first step of HR repair requires recognition and marking of the lesion site. The damage recognition proteins in this pathway are Mre11, Rad50 and Nbs1, which interact and form a functional unit termed the MRN complex, found to be crucial for DSB recognition and early response (Williams et al. 2007). MRN complexes form on both ends of the strand break and are believed to interact by homodimerisation of the Rad50 components, thereby aligning the strands. Additionally, interaction of Nbs1 and the phosphoinositol-3 like kinase ATM leads to the recruitment of ATM and the subsequent phosphorylation of multiple proteins involved in DNA damage response. Comparable to NHEJ, strand ends created by IR need processing and resection, before religation can take place. Proteins such as Exo1, Bloom’s syndrome protein (BLM), BRCA1 and C terminus-binding protein of adenovirus E1A-interacting

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protein (CtIP) have been implicated in end processing during HR (Yun and Hiom 2009). Additionally, 13 genes of the Fanconi anaemia (FANC) family seem to play a role in HR, although their role may depend on BRCA2 function. However, only minimally increased radiosensitivity has been described in cells lacking one of the FANC genes. The FANC genes can be divided into three groups, with eight genes encoding proteins that form a core complex (FANCA, B, C, E, F, G, L, M), two encoding proteins that form another complex that can be ubiquitylated by the core complex (FANCD2 and I), and three encoding proteins that represent downstream targets (FANCD1 [which is the same as BRCA2], J, N). Following DNA damage, the core complex ubiquitylates the D2-I complex, which is then enabled to bind to BRCA2 and regulate its function (Wang 2007). During end processing, single-strand overhangs are created that are bound by replication protein A (RPA). Single-strand binding by RPA prevents the formation of DNA secondary structures, allowing RAD51 multimers to assemble and form a nucleoprotein filament along the strand. Since binding of RAD51 to DNA is weaker than RPA-DNA binding, additional factors are required to replace RPA with RAD51 protein along the strand. BRCA1 and BRCA2 have been attributed to facilitate RAD51 binding, and both proteins have been shown to be required for IR-induced RAD51 foci formation (Zhang et al. 2009). Studies have implicated that the actual loading of RAD51 on to the DNA is mostly due to its direct interaction with BRCA2. The importance of RAD51, BRCA1 and BRCA2, and their involvement in the prevention of genomic instability through HR, is revealed by the early embryonic lethality of mice deficient in any of those proteins. Since a key feature of HR is the use of a homologous DNA sequence as a template for the broken strand to allow for conservative repair, pairing of homologous DNA segments is required. This mechanism has not been fully elucidated yet, but studies suggest that the RAD51-DNA filament collides randomly with duplex DNA until regions of homology are detected (Bianco et al. 1998); several RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, may aid in the search for homology, and mutation or loss of any of these proteins has been shown to severely impair HR (Hartlerode and Scully 2009). Strand invasion by the RAD51-coated single strand leads to the formation of a temporary D loop, and the 3¢ end initiates strand elongation using the complementary double-stranded DNA as a template. It is not yet clear which DNA polymerase is required for this process, but one DNA polymerase, h, has been shown in vitro to be able to perform the D loop extension (McIlwraith et al. 2005). Upon strand invasion and D loop extension of the 3¢ end, several models have been developed to explain ways of repairing and annealing the broken strands. The most commonly applied model proposes that, following extension of the invading strand, the strand may be displaced from the duplex DNA and be reannealed with the processed second strand to form again an intact double-stranded DNA molecule (reviewed in Hartlerode and Scully 2009). However, if the second strand end is also captured by the D loop, a so-called double Holliday junction (HJ) is formed. Once a HJ has been formed, helicases such as Werner syndrome protein (WRN), BLM and RecQ5b move the X structure in any direction in a mechanism termed

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“branch migration” (Bachrati and Hickson 2006). The protein Rad54 has been shown to facilitate branch migration both in 3¢-5¢ and 5¢-3¢ direction using adenosine triphosphate. Eventually, HJs need to be resolved to untie the two DNA duplex structures, either with or without crossovers. BLM has been shown to form a complex with topoisomerase IIIa, which is able to dissolve HJs without crossover products in a processes called HJ dissolution (Wu and Hickson 2003; Dutertre et al. 2002). BLM converges the two HJs, after which topoisomerase IIIalpha decatenates the two strands, resulting in two separate sister chromatid strands and no crossover products. The final ligation step is likely carried out by ligase I, and lack of ligase I significantly reduces HR activity. As an alternative pathway, a protein complex incorporating Mus81 and Eme1 can resolve HJs, producing crossover products. A third HJ resolvase has recently been discovered in human cells. Gen1 can resolve HJs, causing non-crossovers and crossovers with the same efficiency (Ip et al. 2008). 7.2.2.1 Homologous Recombination is Regulated by Checkpoint and Cyclin Dependent Kinases Whereas NHEJ can be used throughout the cell cycle, HR can be used only in the presence of a sister chromatid for accurate repair (Fig. 7.1). The availability of a sister chromatid needs to be sensed to activate HR. In cells, this is accomplished by a high cyclin-dependent kinase (CDK) activity, which regulates the end resection process in cells that are in the S or G2 phases of the cell cycle (Jazayeri et al. 2006). Cells outside these cell-cycle phases will not efficiently resect their DNA ends and thus will rely on NHEJ for repair (Fig. 7.2). Additionally, the HR repair pathway is regulated by the kinase CHK1, which is activated by ATR binding to RPA coated ssDNA regions after resection (Sorensen et al. 2005). The CHK1 kinase phosphorylates a range of HR proteins, which are required for the replacement of RPA with RAD51 to initiate HR (Sleeth et al. 2007).

7.2.3 Other DNA Repair Pathways Repairing IR-Induced Damage Although less toxic than DNA strand breaks, the majority of DNA lesions induced by IR are damaged bases, DNA adducts and intra- and inter-strand cross links. These lesions are mostly repaired by two pathways, mismatch repair (MMR) and base excision repair (BER). BER is a DNA repair pathway that is utilised to remove the majority of DNA adducts induced by both endogenous and exogenous substances. Upon recognition of a base adduct, an adduct-specific DNA glycosylase cleaves the N-glycosidic bond of the damaged base and creates an apurinic/apyrimidinic (AP) site. The AP

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Fig. 7.1 Repair and signalling pathways activated in response to ionising radiation shown relative to phases of the cell cycle. DNA repair (open text) and DNA damage signalling (filled text) pathways active at different cell-cycle stages are shown relative to the G1, S, G2 and M phases of the cell cycle

site then undergoes processing by AP endonuclease 1, which creates a strand gap that is flanked by a 3¢-hydroxyl group on one side and a 5¢-deoxyribose residue on the other (Hegde et al. 2008). The subsequent steps of BER either use the short-patch repair way for single-nucleotide gaps or long-patch repair for gaps spanning between 2 and 15 nucleotides. Both steps are initiated by DNA polymerase β, which is only able to insert a single nucleotide so that the long-patch pathway requires additional nucleotide synthesis by pol d or e. In long-patch BER, a 5¢ nucleotide flap is created that is subsequently removed by flap endonuclease 1 (FEN1) before final ligation can take place. Ligation in the short-patch pathway is carried out by DNA ligase III using XRCC1 as a cofactor, whereas in long-patch BER, DNA ligase I reseals the nick (Tomkinson et al. 2001). MMR repairs base–base mismatches that arise during DNA replication and may otherwise cause point mutations and frameshift mutations. MMR pathway initiation requires the recognition of base mismatches through the MutSa or ß protein dimers, before mismatch excision can be carried out by the exonuclease EXO1. EXO1 is recruited to lesion sites through the binding of MutLa or ß to MutSa and removes several nucleotides beyond the nucleotide mismatch, thus creating a SSB. DNA polymerase d then resynthesises the missing strand segment in a process involving the DNA-binding proteins PCNA and RPA, before a DNA ligase eventually seals the nick (Li 2008). The role of MMR in the response to IR is largely due to

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Fig. 7.2 Double-strand break repair pathways after ionising radiation. NHEJ is preferentially used for repair of simple DSBs throughout the cell cycle, which involves only the core NHEJ complex (Ku70/80, DNA-PKcs, XLF, XRCC4, LigIV) and often resulting in an error-free repair but may also include deletions. About 15% of IR-induced DSBs are severe DSBs, which require ATM for repair. When CDK activity is low (in G1 cells), there will be no resection; the cell will employ NHEJ for repair. With high CDK activity (in S/G2 cells), resection is initiated that will trigger an ATM/ATR-dependent, mostly error-free HR repair

an increase in free 8-oxo-7,8 dihydrodeoxyguanine triphosphate (8-oxodGTP) nucleotides owing to oxidation after IR. These 8-oxodGTPs are incorporated instead of dGTP and are recognised by MMR, leading to G2 arrest by signalling the cell cycle regulation cdc2 pathway. 8-oxoG in DNA is efficiently removed by the human 8-hydroxyguanine glycosylase 1 (hOGG1) via the BER pathway. This fact might explain why there is no significant survival difference between MMRproficient and -deficient cells after exposure to IR (Yan et al. 2001).

7.3 Targeting Main DNA Repair Pathways in Combination with IR as a Therapeutic Strategy RT and many chemotherapeutic agents have anti-cancer activity because of the cytotoxic consequences of DNA damage. RT and cancer drugs employed in the clinic have been used for several decades and are highly efficient in killing proliferating cells. High levels of DNA damage cause cell cycle arrest and cell death. Furthermore, DNA lesions that persist into the S phase of the cell cycle can obstruct replication fork progression, resulting in the formation of replication-associated DNA DSBs. DSBs are generally considered to be the most toxic of all DNA lesions.

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IR and radiomimetic agents, such as bleomycin, cause replication-independent DSBs (Lehmann 2006) that can kill non-replicating cells. Such treatments can also rapidly prevent DNA replication by activation of cell-cycle checkpoints to avoid formation of toxic DNA replication lesions (Painter and Cleaver 1967). Many conventional chemotherapeutic drugs are delivered concurrently with RT in an effort to improve cure rates. These drugs also increase toxicity, so the net effect on therapeutic index is variable. Cisplatin is the most commonly used agent in combination with RT. It interacts with DNA to form inter- and intra-strand cross links, as well as DNA–protein cross links, inhibiting DNA replication and RNA transcription and ultimately inducing mutagenesis or apoptosis (Eastman and Barry 1987). When cisplatin is delivered concurrently with IR, there is an increase in the number of toxic platinum intermediates in the presence of oxygen free radicals generated by IR (Richmond et al. 1984). A third-generation cisplatin analogue, oxaliplatin, has also demonstrated potential radiosensitization (Cividalli et al. 2002) and cytotoxicity in cisplatin-resistant systems (Kraker and Moore 1988). Temozolomide (TMZ) is a DNA-alkylating agent that methylates guanine on the O6 position (Hickman and Samson 1999). These alkylated lesions are usually processed by the DNA repair enzyme O6-methylguanine DNA-methyltransferase (MGMT) that acts by transferring the alkyl groups present on the O6 position of guanine to a cysteine residue in its active site; hence, tumours not expressing MGMT are preferentially radiosensitized (Hermisson et al. 2006). TMZ has a favourable toxicity profile, and its combination with RT for patients with glioblastoma is a highly efficacious and well-tolerated therapeutic regimen (Stupp et al. 2005). 5-Fluorouracil (5-FU) and capecitabine, an oral prodrug of 5-FU, are halogenated pyrimidine nucleoside analogues commonly used with IR in the clinic. Gemcitabine is a pyrimidine analogue with radiosensitizing effects against several different cancer cell lines. Clinical trials have shown promising results for the treatment of non-small cell lung cancer (NSCLC) and pancreatic cancer with the combination of RT and gemcitabine (Blackstock et al. 2001; Talamonti et al. 2006). Cisplatin and gemcitabine are among the most effective clinical radiosensitizers, and recent reports have suggested that these drugs can act in part by inhibiting HR and NHEJ (Wachters et al. 2003; Diggle et al. 2005). These observations support the concept that the pre-determination of the repair capacity of tumour cells may help select appropriate agents for use in combination with radiotherapy (Fan et al. 2004; Taneja et al. 2004). Some tumour cells may have acquired DNA repair defects during carcinogenesis because loss of DNA damage surveillance contributes to a hypermutable phenotype (Loeb et al. 2003, 2008). Thus, therapeutic targeting of specific components of DNA repair pathways in cancer cells should enhance the efficacy of conventional anti-cancer treatments. DNA repair gene expression correlates with radiosensitivity (Bishay et al. 2001; Djuzenova et al. 2004). As DSBs are considered the most important type of lesion for the biological effects of IR, the proteins that have been targeted predominantly for radio-gene therapy strategies are those that play a critical role in DSB repair pathways (Collis and DeWeese 2004). Furthermore, Table 7.1 lists the chemical

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Table 7.1 Examples of radiosensitization effected by pharmacological inhibitors of DNA d amage repair (see main text for supporting references) Pharmacological agent Target Radiosensitization effect observed NHEJ inhibitors LY294002 DNA-PK Radiosensitization of tumour cells and tumour xenografts NU7026 DNA-PK Potentiation of IR cytotoxicity in DNA-PK proficient but not in DNA-PK deficient cells. Additive effect with PARP inhibitor AG14361 IC87102 DNA-PK Radiosensitiser in tumour cells and xenografts IC87361 DNA-PK Radiosensitiser in tumour cells and xenografts Wortmannin DNA-PK Radiosensitization in radioresistant bladder tumour cell lines and a xenograft model of hepatocarcinoma BER inhibitors Methoxyamine

APE1

Potentiation of the radiosensitization of 5-iodo-2¢-deoxyuridine in cancer cells

PARP inhibitors NU1025

PARP

NU1085

PARP

ABT-888 AG14361

PARP PARP

AG014699 GPI15427

PARP PARP

Potentiation of cytotoxicity of IR in the murine leukaemia cell line L1210 and in the Chinese hamster ovary-K1 cells Potentiation of cytotoxicity of IR in the murine leukaemia cell line L1210 Radiopotentiation in xenografts of colorectal and lung tumours Radiopotentiation in cancer cell lines and in colon carcinoma subcutaneous xenografts when administered intra-peritoneally Radiopotentiation in xenografts of colorectal and lung tumours Radiopotentiation in a human xenograft head and neck model

Cell-cycle checkpoints inhibitors KU-0055933 ATM Enhances the effects of IR in different cell lines Caffeine ATR Radiopotentiation in different cancer cell lines depending on the p53 status Pentoxyfylline ATR Radiopotentiation in different cancer cell lines depending on the p53 status. Little improvement in tumour response rates combined to RT in NSCLC and in patients with brain metastasis Staurosporine CHK1 Radiopotentiation in different cancer cell lines UCN-01 CHK1 Radiopotentiation in murine fibrosarcoma and in mutated-p53 carrying NSCLC models Flavopiridol CDK Radiopotentiatio in colon and gastric cancer cells CYC-202 CDK Radiopotentiatio in EBV-positive nasopharyngeal xenograft models BMS-387032 CDK Enhanced radiosensitization in quiescent and hypoxic NSCLC cells MGMT inhibitors O6-BG

Enhanced radiosensitization in a panel of four primary human glioblastoma cell lines with heterogeneous MGMT protein expression, normal human astrocytes, and U87 xenografts, concurrent with TMZ

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inhibitors of DNA damage repair that have been most recently developed and used as radiosensitizers in experiments performed in vitro and in clinical trials. Because of the increasing accuracy in the focused delivery of IR to tumours using new techniques for high-precision conformal radiotherapy (Purdy 2008), targeting of DNA repair proteins would lead to a selective killing of cancer cells in the irradiated field, reducing the risk of toxicity to the normal tissue due to the inhibition of mechanisms of repair of DNA damage. Expression levels of several proteins involved in NHEJ have been targeted, including Ku70, Ku80 and DNA-PK (consisting of DNA-PKcs and the Ku70/80 heterodimer) (Belenkov et al. 2002; Omori et al. 2002; Sak et al. 2002), with strategies such as RNA interference, anti-sense and novel inhibitory small molecules. Inhibition of DNA-PK has led to increased radiosensitization in vitro and in vivo in lung, colon, prostate and brain cancer models but, unfortunately, also in normal human fibroblasts (Collis et al. 2003; Peng et al. 2005). This latter observation might be relevant to predict possible normal tissue toxicity and an unfavourable therapeutic ratio. Novel small-molecule inhibitors of DNA-PK have been tested both in vitro and in vivo. LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) is a competitive inhibitor of DNA-PK that caused significant radiosensitization in in vitro studies and in tumour xenografts (Gupta et al. 2003; Okayasu et al. 2003). NU7026 (2-(morpholin-4-yl)-benzo[h]chromen-4-one) is a potent and specific inhibitor of DNA-PK that potentiates IR cytotoxicity in DNA-PK-proficient, but not in DNA-PK-deficient cells, and has additive effects as a radiosensitizer when used in combination with the PARP inhibitor, AG14361 (Veuger et al. 2003, 2004). Novel DNA-PKcs inhibitors, the arylmorpholines IC87102 and IC87361, are potent radiosensitizers in tumour xenografts that enhance the cytotoxic effects of IR in both tumour cells and tumour microvasculature (Shinohara et al. 2005). The steroid-like molecule wortmannin is an irreversible inhibitor of DNA-PK, which caused significant potentiation of toxicity of IR in pre-clinical studies, in cell lines (Ortiz et al. 2004), and in a C3H/HeJ mice bearing syngeneic hepatocarcinoma (Kim et al. 2007). Inhibition of DNA-PK with anti-sense oligodeoxynucleotide is a different strategy used to inhibit DSB rejoining and resulting in radiosensitization of NSCLC cell lines (Sak et al. 2002). Similar approaches have been used to disrupt HR repair by interfering with protein expression to potentiate the lethality of IR. Investigators have observed increased radiosensitization in vitro and in vivo by treating lung, glioma and prostate tumour cells with siRNA or anti-sense to RAD51 (Collis et al. 2001; Fan et al. 2004). Furthermore, peptides exist that appear to disrupt the functions of XRCC3 (Connell et al. 2004) and histone H2AX (Taneja et al. 2004), although this protein’s role in radiosensitization is yet to be established. Several pre-clinical studies using anti-sense and RNA interference have shown that downregulation of BER enzymes sensitises cells to various anti-cancer agents. In particular, APE1 has been described to have a role in the protection of the toxic effects caused by bleomycin and IR treatments through its 3¢-phosphoglycolate diesterase activity. Downregulation of APE1 sensitises cells to alkylating agents and IR (Tell et al. 2005). Methoxyamine is a small molecule that specifically targets

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BER by binding directly to AP sites and preventing their processing by APE-1 (Liu and Gerson 2004). Combining methoxyamine with 5-iodo-2¢-deoxyuridine, a halogenated thymidine analogue that has been found to radiosensitize cancer cells in pre-clinical studies, has been shown to potentiate the radiosensitizing effect exerted by 5-iodo-2¢-deoxyuridine alone (Taverna et al. 2003). The PARP family of proteins, composed of at least 18 members, is characterised by the enzymatic property of poly(-ADP-ribosyl)ation, a function that modulates the catalytic activity and protein–protein interactions of several target proteins involved in a range of several cellular processes (D’Amours et al. 1999). PARP1 is responsible for at least 80% of total cellular PARP activity and together with its nearest relative PARP2 in the family is devoted to the DNA damage response (Ame et al. 2004). PARP1 has a role in the sensing and repair of DNA SSBs; there is a lot of interest in developing strategies for targeting this protein in cancer treatment. In fact, PARP1 has been shown to be involved in several steps of DNA damage repair, the completion of BER (Ahnstrom and Ljungman 1988), the recruitment of the MRN complex to DNA damage sites (Haince et al. 2008) and the activation of ATM (Haince et al. 2007). PARP inhibitors have been shown to be especially useful not only when combined with IR or chemotherapy but also as single agents in cancer cells with defects in HR (see Sect. 7.4). In vitro studies have suggested that PARP inhibitors enhance the cytotoxicity of IR (Fernet et al. 2000; Veuger et al. 2003; Brock et al. 2004; Calabrese et al. 2004; Chalmers et al. 2004; Noel et al. 2006). PARP1 knockout mice are hypersensitive to IR and alkylating agents (Molinete et al. 1993; de Murcia et al. 1997). Overexpression of human PARP in transfected hamster cells leads to increased poly(-ADP-ribosyl)ation and cellular sensitization to gamma irradiation (Van Gool et al. 1997). PARP inhibitors appear to be effective as radiosensitizers especially in cells in S or G2 cell-cycle phases, and non-cycling cells exhibit minimal sensitivity (Chalmers et al. 2004; Noel et al. 2006). Although radiosensitization is partly due to the inhibition of SSB repair, it is likely that DSB repair, which may be more cytotoxic, is also affected. In fact, a role for PARP in NHEJ repair has been suggested. PARP can bind DSB, DNA-PKcs and Ku70/80. Furthermore, PARP together with XRCC4 and ligase III is actively involved in DNA end rejoining, independent of the complex DNA-PK/XRCC4-ligase IV (Audebert et al. 2004). In this respect, it is noteworthy that by combining DNA-PK inhibitors and PARP inhibitors, 90% of DSB repair after IR can be prevented with an additive effect of the two compounds together (Boulton et al. 1999; Veuger et al. 2003). This is explained by PARP1 being critical in a backup NHEJ pathway, the so-called B-NHEJ pathway, active in cells defective in the DNA-PK dependent NHEJ pathway (Wang et al. 2005, 2006). Although PARP activity seems important for the repair of both SSBs and DSBs caused by IR, one study has found that a PARP1 knockout cell line is immune to the radiosensitization effects of PARP inhibitors at low doses of radiation (Chalmers et al. 2004). This is likely explained by a differential role of PARP1 in response to low dose IR, which would be relevant for non-targeted tissue receiving lower IR dose during conventional RT course.

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Several small-molecule PARP inhibitors have been synthesised, and novel highly specific inhibitors are produced by a number of pharmaceutical companies. Quinazolin-4-[3H]one (e.g. NU1025) and benzimidazole-4-carboxamide (e.g. NU1085) are some of the first potent inhibitors of PARP1 and were shown to have the ability to potentiate the in vitro cytotoxicity of a range of agents, including TMZ and IR (Boulton et al. 1995, 1999; Bowman et al. 1998). In a panel of human common tumour cell lines, both NU1085 and NU1025 have been shown to enhance the cytotoxicity of TMZ and topotecan, an inhibitor of topoisomerase I, independent of p53 status and tissue of origin (Delaney et al. 2000). Radiopotentiation has also been reported with ABT-888 in xenografts of colorectal and lung tumours (Albert et al. 2007; Donawho et al. 2007), with AG14361 in cancer cell lines and when administered intra-peritoneally in colon carcinoma subcutaneous xenografts (Calabrese et al. 2004), and with AG014699 (Albert et al. 2007; Thomas et al. 2007). GPI 15427 enhanced radiation treatment in a human xenograft head and neck model (Khan et al. 2010). In addition, oral administration of GPI 15427 increases the anti-tumour activity of TMZ against intra-cranial melanoma, glioma and lymphoma (Tentori et al. 2003). Potentiation of TMZ cytotoxicity has been confirmed in pre-clinical models with the other novel inhibitor Ino-1001 (Cheng et al. 2005). Chemopotentiation of TMZ, irinotecan and cisplatin by CEP-6800 a new PARP inhibitor, has recently been reported in tumour xenografts (Miknyoczki et al. 2003). The first PARP inhibitor to enter cancer clinical trials was AG014699 in combination with TMZ in patients with metastatic malignant melanoma. A clinical report of PARP1 overexpression in poor prognosis malignant melanoma (Staibano et al. 2005) suggested that this enzyme is a potential therapeutic target in melanoma, consistent with data from other malignant tissues (Hirai et al. 1983; Shiobara et al. 2001). It is not yet clear whether it will be possible to exploit this overexpression as a biomarker to select patients who might benefit from treatment with PARP inhibitors. Owing to the promising lack of toxicity of the combination treatment recorded in this phase I trial (Plummer et al. 2005, 2008), a phase II study as first-line therapy in metastatic melanoma was commenced, and first results have been reported in abstract form (Plummer R et al. First and final report of a phase II study of the poly(ADP-ribose) polymerase (PARP) inhibitor, AG014699, in combination with temozolomide (TMZ) in patients with metastatic malignant melanoma (MM), 2006, in 2006 ASCO Annual Meeting, Atlanta, GA). Response rates of the combination therapy were encouraging, but the haematological toxicity of TMZ was exacerbated with one toxic death, three patients hospitalised because of myelosuppression and a TMZ dose reduction was required in 12 of the 40 patients. Alternative schedules of TMZ are available to enhance the anti-tumour effects of combination regimens while keeping toxicity profiles within acceptable parameters. A number of clinical trials of PARP inhibitors combined with chemotherapy are currently underway or in development, but data are not yet available (Helleday et al. 2008). It has been recently suggested to combine RT with TMZ and PARP inhibitors in the adjuvant setting of glioblastoma (Chalmers 2009), since the specificity of the radiosensitizing effects of PARP inhibitors for cells undergoing DNA replication

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(Dungey et al. 2008). A phase I/II clinical trial has just been started combining the PARP inhibitor ABT-888, RT and TMZ in patients with newly diagnosed glioblastoma multiforme (NCT00770471). Another phase I trial is currently ongoing with ABT-888 and conventional whole-brain RT in patients with brain metastases (NCT00649207). The major concern about these therapeutic regimens is the toxicity documented in vitro of combination of PARP inhibitors and TMZ, not only in cells undergoing DNA replication but in resting cells as well (Tentori et al. 2001). Although no significant toxicities have been reported so far, results of ongoing and future clinical trials are awaited to carefully monitor early effects on normal tissues, particularly gut epithelium and bone marrow. It should also be noted that, analogous to the effects of IR on certain normal tissues, covert toxicity to differentiated resting cells may not become apparent until months or years have passed since treatment with PARP inhibitors (Sharma and Dianov 2007). Targeting cell-cycle checkpoint-associated proteins, such as ATM and ATR (Canman et al. 1998; Cliby et al. 1998; Falck et al. 2001), in tumour cells in combination with RT, is an attractive therapeutic concept. Defects in DNA damage checkpoint pathways result in sensitivity to a range of anti-cancer treatments. Loss of ATM results in sensitivity to IR (Taylor et al. 1975). KU-0055933 (2-morpholin- 4-yl-6thianthren-1-yl-pyran-4-one) is a small molecule ATP competitive inhibitor of ATM kinase that significantly enhanced the effects of IR or DSB induced by etoposide (Hickson et al. 2004). As tumour cells lack a normal G1 cell-cycle checkpoint and rely more on preserved S and G2 for survival when compared to normal cells (Kawabe 2004), several studies have been conducted to test G2-checkpoint inhibitors, such as caffeine (1,3,7-trimethylxanthine), the derivative pentoxifylline, and the natural alkaloid staurosporine, as radiosensitizers (Eastman 2004). The radiosensitizing effects of caffeine were influenced by the p53 status in different cancer cell lines (Valenzuela et al. 2000). In the same fashion as for caffeine, p53 mutations are a general requirement for radiosensitization by pentoxyfilline, depending upon the location of the p53 mutation (Binder et al. 2002). Furthermore, pentoxifylline has been combined with RT to treat NSCLC patients (Kwon et al. 2000) and with standard-dose whole-brain RT to treat patients with brain metastasis (Johnson et al. 1998), with modest improvement in tumour response rates. UCN-01 is a staurosporine analogue CHK1 inhibitor that exhibits a potent radiosensitivity effect in murine fibrosarcoma (Tsuchida and Urano 1997) and mutated-p53 carrying NSCLC models (Xiao et al. 2002). Clinical trials are underway to test the efficacy of the more selective CDKs antagonists, such as the synthetic flavone flavopiridol, as single agents or in combination with other treatments (Kawabe 2004). In colon and gastric cancer cells, flavopiridol has been shown to potentiate the effect of IR (Jung et al. 2003). A phase I study involving patients with inoperable pancreatic cancer treated with flavopiridol on a twice-weekly schedule, concurrently with standard daily fractionated RT, has just been completed, but data have not been published yet (Ma et al. 2003). Several newer compounds have been identified that inhibit CHK1 with varying degrees of specificity (Noble et al. 2004). The 2,6,9-trisubstituted purine CYC202-roscovitine is a CDK inhibitor that enhanced the anti-tumour efficacy of RT in EBV-positive nasopharyngeal xenograft models (Hui et al. 2009). Another potent

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CDK inhibitor, the aminothiazole BMS-387032, has caused enhanced radiosensitization in quiescent and hypoxic NSCLC cells (Kodym et al. 2009). MGMT, the protein responsible for the repair of TMZ-induced lesions, is involved in the mechanism of resistance to several anti-cancer agents, not only TMZ and other methylating agents, such as dacarbazine and procarbazine, but also chloroethylating agents, such as chloroethylnitrosoureas, viz. BCNU or carmustine, CCNU or fotemustine (Friedman et al. 2002). In this respect, it has been reported that the survival benefit observed in patients treated with TMZ and RT was significantly greater for patients whose tumours contained a methylated MGMT promoter (Hegi et al. 2005). For this reason, MGMT inhibitors have been used in the combined regimen of RT and methylating (Chakravarti et al. 2006) or chloroethylating agents (Palanichamy and Chakravarti 2009). Inhibitors of MGMT consist of pseudosubstrates to act as a suicide inhibitor of DNA methyltransferase activity. Using a panel of four primary human glioblastoma cell lines with heterogeneous MGMT protein expression, normal human astrocytes and U87 xenografts, it has been shown that the MGMT inhibitor O6-benzylguanine (O6-BG) can enhance the anti-tumour effects of concurrent radiation and TMZ. A clinical phase III trial for newly diagnosed glioblastoma, of RT and BCNU with and without O6-BG, is currently ongoing (NCT00017147). It is not yet known whether radiation therapy and carmustine are more effective with or without O6-BG (Palanichamy and Chakravarti 2009). Although exciting in concept, clinical trial data for many of these agents in combination with RT is still lacking. Biomarker studies are needed to identify tools to assess the efficacy of DNA repair inhibitors in combination with RT in patient tissues. In this respect, detection and quantification of intra-nuclear focal complexes of activated checkpoint or DNA repair proteins could help in determining genetic instability level or DNA repair activity in treated tissues (Fan et al. 2004; Qvarnstrom et al. 2004). For example, gH2AX foci form in a dose-dependent manner at the site of DNA breaks within minutes of irradiation. The number and resolution of these foci over time is an indicator of global DNA-DSB repair and correlates with tumour cell radiosensitivity under oxic and hypoxic conditions (Banath et al. 2004).

7.4 Exploiting DNA Repair Defects for Selective Cancer Therapy The implementation of microarray and proteomic technologies has started to clarify the so-called molecular signature of tumours, the complex network of genes that are activated and deregulated in different cancer types. This knowledge has been pivotal for the increasing development of tailored cancer treatments we have had in the latest years, and thus for cancer cures. In this respect, the molecular characterisation of a sub-set of tumours harbouring specific defects in the DNA repair pathway has unravelled the mechanisms that lead to DNA damage and repair, thus allowing the design of new therapeutic strategies, and leading to the possibility of tailoring treatment according to the particular tumour DNA repair defect.

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Cells lacking or expressing defective variants of certain proteins involved in the sensing/detection, signalling, repair and cell-cycle checkpoints tend to be sensitive to DNA-damaging agents (Moses 2001; Petrini and Stracker 2003; Thompson and Schild 2002). Quite often, these defects will manifest as genetic disorders associated with an elevated pre-disposition to cancer and sensitivity to DNA damaging agents such as UV and IR (Shiloh 2003). For example, patients who possess defects in ATM, the gene defective in the cancer pre-disposing disorder ataxia telangiectasia (Petrini and Stracker 2003; Shiloh 2003), are hypersensitive to IR. If one can mimic this phenotype specifically within the tumour cell microenvironment, then theoretically a much greater amount of tumour eradication could be achieved and may permit a reduction in total radiation exposure, reducing normal cell toxicity side effects. This simple paradigm is the basis of an increasing number of different strategies that aim to target specific DNA repair proteins. Other genetic syndromes exist with defective DNA repair pathways. Besides the previously described HR, NHEJ, BER and MMR, other DNA repair pathways exist, such as nucleotide excision repair (NER) and translesion synthesis (TLS) repair. Coordination of NER, HR and TLS repair is thought to be defective in cells from patients with the genetic disorder Fanconi anaemia (Kennedy and D’Andrea 2005). NER repair is defective in xeroderma pigmentosum cells and Cockayne syndrome cells (Lehmann 2003). MMR is defective the Lynch syndrome or in children with Turcot syndrome and in tumour cells derived from adult patients with hereditary non-polyposis colorectal cancer (Hegde et al. 2005). Although these syndromes are rare, the DNA repair pathways disrupted by germ-line mutations in these individuals are often the same pathways disrupted by somatic mutation or epigenetic inactivation in cancers from the general population. For these sporadic cancers, a knowledge of which DNA repair mechanism is disrupted provides important clues to the behaviour of the cancer and may help to predict the specific treatment sensitivity spectrum. Tumour progression in general is often associated with progressive impairment of DNA damage repair (Seoane et al. 2008). Furthermore, during chemotherapy or RT, genomic instability is further amplified and the cell capability to effectively carry out the DNA damage repair progressively deteriorates (Wynne et al. 2007; Borst et al. 2008). We could therefore conclude that a large majority of sporadic cancers to be treated have defects in one or more elements of the DNA damage repair machinery. As discussed previously, most of the current small-molecule inhibitors of DNA repair have so far been tested in early clinical trials as sensitizers of tumour cells to chemotherapy. However, DNA damage also occurs spontaneously in cells in the absence of treatments, and DNA repair pathways are therefore essential for the survival of untreated cells. The “synthetic lethality” principle, described over a half century ago (Dobzhansky 1946), is defined as the situation when mutation in either of two genes individually has no effect, but combining the mutations leads to cell death. Targeting the redundant DNA repair pathway activated in tumour cells bearing specific defects in one or more DNA damage response pathways due to cancer cells’ rapid turnover or to germ-line mutations leads to selective tumour

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cell killing and thus represents a great opportunity for the exploration of this “weakness” therapeutically (Helleday et al. 2008). The concept of synthetic lethal interactions can be utilised to advocate the use of DNA repair inhibitors as monotherapies. DNA repair is an ideal target for inhibition in cancer cells, as the inhibitors should be exclusively toxic to cancer cells and therefore be associated with minimal side effects for patients (Kaelin 2005). As mutations in checkpoint and DNA repair pathways are associated with cancer, it should be straightforward to exploit DNA repair inhibitors for the treatment of tumours carrying specific defects in DNA repair or damage signalling. Genomewide screens in model organisms, such as the budding yeast Saccharomyces cerevisiae, have helped to identify protein interaction networks, which serve to elucidate protein functions within highly complex cellular processes, and homologous synthetic lethal interactions with mutations in DNA damage response genes found in human tumours. Such knowledge can prove extremely useful in identifying suitable targets for monotherapy, but perhaps more significantly, those that could be used in combination therapies. Unfortunately, the poorly defined biochemical properties of some of the proteins involved in the repair mechanisms are still a significant barrier to their exploitation as potential targets for drug intervention in the immediate future (Helleday et al. 2008). Indeed, DNA repair inhibitors have been demonstrated to work as single agents in patients with DNA repair-defective tumours. Tumour inactivation of DNA damage signalling and DNA repair are often relatively early events during carcinogenesis, suggesting that non-toxic DNA repair inhibitors may be considered in the treatment of patients with pre-malignant or early neoplastic lesions, such as those arising in patients with inherited mutations in BRCA1 or BRCA2 genes, or intestinal lesions in patients with defects in hMLH1 and hMSH2 genes or changes in their methylation status. The most notable example so far is the use of PARP inhibitors to treat patients with inherited breast and ovarian cancers that lack wild-type copies of the BRCA1 or BRCA2 genes (Bryant et al. 2005; Farmer et al. 2005). BRCA1- and BRCA2-mutated cells are defective in HR repair (Moynahan et al. 1999, 2001), which is required for efficient repair of replication-associated DSBs (Arnaudeau et al. 2001). These recombination-defective cells are 100–1,000-fold more sensitive to PARP inhibitors than are the heterozygote or the wild-type cell lines, indicating their potential to be exploited as specific treatments of BRCA1 or BRCA2 defective tumours (Bryant et al. 2005; Farmer et al. 2005). One explanation for this sensitivity is that PARP inhibitors induce SSBs that can result in DSBs as a result of stalled replication forks. Such lesions would normally be repaired by HR, but this is prohibited in BRCA1- or BRCA2-deficient cancer cells (Bryant et al. 2005; Fisher et al. 2007; Helleday et al. 2005; Schultz et al. 2003). PARP activity is also required for the actions of the protein checkpoint with forkhead-associated FHA and ring finger (CHFR) (Ahel et al. 2008) and the reactivation of stalled replication forks (Bryant et al. 2009; Yang et al. 2004). These functions might also explain the hypersensitivity to PARP inhibitors in recombination-defective cells. Besides germ-line mutations/deletions of the BRCA1 gene, methylation of the BRCA1 promoter region can cause diminished BRCA1 transcription in breast

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tumours (Catteau et al. 1999; Esteller et al. 2000; Matros et al. 2005; Rice et al. 2000). Transcriptional inhibitors, ID4 (Turner et al. 2007) and HMGA1 (Baldassarre et al. 2003), have also been implicated in suppressing BRCA1 transcription. The condition of sporadic tumours displaying a phenotype similar to the BRCA germ-line mutation in the absence of a mutation in the BRCA genes is named “BRCAness” (Turner et al. 2004). These sporadic tumours with the BRCAness phenotype should be very sensitive to the treatment with PARP inhibitors used as monotherapy. Furthermore, it has been shown that hypoxia causes the downregulation of HR proteins such as BRCA1 and RAD51 (Bindra et al. 2005; Chan et al. 2008). In this regard, it has been shown that the novel PARP inhibitor ABT-888 radiosensitizes malignant human cell lines under hypoxia. It would be interesting, for therapeutic purposes, to investigate whether PARP inhibitors can selectively kill hypoxic regions of tumours (Liu et al. 2008). Translation of these observations has led to a phase II clinical trial of monotherapy using the PARP inhibitor, AZD2281 (AstraZeneca, London, UK), currently recruiting patients with breast and ovarian cancer, who harbour mutations in BRCA1 or BRCA2 genes. Interestingly, in this trial, responses were much more frequent in patients whose tumours were cisplatin-sensitive than in cisplatin-refractory cases. Resistance of BRCA2-deficient cell lines to PARP inhibition has been shown to be associated with restoration of function of the HR pathway, and the observation of a similar mechanism conferring resistance to platinum-based cytotoxic agents may explain this cross-resistance (Edwards et al. 2008). This phase II trial follows a successful phase I dose escalation trial with the same drug, which has shown excellent toxicity data (Fong et al. 2009). A separate phase II trial with the PARP-1 inhibitor AG014699 (Pfizer GRD, La Jolla, CA) is due to open to recruitment of known carriers of BRCA1 or BRCA2 mutations with locally advanced or metastatic cancers of the breast or ovaries. It should be noted, however, that not all patients with mutations in BRCA1 or BRCA2 respond to PARP inhibitors as a monotherapy. The reasons for this are currently under investigation. Cells defective in recombination-related proteins other than BRCA1 or BRCA2, such as RAD51, RAD54, XRCC2, XRCC3, DSS1, RPA1, ATM, ATR, CHEK1, CHEK2, NBS1 and components of the Fanconi anaemia repair pathway, also show increased sensitivity to PARP inhibition (Bryant and Helleday 2006; Bryant et al. 2005; McCabe et al. 2006). This suggests that PARP inhibitors might also be suitable in treating several types of tumours with defects in HR. Besides defects in genes involved in HR, such as BRCA1 and 2, mutations in genes encoding important checkpoint proteins involved in DNA damage repair might be exploited to improve cancer treatment. For example, the p53 gene is mutated in over 50% of human cancers, and cells without the p53-mediated G1 arrest rely on a G2/M cell cycle arrest to avoid mitotic catastrophe. However, if the G2/M checkpoint is targeted by, for example, CHK1 inhibitors, p53-deficient cancer cells will lose their ability to arrest in G2/M following radiation or chemotherapy (Chen et al. 2006). Thus, the combination of CHK1 inhibitors and RT should be selectively toxic to p53-defective tumour cells, since normal cells with wild-type p53 would be able to arrest in G1 (Koniaras et al. 2001). Furthermore, ATM is frequently mutated and

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deregulated in human cancers (Sommer et al. 2003). Since the role of ATM in repairing DNA following exposure to agents inducing DNA strand breaks, it is expected that ATM-defective tumours are radiosensitive (Mamon et al. 2003; Zhang et al. 2004). Thus, tumours defective in ATM function would be good candidates for RT. Other genes involved in DNA damage repair might be deficient in sporadic cancers. HR is frequently disrupted in breast and ovarian cancer (Chen et al. 1998), NER is potentially inactivated in testicular cancer (Koberle et al. 1999), and MMR in sporadic colon cancer (Kane et al. 1997; Peltomaki 2001). A biomarker for these specific DNA repair defects might help in predicting, at least in part, specific drug and radiation sensitivity. Reliable biomarkers are particularly needed for the selection of patients that might respond to treatments with DNA-damaging agents as monotherapy. This is particularly important as cancers in patients that have no DNA-repair defect will not respond. The most reliable biomarkers will likely be those that identify loss of specific post-translational modifications present in the DNA damage response and repair pathways, or those that indicate increased activity of the targeted pathway. For example, a biomarker that indicates a failure of BRCA1 and 2 function in tumour cells may allow the application of PARP inhibitors not only to patients with BRCA germ-line mutations but also to a wide spectrum of sporadic human malignancies deficient in expression of HR-related proteins.

7.5 Conclusions Most cancer therapies commonly applied at present, particularly IR and certain classes of cytotoxic chemotherapies, cause cell death by damaging DNA. The therapeutic index refers to the amount of tumour cell kill relative to normal tissue damage. Molecularly targeted agents have been developed over the past decade to try to improve the therapeutic index in the treatment of cancer. DNA repair inhibitors possess a risk of causing damage to normal tissues. For this reason, it would be preferable to develop DNA repair inhibitor strategies to enzymes/pathways involving key proteins, such as the BER pathway or PARP, which are aberrant in cancer cells. Basic scientific discoveries and the use of high-throughput screening have contributed to the elucidation of pathways involved in DNA damage and the identification of drug targets for combination treatment with conventional radiotherapy. Molecular/genetic and biochemical/proteomic methods are likely to help in tailoring appropriate treatments to future patients, according to their specific “tumour genotype,” probed by reliable markers. Although laboratory studies need to be conducted to establish the most rational dosage schedule of the treatment combination to optimise activity and reduce toxicity of new therapeutic regimens, only well-designed clinical trials can inform us whether or not the therapeutic index is improved for any potential new radiation-modulating drug. When extra polating data from controlled bench experiments to the clinic, it should be remembered that clinical RT generally consists of multiple repeated fractions of

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1.5–3 Gy/day (most commonly 1.8–2 Gy/day) delivered over weeks. Furthermore, different IR-induced gene expression patterns are also observed between cells grown in xenograft tumours relative to cells cultured in vitro (Khodarev et al. 2001). Basic research into the understanding of the nature of toxic lesions created in DNA and into the development of new DNA targeting drugs should eventually lead to the development of more effective therapeutic strategies targeting DNA repair to treat cancer. Acknowledgements The authors’ research programmes are supported by Cancer Research UK, the Swedish Cancer Society, the Swedish Children’s Cancer Foundation, the Swedish Research Council, the Swedish Pain Relief Foundation, the UK Breast cancer campaign, the UK Medical Research Council, the Higher Education Funding Council for England and the NIHR Biomedical Research Centre, Oxford.

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Chapter 8

Gene Therapy and Radiation Svend O. Freytag, Kenneth N. Barton, Farzan Siddiqui, Mohamed Elshaikh, Hans Stricker, and Benjamin Movsas

Abstract Owing to a low efficiency of gene transfer when delivered systemically, gene therapy may find its greatest utility in the clinic when combined with locoregional cancer treatment such as radiation therapy. Although a variety of gene therapy strategies have been combined with radiation in preclinical models, only a handful have been translated into the clinic. Overall, combining gene therapy with radiation therapy has been well tolerated. Most of the gene therapy-related adverse events have been mild to moderate, transient, produced no noticeable symptoms to the patient, and did not exacerbate the side effects of radiation therapy. Several strategies have demonstrated antitumor activity in early-stage trials, and at least two have progressed to phase 3. Future developments will be driven by a better understanding of the radiation response and molecular basis for tumor radioresistance. Keywords Gene therapy • Radiation therapy • Radiosensitizer • Adenovirus • Suicide gene therapy • Oncolysis • p53 • TNFa, EGFR • Ku70

8.1 Introduction Radiation therapy is one of the three major forms of cancer treatment and it is currently being used to treat approximately half of all cancer patients. When the cancer is detected early and localized, radiation therapy alone often results in very good tumor control. However, radiation therapy tends to be less effective against more advanced cancers owing to a large local tumor burden and/or acquired resistance to radiation. In the past, the most common strategy used to improve the outcome of radiation therapy has been dose escalation. Although gains have been made in several indications, there is a limit to the radiation dose that can be delivered to the

S.O. Freytag (*) Department of Radiation Oncology, Henry Ford Health System, One Ford Place, 5D, Detroit, MI 48202, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_8, © Springer Science+Business Media, LLC 2011

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tumor owing to damage to normal surrounding tissue. Further improvements in conformal techniques, which allow for the use of higher radiation doses, are likely to be incremental. Hence, in the future, we should focus our efforts on developing ways to make the prescribed radiation dose more effective (i.e., radiosensitization). Indeed, the gain achieved by radiosensitization can be many times greater than what is possible with dose escalation. Gene therapy is an investigational approach that has the potential to improve the effectiveness of cancer radiotherapy. Owing to a low efficiency of gene transfer when delivered systemically, gene therapy has not shown significant activity against metastatic disease in the clinic. By contrast, delivering genes to localized tumors is rather straightforward, and, therefore, gene therapy may find greater applicability when used as an adjuvant to loco-regional cancer treatments such as radiation therapy. A major attraction of gene therapy strategies is that they can be engineered to target the molecular defects that drive the abnormal behavior of malignant cells. Hence, rational therapies that specifically target the basis of tumor radioresistance can be developed. Here, we review the gene therapy strategies that have been combined with radiation therapy in the clinic as well as some new approaches that are currently under development.

8.2 Gene Therapy Strategies That Have Been Combined with Radiation Therapy in the Clinic 8.2.1 Suicide Gene Therapy Suicide gene therapy involves the tumor-targeted delivery of genes encoding metabolic enzymes that convert systemically delivered, innocuous prodrugs into toxic metabolites. A strength of suicide gene therapy strategies is that many of them demonstrate a bystander effect, whereby neighboring cells not expressing the suicide gene are killed due to the local diffusion of toxic metabolites (Moolten and Wells 1990; Culver et al. 1992; Huber et al. 1994). One of the first suicide genes studied, herpes simplex virus thymidine kinase (HSV-1 TK), converts nucleoside analogs such as ganciclovir (GCV) and acyclovir (ACV) into their corresponding monophosphates, which are potent DNA chain terminators and kill cells by inhibiting DNA replication. Kim et al. hypothesized that the HSV-1 TK/GCV suicide gene system might function as a tumor cell radiosensitizer by inhibiting the repair of potentially lethal DNA damage (PLD) following irradiation. Using human glioma cells stably expressing HSV-1 TK, sensitization enhancement ratios (SERs) of 1.3–1.9 were observed in vitro and better tumor radiocurability was achieved in vivo (Kim et al. 1994, 1995, 1997). Similar results were obtained by others (Vlachaki et al. 2001; Chhikara et al. 2001). Subsequently, mutant forms of HSV-1 TK with more favorable catalytic properties were developed making them preferable over the wild-type enzyme for suicide gene therapy (Black et al. 1996; Qiao et al. 2000; Black et al. 2001).

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The same group proposed that another suicide gene, cytosine deaminase (CD), might also function as a tumor cell radiosensitizer (Khil et al. 1995). CD is expressed in bacteria (bCD) and yeast (yCD), but not humans, and converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). 5-FU is used in the clinic in various indications and is a well-known radiosensitizer. The mechanism of 5-FU radiosensitization is believed to be mediated through the inhibition of thymidylate synthase (TS) by 5-FdUMP, resulting in the depletion of dTMP pools and increased DNA strand breaks as well as redistribution of cells in early S phase, a radiosensitive phase of the cell cycle (McGinn et al. 1996; Hwang et al. 2000). Using human colorectal adenocarcinoma cells stably expressing bCD, SERs of up to 2.4 were observed in vitro and significant improvements in tumor radiocurability were observed in vivo (Khil et al. 1995; Gable et al. 1997; Hanna et al. 1997; Stackhouse et al. 2000). Owing to its more favorable catalytic properties, yCD proved to be a better suicide gene than bCD (Kievit et al. 1999; Hamstra et al. 1999; Kievit et al. 2000). Hence, yCD was used in future studies. Because the CD/5-FC system induces DNA strand breaks and the HSV-1 TK/ GCV system can inhibit the repair of those breaks, Freytag et al. were the first to propose combining the two suicide gene systems (Rogulski et al. 1997a). They, and others (Aghi et al. 1998; Uckert et al. 1998), generated a novel bCD/HSV-1 TK fusion gene that could confer sensitivity to both 5-FC and GCV. Implementation of both suicide gene systems simultaneously without, or with, radiation proved to be far superior than either system alone in vitro and in vivo (Rogulski et al. 1997a, b, 2000a; Aghi et al. 1998; Uckert et al. 1998; Freytag et al. 1998, 2002, 2007a; Kim et al. 1998; Xie et al. 1999; Boucher et al. 2006). When used simultaneously, SERs of 2.4 could be achieved in vitro under conditions that resulted in SERs of 1.3–1.4 when the suicide gene systems were used independently. Significantly better tumor control was observed in preclinical models when both suicide gene systems were implemented. The safety and efficacy of combining CD/5-FC and/or HSV-1 TK/GCV suicide gene therapies with radiotherapy have been evaluated in several trials of newly diagnosed prostate cancer and primary glioblastoma (Rainov 2000; Teh et al. 2001, 2004; Freytag et al. 2003, 2007b; Immonen et al. 2004; Barton et al. 2008). In the prostate trials, the suicide genes were delivered intraprostatically via a replicationdefective or replication-competent adenovirus up to a dose of 2 × 1012 viral particles (vp)/injection. Up to three adenovirus injections were given. Overall, the combined therapy was well tolerated. The most frequent gene therapy-related side effects included mild to moderate (grade 1/2) flu-like symptoms (~33%), transaminitis (~33%), and hematological events (as high as 90%). All adverse events were transient and self-limiting. The flu-like symptoms and transaminitis are likely due to dissemination of the adenovirus beyond the prostate. Hematological events are likely due to the 5-FC and GCV prodrug therapies. Importantly, the gene therapy did not exacerbate the side effects of radiation therapy (Freytag et al. 2003). Provocative signs of efficacy have emerged. Two trials demonstrated better-thanexpected biopsy results at 2 years. In one study, a possible treatment effect was observed in men with intermediate-risk (Gleason = 7 or PSA 10–20 ng/mL), but not

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high-risk (Gleason ³ 8 or PSA > 20 ng/mL) features (Freytag et al. 2007b). Based on these results, a randomized, controlled phase 2/3 has been opened in newly diagnosed, intermediate-risk prostate cancer. In the second study, a possible treatment effect (100% adenocarcinoma free) was observed across all prognostic risk groups (Teh et al. 2004). These latter results should be interpreted cautiously as only two biopsy cores, rather than the standard 6–14, were taken in posttreatment biopsies. This is not standard practice and likely overestimates the true activity of the investigational therapy. In primary glioblastoma, HSV-1 TK suicide gene therapy has been combined with radiotherapy in two randomized trials (Rainov 2000; Immonen et al. 2004). In this setting, patients on the investigational therapy arm received a local injection of a replication-defective retrovirus or replication-defective adenovirus expressing HSV-1 TK following surgical resection of the primary tumor. They then received 2 weeks of GCV along with a standard course of radiotherapy. Patients on the control arm received standard therapy only (surgery and radiotherapy). Although the combined therapy was well tolerated, neither strategy produced convincing evidence of efficacy. In the trial that utilized the replication-defective retrovirus (248 patients), median survival on the investigational arm was 365 days vs. 354 days on the control arm. In the smaller trial that utilized the replication-defective adenovirus (36 patients), median survival on the investigational therapy arm was 62.4 weeks vs. 37.7 weeks on the control arm, raising the possibility that the gene therapy could in fact improve survival. However, these preliminary results were not confirmed in a larger, four-arm study of 251 patients. Here, patients were randomized to receive surgery followed by radiation ± temozolomide (control arms), or surgery followed by HSV-1 TK gene therapy (Cerepro) and radiation ± temozolomide (investigational arms). Surgery followed by chemoradiation (with temozolomide) is the standard of care for primary glioblastoma. The primary end point was survival, which was defined as death or re-intervention, the latter of which opens up the possibility of investigator bias and would likely drive the survival data. When comparing the two arms that received chemoradiation, median survival was essentially identical (~600 days). As a result, the European Medicines Agency rejected marketing authorization for Cerepro.

8.2.2 p53 Gene Therapy The p53 tumor suppressor gene codes for a sequence-specific DNA-binding protein that has pleotropic functions including the regulation of cell cycle arrest and apoptosis and is mutated in at least 60% of human cancers. Exposure of normal cells to DNA damaging agents, such as radiation, induces p53 expression and cell cycle delays in G1 and G2 permitting the repair of damaged DNA before cells enter S phase or mitosis. Following insult, p53 can induce cells down the apoptotic pathway and may be a major determinant of the sensitivity of cells to chemotherapeutic agents and radiation (Lowe et al. 1993, 1994; Hamada et al. 1996). Because p53 can induce apoptosis following irradiation, several groups proposed using p53 gene

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therapy as a tumor cell radiosensitizer (Gallardo et al. 1996; Spitz et al. 1996; Badie et al. 1998; Li et al. 1999; Sah et al. 2003). Adenovirus-mediated transfer of the wild-type p53 gene was found to potentiate the effects of radiation in a variety of human cancer cell lines in vitro. The magnitude of the radiosensitization effect (i.e., SERs) was rather modest (1.1–1.3) and lower than what can be achieved with suicide gene therapy. As expected, the increased radiosensitivity correlated with the induction of p53-dependent apoptosis. Based on these preclinical results, a number of p53 gene therapy trials were conducted targeting cancers of the head and neck, lung, ovary, urinary bladder, and brain (Clayman et al. 1998, 1999; Swisher et al. 1999, 2003; Weill et al. 2000; Nemunaitis et al. 2000; Lang et al. 2003; Pagliaro et al. 2003; Wolf et al. 2004; Yoo et al. 2009). These trials used adenovirus-mediated p53 gene therapy as a single agent or in combination with chemotherapy and/or radiotherapy. Overall, p53 gene therapy was well tolerated. Gene therapy-related side effects included local inflammation and mild flu-like symptoms. Preliminary signs of efficacy were observed in a phase 2 trial of non-small lung cancer (NSLC) (Swisher et al. 2003). Nineteen patients received 60 Gy of radiation over a 6-week period along with three intratumoral injections of a p53-expressing replication-defective adenovirus (INGN 201, Advexin). Following the gene therapy, 12 (63%) biopsies showed no viable tumor, which is better than expected (17–20%) for patients receiving radiotherapy only. Seventeen patients were evaluable by computed tomography (CT) and bronchoscopy at 3 months. The overall (p53-injected and noninjected) tumor response was complete response in 1 (5%) patient, partial response in 5 (26%) patients, stable disease in 1 (5%) patient, and progressive disease in 11 (58%) patients. With a median follow-up of 37 months, there was 47% overall survival at 1 year and 26% at 3 years. Five patients were alive without evidence of disease at 34–48 months.

8.2.3 Tumor Necrosis Factor Alpha (TNFa) Gene Therapy TNFa is a multifunctional cytokine that demonstrated antitumor activity over 30 years ago (Carswell et al. 1975; Creasey et al. 1986; Asher et al. 1987). However, the development of recombinant TNFa as a cancer therapeutic was hampered by its severe toxicity when delivered systemically (Chapman et al. 1987; Feinberg et al. 1988; Spriggs et al. 1988). Weischelbaum et al. were the first to propose using TNFa gene therapy as a tumor radiosensitizer (Weichselbaum et al. 1994; Hallahan et al. 1995; Chung et al. 1998). Their approach was somewhat unique in that they placed the TNFa gene under the control of the radiation-inducible Egr-1 promoter. Up to tenfold greater TNFa levels were observed in tumors following irradiation. Combining adenovirus-mediated TNFa gene therapy and radiation resulted in significant tumor control under conditions that produced only modest effects with either single modality. Although it was initially thought that TNFa might actually increase the intrinsic radiosensitivity of tumor cells, it is now believed that most of TNFa antitumor activity is mediated through its effects on the tumor vasculature (Staba et al. 1998).

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Adenovirus-mediated TNFa gene therapy (TNFerade) is another gene therapy strategy that has progressed to phase 3. Early-stage trials of solid cancers demonstrated that multiple injections of TNFerade were generally well tolerated up to a dose of 4 × 1011 particle units (pu)/injection (Senzer et al. 2004; Mundt et al. 2004; McLoughlin et al. 2005). The most common gene therapy-related side effects were mild to moderate injection-site pain and flu-like symptoms (~20%). Objective tumor responses were observed in a significant fraction of patients (up to 50%). Some patients had synchronous lesions that allowed for a comparison of the tumor response following the gene therapy plus radiotherapy vs. radiotherapy alone. In one study, four of five (80%) patients showed a greater response in the index lesion that received the same radiation dose as the control lesion. Based on these phase 1 results, a randomized, controlled phase 2/3 trial was opened in locally advanced pancreatic cancer comparing TNFerade plus chemoradiation vs. chemoradiation alone. The study called for 330 patients that were enrolled using a 2:1 (investigational arm:control arm) randomization scheme. Patients on the investigational therapy arm received five weekly intratumoral injections of TNFerade concomitant with a standard 5½ week course of 5-FU-based chemoradiation, whereas patients on the control arm received only standard chemoradiation. The primary end point was survival. At the first interim analysis (92 of expected 276 deaths), median survival on both the investigational therapy and control arms was 9.9 months (Chang et al. 2009). There was preliminary evidence of a “late effect” with 30.5% of patients being alive on the investigational therapy arm at 18 months vs. 11.3% on the control arm. Final results showed no difference in survival between the two arms and further development of TNFerade for pancreatic cancer was terminated.

8.3 Gene Therapy Strategies That Have Been Combined with Radiation in Preclinical Models 8.3.1 Replication-Competent Oncolytic Adenoviruses There are several possible mechanisms by which replication-competent adenoviruses may enhance the therapeutic effects of radiation. E1A expression is required for viral replication and it is well-established that E1A can sensitize cells to both chemotherapeutic agents and radiation (Lowe et al. 1993, 1994; Shao et al. 1997; 2001). Another hypothesis is that expression of the adenovirus E4-34K protein, which is known to disrupt V(D)J recombination through its interaction with DNAdependent protein kinase (DNA-PK), may block the repair of double-strand breaks (DSB) following irradiation (Collis et al. 2003a, b). DSB correlate closely with radiation lethality and cells defective in DSB repair and V(D)J recombination exhibit a radiation-sensitive phenotype (Rooney et al. 2002). A variety of replication-competent, oncolytic adenoviruses have been combined with radiation in preclinical models including Ad5-CD/TKrep (Freytag et al. 1998,

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2002; Rogulski et al. 2000b), Ad5-yCD/mutTKSR39rep-ADP (Freytag et al. 2007a), ONYX-015 virus (Rogulski et al. 2000a), CV706 (Chen et al. 2001), Ad5-D24RGD (Lamfers et al. 2002), KD1, KD3, and VRX-007 (Toth et al. 2003). Ad5-CD/TKrep and Ad5-yCD/mutTKSR39rep-ADP are armed with therapeutic “suicide” genes and have progressed into late-stage clinical trials, which has been discussed previously, whereas the other oncolytic adenoviruses lack a therapeutic gene. All exhibited additive or greater antitumor activity when combined with radiation in preclinical models.

8.3.2 Targeting Signal Transduction and Apoptotic Pathways One of the hallmark characteristics of the malignant cell is the dysregulation of growth- and survival-related signal transduction pathways that confer resistance to conventional cancer treatments such as chemotherapy and radiation therapy. Not surprisingly, components of these pathways including the epidermal growth factor receptor (EGFR) (Lammering et al. 2001, 2003), Raf-1 kinase (Soldatenkov et al. 1997; Kasid and Dritschilo 2003), NF-kB (Jung et al. 1995; Pajonk et al. 1999; Mukogawa et al. 2003), Bcl-2 (Arafat et al. 2000, 2003), mda-7/IL-24 (Kawabe et al. 2002; Su et al. 2003; Yacoub et al. 2003), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Chinnaiyan et al. 2000; Belka et al. 2001), have been the targets of many gene therapy strategies. Ablation of signal transduction activity using a dominant negative inhibitors (EGFR-CD533 with EGFR, IkB mutants with NF-kB, Bax with Bcl-2) or antisense oligonucleotides (Raf-1) demonstrated SERs of 1.3–2.4 in vitro and increased tumor radiocurability in vivo (Jung et al. 1995; Soldatenkov et al. 1997; Pajonk et al. 1999; Lammering et al. 2001, 2003; Kasid and Dritschilo 2003; Mukogawa et al. 2003). Given that EGFR, Raf-1, and NF-kB signaling are dysregulated in a wide spectrum of human cancers, these targeted gene therapies have the potential to have widespread applicability in the clinic. However, if these gene therapy strategies do not exhibit a bystander effect, they will be limited by the efficiency of gene delivery in vivo. The safety of administering a replication-defective adenovirus expressing mda-7/IL-24 (INGN241) as a single agent has been evaluated in advanced cancers (Tong et al. 2005). Multiple (twice weekly × 3) injections proved to be safe up to a dose of 2 × 1012 vp/injection. Significant tumor apoptosis was observed along with immuneactivating events. Clinical trials combining adenovirus-mediated mda-7/IL-24 gene therapy with radiation therapy are pending.

8.3.3 Targeting DNA Repair Pathways Genes involved in the repair of radiation-induced DNA damage are ideal targets for cancer therapies designed to increase tumor cell radiosensitivity. Double-strand DNA breaks are particularly lethal and are repaired by homologous recombination,

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single-strand annealing and nonhomologous end joining (NHEJ), the latter of which predominates in human cells. A number of genes involved in these DNA repair pathways have been identified including Ku (Ku70 and Ku80), DNA-PK, XRCC4, DNA ligase IV, RAD51, RAD 52, RAD 54, BCRA1, BRCA2, and others. These repair pathways are intimately linked to cell cycle checkpoints that involve the sensors of radiation-induced DNA damage, namely, ATM, ATR, DNA-PK, and their downstream effector, p53. Hence, several have been the targets of gene therapy strategies. Ablation of Ku, ATM, ATR, and DNA-PK expression by antisense strategies resulted in marked radiosensitization in vitro and improved tumor radiocurability (Nussenzweig et al. 1997; Marangoni et al. 2000; Li et al. 2003; Collis et al. 2003b). Further investigations of these gene therapy strategies are warranted.

8.3.4 Other Strategies Other gene therapy strategies that have been combined with radiation include nitric synthase (iNOS; Wang et al. 2004), phosphatase and tensin homolog (PTEN; Tomioka et al. 2008), and interference of genes that play a role in autophagy (Apel et al. 2008). Nitric oxide (NO) has many activities and is known to sensitize oxic tumor cells to irradiation. Adenovirus-mediated iNOS gene transfer into human colorectal cells resulted in respectable radiosensitization with SERs of ~1.6. An advantage of iNOS gene therapy is that it exhibits a bystander effect. PTEN is a tumor suppressor that negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway. Delivery of PTEN by microcapsules was shown to improve the radiocurability of prostate xenografts. Authophagy or “selfeating” is frequently activated in tumor cells treated with chemotherapy or irradiation. Inhibition of genes involved in autophagy using small interfering RNAs (siRNAs) potentiated the effects of irradiation in vitro. All of these approaches warrant further investigation.

8.4 Conclusion With the exception of perhaps ex vivo vaccine-based strategies, gene therapy has not demonstrated significant activity against metastatic disease in the clinic. This is not surprising given the low efficiency of gene transfer in vivo when delivered systemically. Until this limitation is overcome, it makes more sense to apply gene therapy as an adjuvant (meaning auxiliary) to loco-regional cancer treatments such as radiation therapy. As discussed above, a variety of gene therapy strategies designed to improve the effectiveness of radiation therapy have been developed to date. Of those translated into the clinic, all have been well tolerated and have not exacerbated the side effects of radiation therapy. Several have generated provocative

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antitumor activity in early-stage trials. These preliminary results await confirmation in larger randomized trials, which are in progress. In the future, we should focus our efforts on improving the efficiency of gene transfer (both locally and systemically) as well as targeting pathways that play a role in the radiation response and tumor radioresistance. Much has been learned about these pathways in recent years (discussed elsewhere) and this knowledge will undoubtedly lead to the development of new gene therapy strategies designed to make the tumor cell more susceptible to the therapeutic effects of radiation. However, as with all cancer therapies, it will be important that these new strategies specifically target the malignant cell while sparing normal tissue. Although much work lies ahead, we believe combining gene therapy with radiotherapy will someday earn a place in the management of human cancer. Acknowledgment This work was supported by a grant (P01 CA097012) from the National Institutes of Health to SOF.

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Shao R, Karunagaran D, Zhou B et al (1997) Inhibition of nuclear factor-kappa B activity is involved in E1A-mediated sensitization of radiation-induced apoptosis. J Biol Chem 272:32739–32742 Shao R, Tsai E, Wei K et al (2001) E1A inhibition of radiation-induced NF-kappa B activity through suppression of IKK activity and I-kappa B degradation, independent of Akt activation. Cancer Res 61:7413–7416 Soldatenkov V, Dritschilo A, Wang F et al (1997) Inhibition of Raf-1 protein kinase by antisense phosphorothioate oligodeoxyribonucleotide is associated with sensitization of human laryngeal squamous carcinoma cells to gamma radiation. Cancer J Sci Am 3:13–20 Spitz F, Nguyen D, Skibber J et al (1996) Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clin Cancer Res 2:1665–1671 Spriggs DR, Sherman ML, Michie H et al (1988) Recombinant tumor necrosis factor administered as a 24-hour intravenous infusion. A phase I and pharmacologic study. J Natl Cancer Inst 80:1039–1044 Staba M, Mauceri H, Kufe D et al (1998) Adenoviral TNF-a gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft. Gene Ther 5:293–300 Stackhouse M, Pederson L, Grizzle W et al (2000) Fractionated radiation therapy in combination with adenoviral delivery of the cytosine deaminase gene and 5-fluorocytosine enhances cytotoxic and antitumor effects in human colorectal and cholangiocarcinoma models. Gene Ther 7:1019–1026 Su Z, Lebedeva I, Sarkar D et al (2003) Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 22:1164–1180 Swisher S, Roth J, Komaki R et al (2003) Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery of adenoviral p53 (INGN 201) and radiation therapy. Clin Cancer Res 9:93–101 Swisher S, Roth J, Nemunaitis J et al (1999) Adenovirus-mediated p53 gene transfer in advanced non-small cell lung cancer. J Natl Cancer Inst 91:763–771 Teh B, Aguilar-Cordova E, Kernen K et al (2001) Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer – a preliminary report. Int J Rad Oncol Biol Phys 51:605–613 Teh BS, Ayala G, Aguilar L et al (2004) Phase I–II trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys 58:1520–1529 Tomioka A, Tanaka M, De Velasco M et al (2008) Delivery of PTEN via a novel gene microcapsule sensitizes prostate cancer cells to irradiation. Mol Cancer Ther 7:1864–1870 Tong A, Nemunaitis J, Su D et al (2005) Intratumoral injection of INGN 241, a nonreplicating adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer patients. Mol Ther 11:160–172 Toth K, Tarakanova V, Doronin K et al (2003) Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein. Cancer Gene Ther 10:193–200 Uckert W, Kammertons T, Haack K et al (1998) Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. Hum Gene Ther 9:855–865 Vlachaki M, Chhikara M, Aguilar L et al (2001) Enhanced therapeutic effect of multiple injections of HSV-TK + GCV gene therapy in combination with ionizing radiation in a mouse mammary tumor model. Int J Radiat Oncol Biol Phys 52:1008–1017 Wang Z, Cook T, Alber S et al (2004) Adenoviral gene transfer of the human inducible nitric oxide synthase gene enhances the radiation response of human colorectal cancer associated with alterations in tumor vascularity. Cancer Res 64:1386–1395 Weichselbaum R, Hallahan D, Beckett M et al (1994) Gene therapy targeted by radiation preferentially radiosensitizes tumor cells. Cancer Res 54:4266–4269

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Chapter 9

Molecular Targeted Drug Delivery Radiotherapy Eugenia M. Yazlovitskaya and Dennis E. Hallahan

Abstract This chapter discusses prosurvival signal transduction pathways, such as PI3K/Akt and MAPK/ERK signaling, induced by ionizing radiation in the tumor microvasculature. Molecular targeting of these radiation-induced signaling pathways provides a means to enhance tumor control. Preclinical studies show that this approach improves the efficacy of radiotherapy in mouse models of cancer. Keywords Signal transduction pathways • Molecular targeting • Radiotherapy

9.1 Introduction Roughly 50% of cancer patients are treated with ionizing radiation (IR) therapy (Owen et al. 1992). Despite this wide use and effectiveness, radiotherapy has limitations, such as restricted tolerance of normal tissues and tumor radioresistance. One approach to enhance the therapeutic ratio of ionizing radiation is to combine it with chemotherapy (Lawrence et al. 2003; Wilson et al. 2006). In general, radiotherapy and chemotherapy target deregulated cancer cells, and the initial rationale for the combination of radiation and chemotherapeutic agents was based on the “spatial cooperation” concept (Hall and Giaccia 2006). More recently, a new paradigm had emerged from the findings that the host component of cancer (e.g., microvasculature, stroma, and immune system) is an important target for the effects of radiation and cytotoxic drugs (Lu et al. 2005; Kim et al. 2006; Heath and Bicknell 2009). For example, the tumor vasculature is particularly interesting because of its resistance to clinically relevant low IR doses of 2–5 Gy (Geng et al. 2001; Edwards et al. 2002; Tan et al. 2006; Linkous et al. 2009).

D.E. Hallahan (*) Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Suite 200, St. Louis, MO 63130, USA e-mail: [email protected]

T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_9, © Springer Science+Business Media, LLC 2011

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9.2 Role of Vasculature in Development and Treatment of Solid Tumors Recent preclinical studies suggest that the combination of radiotherapy with antiangiogenic or vascular targeting agents enhances the therapeutic effect of ionizing radiation (Siemann and Horsman 2004; Wachsberger et al. 2004; Kobayashi and Lin 2006). Using tumor vascular endothelium for molecular targeted therapy has specific advantages. Formation of new blood vessels is very limited in developed tissues, thus, antiangiogenic drugs targeting proliferating tumor vascular endothelium causes little normal tissue toxicity. Most importantly, each tumor capillary supplies hundreds of tumor cells leading to potentiation of therapeutic effect (Wachsberger et al. 2003). The response of tumor microvasculature to radiation is dependent upon the dose and time interval after treatment. Tumor blood flow decreases when tumors are treated with single doses in the range of 20–45 Gy (Song et al. 1972). One of the underlying reasons for this response is that radiation doses in the range of 15–20 Gy given as a single treatment induce apoptosis in tumor vascular endothelial cells (Schwentker et al. 1998; Garcia-Barros et al. 2003, 2004). This apoptosis was shown to be dependent upon acid sphingomyelinase-mediated production of ceramide (Paris et al. 2001; Garcia-Barros et al. 2003, 2004), FGF signaling pathway (Paris et al. 2001), and/or Bax (Garcia-Barros et al. 2003, 2004). Tumor blood vessels show less response to radiation doses in the range of 2–3 Gy, which is typically used to treat cancer (Geng et al. 2001; Edwards et al. 2002; Tan et al. 2006; Linkous et al. 2009). Endothelial cells grown in culture are also resistant to the low doses of IR (Edwards et al. 2002; Tan et al. 2006; Yazlovitskaya et al. 2008). Molecular mechanisms of this radioresistance are not known; however, some recent studies suggest the involvement of prosurvival signaling pathways, such as activation of the receptor tyrosine kinases (RTK) (Geng et al. 2001; Kim et al. 2004; Yazlovitskaya et al. 2008), Src pathway (Cuneo et al. 2006), PI3K/Akt (Edwards et al. 2002; Tan and Hallahan 2003; Tan et al. 2006; Yazlovitskaya et al. 2008; Linkous et al. 2009), and ERK ( Maclachlan et al. 2005; Yazlovitskaya et al. 2008; Linkous et al. 2009). Therefore, blocking of radiation-induced activation of these pathways could potentially abrogate radioresistance of the tumor vasculature and potentiate tumor response to radiotherapy.

9.3 Radiation-Induced Prosurvival Signal Transduction Pathways Exposure of cells to ionizing radiation induces multiple cellular and biological effects by either direct DNA damage or through the formation of free radical species (Fig. 9.1a). Accumulating evidence suggests that these initial events activate multiple signaling pathways (Valerie et al. 2007). A balance exists in the

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activation of prosurvival and cell killing pathways which determine cellular response to radiation, ultimately controlling cell metabolic, proliferative, or death processes (Fig. 9.1a) (Schmidt-Ullrich et al. 2000; Dent et al. 2003a; Valerie et al. 2007). The main signal transduction pathways activated by ionizing radiation in a

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Fig. 9.1 Radiation-induced signal transduction pathways. (a) Schematic representation of r adiation-induced signal transduction pathways, including prodeath and prosurvival paths, and their interaction. (b) Schematic representation of metabolic transformations of cellular phospholipids (PC, LPC, PA, LPA) by lipases (cPLA2, LPLD/ATX, PLD) and subsequent activation of receptors (GPCR, RTK). (c) (Adapted from Hirabayashi et al. 2004.) Proposed model for cPLA2a activation. Ca2+ binding to the C2 domain promotes cPLA2a translocation from the cytosol to the membrane containing phosphatidylcholine (PC). Phosphorylation on serine residues and/or binding to anionic phospholipids stabilize the association of cPLA2a with the membrane and increase its catalytic activity

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vascular endothelium are phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ ERK) pathways. Radiation-induced activation of these pathways occurs very early, at 1–15 min after exposure (Hallahan et al. 1992; Edwards et al. 2002; Dent et al. 2003b; Maclachlan et al. 2005; Tan et al. 2006; Yazlovitskaya et al. 2008; Linkous et al. 2009).

9.3.1 PI3K/Akt Pathway The PI3K/Akt signal transduction pathway has been implicated in survival signaling in various cell types (Brazil and Hemmings 2001; Vivanco and Sawyers 2002). In vascular endothelial cells, PI3K/Akt can be activated through a family of receptor tyrosine kinases (RTKs) that are activated by growth factors (Burgering and Coffer 1995; Mazure et al. 1997). For example, Akt signaling participates in angiogenesis following VEGF stimulation of endothelial cells and regulates capillarylike tubule formation (Gerber et al. 1998). In addition, Akt can be activated independently of growth factors or PI3K signaling (Bianco et al. 2003). Recently, it has been shown that radiation induces phosphorylation of Akt within endothelial cells (Tan and Hallahan 2003). Low dose of irradiation (2–3 Gy) activates the PI3K/ Akt-mediated cell viability pathway, which also involves inhibitory phosphorylation of the proapoptotic kinase GSK-3b (Tan et al. 2006). Taken together, growing evidence suggests that radiation-induced PI3K/Akt pathway is a vital target for enhancement of the radiation response in tumor vascular endothelium. Inhibition of PI3K/Akt pathway at the various steps of activation prior to IR leads to the potentiation of radiation-induced cell death in vascular endothelial cells. Inhibition of RTKs or inhibition of their ligands was studied (Kim et al. 2004, 2006). Much of the effort has been directed at compounds that specifically inhibit VEGF, such as neutralizing antibody (Gorski et al. 1999), or VEGFR, such as DC101 and SU5416 (Geng et al. 2001). The broad-spectrum RTK inhibitors have the advantage of targeting multiple RTKs that may be present in the tumor, its microenvironment, or both. One such small molecule is PTK787 that targets VEGFR2, PDGF, and c-Met (Wood et al. 2000). Other examples are SU11248 and SU6668. SU11248 is a selective inhibitor of VEGFR2 and PDGFR, as well as Kit and VEGFR1 (Mendel et al. 2003). SU6668 was designed to inhibit kinase activity of FGF, PDGF, and VEGF receptors and have demonstrated significant radiation sensitization effects in preclinical models (Lu et al. 2004). Inhibition of PI3K, Akt, and their down-stream targets was evaluated. Inhibition of p110 subunit of PI3K (Geng et al. 2004) either using specific chemical inhibitors (wortmannin, LY294002, IC486068) or overexpression of the mutant p85 component of PI3K-enhanced radiation-induced apoptosis and minimized capillary tubule formation (Edwards et al. 2002; Tan and Hallahan 2003; Geng et al. 2004; Tan et al. 2006). Chemical inhibition of Akt with ALX-349 as well as overexpression of dominant-negative mutants Akt or its down-stream target GSK-3b also lead to

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radiosensitization of vascular endothelium (Tan et al. 2006). Another down-stream target of PI3K/Akt signaling, mTOR, was shown to be a promising molecule for radiosensitization. Inhibitors of mTOR RAD001 and rapamycin were potent radiosensitizers of vascular endothelial cells in vitro and led to improved tumor-growth delay in vivo (Shinohara et al. 2005; Albert et al. 2006).

9.3.2 MAPK/ERK Pathway Activation of “classical” MAP kinases, ERK1 and 2, by IR is dependent on the cell type, the expression of multiple growth factor receptors, and genetic alterations (Dent et al. 2003b; Lammering et al. 2004). In some cases, Inhibition of MEK1/2, upstream kinases regulating ERK1/2 activity, enhances cell killing by radiation due to increased G2/M arrest and apoptosis (Abbott and Holt 1999); while in some cell types, activation of ERK pathway following irradiation has been shown to promote radiosensitivity by abrogating G2/M checkpoint (McKinstry et al. 2002). Radiationinduced ERK activation was linked to increased expression of radioprotective transcription factors and DNA repair proteins (Amorino et al. 2003; Yacoub et al. 2003). More importantly, ERK activation in endothelial cells plays a radioprotective role resulting in increased survival of irradiated cells (Maclachlan et al. 2005; Yazlovitskaya et al. 2008; Linkous et al. 2009). The cytotoxic effect of radiation can be enhanced by the inhibition of radiation-induced activity of both Akt and ERK (Dent et al. 2003b; Taira et al. 2006).

9.3.3 Phospholipids and Cytosolic Phospholipase A2 (cPLA2) Mechanisms of radiation-induced activation of PI3K/Akt and MAPK/ERK pathways in vascular endothelium involve biologically active lipids and proteins, such as phospholipases, lipid kinases, and phosphatases, which regulate production and activity of these lipids (Cabral 2005; Farooqui and Horrocks 2006). One group of cellular lipids involved in signal transduction comprises the phospholipids. Major phospholipids found in membranes of mammalian cells are phosphatidylcholine (PC) and phosphatidylinositol (Ivanova et al. 2004). Phospholipid metabolism is regulated through G-protein- and tyrosine kinase-mediated enzymes, including phospholipases A, C, and D (PLA, PLC, PLD) as well as cyclooxygenases 1 and 2, lipid kinases and phosphatases (Ivanova et al. 2004). These enzymes convert lipids into bioactive signaling molecules, such as arachidonic acid (AA), phosphatidic acid (PA), diacylglycerol, phosphorylated phosphatidylinositols, lysophosphatidic acid (LPA), and lysophosphatidylcholine (LPC). Phospholipases are enzymes which hydrolyze phospholipids. Phospholipase A2 (PLA2) hydrolyzes phospholipids at the sn-2-acyl ester bond, generating free fatty acids and lysophospholipids (Chakraborti 2003) (Fig. 9.1b). The PLA2 superfamily can be divided into ten groups by gene sequences. Based on

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biological properties, the classification of PLA2s can be simplified into three main types: the cytosolic (cPLA2), the secretory (sPLA2) and the intracellular Ca2+independent (iPLA2) (Chakraborti 2003). The cPLA2, or group IV PLA2, includes at least four paralogs in mammals: PLA2a, -b, -g, and -d (Chakraborti 2003; Hirabayashi et al. 2004). cPLA2a is the most ubiquitously expressed enzyme in the group. The protein contains N-terminal regulatory C2 domain, which is involved in the Ca2+-dependent phospholipid binding, and two catalytic domains A and B (Nalefski et al. 1994) (Fig. 9.1c, adapted from Hirabayashi et al. 2004). Upon membrane binding, conformational changes remove the lid from the active site and allow the fatty acid chain of a substrate molecule (PC) to enter the active site (Fig. 9.1c, adapted from Hirabayashi et al. 2004). Regulatory mechanisms of cPLA2a activity are complex and include subcellular localization, intracellular Ca2+ content, phosphorylation, protein–protein interaction, and cleavage (Chakraborti 2003; Hirabayashi et al. 2004). The specific structural changes and regulatory mechanisms involved in activation of cPLA2 serve as a foundation for the design of chemical inhibitors of cPLA2 (Farooqui et al. 2006). We have recently determined that activation of cPLA2 is an initial event (within 2 min) required for radiation-induced activation of Akt and ERK1/2 in vascular endothelial cells leading to the increased cell viability (Fig. 9.2a) (Yazlovitskaya et al. 2008). We studied cPLA2 inhibitors methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethyl ketone (AACOCF3). AACOCF3, a cellpermeable trifluoromethyl ketone analog of arachidonic acid, is potent cPLA2 inhibitor with IC50 = 1.5 mM (Farooqui et al. 2006). NMR studies have demonstrated

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Fig. 9.2 Schematic representation of interaction of radiation-induced prosurvival responses in vascular endothelial and tumor cells. Radiation-induced signaling pathways involve activation of cPLA2 followed by the increased production of LPC (a). LPC in turn activates RTK or GPR4 (b). LPC can be converted to LPA by LPLD resulting in activation of LPA1-7 receptors (c)

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that the carbon chain of AACOCF3 binds in a hydrophobic pocket of cPLA2 and the carbonyl group of AACOCF3 forms a covalent bond with serine 228 in the enzyme active site (Street et al. 1993; Trimble et al. 1993; Farooqui et al. 2006). AACOCF3 has been used extensively to study the role of cPLA2 in platelet aggregation and inflammation-associated apoptosis (Fabisiak et al. 1998; Calzada et al. 2001; Duan et al. 2001; Kirschnek and Gulbins 2006). MAFP is a potent irreversible inhibitor of cPLA2 with IC50 = 0.5 mM (Farooqui et al. 2006). Since at concentration of 5 mM it also completely inhibits iPLA2, MAFP is considered less selective inhibitor of cPLA2 than AACOCF3. In our study, inhibition of cPLA2 with both inhibitors significantly enhanced radiation-induced cytotoxicity of human umbilical vein endothelial cells (HUVEC) due to mitotic catastrophe and delayed programmed cell death (Yazlovitskaya et al. 2008). This effect was confirmed using HUVEC transfected with shRNA for cPLA2a and primary cell cultures from cPLA2a−/− mice. Endothelial functions were also affected by inhibition of cPLA2 during irradiation resulting in attenuated cell migration and tubule formation (Yazlovitskaya et al. 2008). When combined with radiation, the inhibition of cPLA2 with AACOCF3 disrupts the biological functions of the tumor vasculature, enhances destruction of tumor blood vessels and suppresses tumor growth (Fig. 9.3, reproduced from Linkous et al. 2009). This confirmed that cPLA2 contributes to vascular endothelial cell radioresistance and presents a potential molecular target for tumor sensitization to radiotherapy.

9.3.4 Signaling by the cPLA2 Products LPC and LPA Since the most represented phospholipid in mammalian membranes is PC, the main lysophospholipid produced by activated cPLA2a is lysophosphatidylcholine (LPC) (Figs. 9.1b and 9.2a). Alternatively, LPC could be generated in the reaction mediated by lecithin-cholesterol acyltransferase that transfers fatty acid residue from PC to cholesterol (Prokazova et al. 1998). This biologically active lipid works as the second messenger in the signal transduction pathways, regulating a number of cellular responses (Prokazova et al. 1998; Chakraborti 2003). It is noteworthy that cellular response to exogenously added LPC critically depends on LPC concentration. Doses less than 25 mM usually trigger cell survival and proliferation (Prokazova et al. 1998; Fujita et al. 2006; Gwak et al. 2006). LPC signaling could be mediated by receptor activation or be receptor independent. LPC may bind to the plasma membrane in receptor-independent manner or directly enter the lipid bilayer (Prokazova et al. 1998). Receptor-dependent LPC signaling could involve LPC-specific G-protein-coupled receptors (GPCRs), G2A and GPR4 (Figs. 9.1b and 9.2b) (Xu 2002). LPC stimulates proliferation in HUVEC by transactivating the VEGFR 2 and activating Akt and ERK1/2 (Figs. 9.1b and 9.2b) (Fujita et al. 2006). Increased levels of LPC has been linked directly to cytokine and chemokine production in endothelial cells by activating MAPK and PI3K/Akt pathways, thus regulating the chemotaxis of leukocyte subpopulations

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d

C

LLC 1600

Tumor volume (mm3)

Tumor volume (mm3)

c

Fig. 9.3 Inhibition of cPLA2 with AACOCF3 attenuates tumor vascularity and decreases tumor size in irradiated mouse models. (Reproduced from Linkous et al. 2009.) Using heterotopic tumor models of Lewis lung carcinoma (LLC) (a–c) or H460 large cell carcinoma of the lung (d), mice were treated intraperitoneally with vehicle DMSO (control) or 10 mg/kg AACOCF3 and tumors were irradiated 30 min later with 3 Gy. (a, b) Treatment was repeated for 5 consecutive days. Twenty-four hours after the final treatment, tumor blood flow was analyzed by three-dimensional Power Doppler sonography. Shown are representative images of tumor blood flow (a). Shown is a bar graph of the average percent vascular index with SEM from group of three to five animals; *p < 0.05 (b). (c, d) Tumor volumes were calculated using external caliper measurements. Shown is a bar graph of the mean LLC tumor volumes in each of the treatment groups of four to six mice (c). Shown is a bar graph of the average H460 tumor volumes 13 days postinjection with SEM from groups of four to six mice; *p < 0.05 (d)

during inflammation (Murugesan et al. 2003). LPC displays biphasic regulation of inflammatory factors in endothelial cells, causing activation of NF-kB at low concentrations and inhibition at higher concentrations (Sugiyama et al. 1998). In our recent studies, we have demonstrated that IR-induced activation of cPLA2

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in vascular endothelial cells led to an increased production of LPC within 3 min after IR, followed by activation of VEGFR2, Akt, and ERK1/2 (Yazlovitskaya et al. 2008). Inhibition of cPLA 2 prevented this increase and following prosurvival signaling transduced by Akt and ERK. This observation confirms that cPLA2-dependent radioresistance of vascular endothelial cell is mediated by LPC and suggests that LPC is a vital target for radiosensitization of tumor vasculature. Another bioactive phospholipid with known angiogenic properties is lysophosphatidic acid (LPA) (Folkman 2001; Kishi et al. 2006; Ptaszynska et al. 2008). Two major pathways of LPA production depend upon cPLA2 activity (a) lysophospholipids generated by cPLA2 (such as LPC) are subsequently converted to LPA by lysophospholipase D/autotaxin (LPLD/ATX); (b) phosphatidic acid (PA) generated by phospholipase D (PLD) or diacylglycerol kinase is subsequently converted to LPA by cPLA2 (Figs. 9.1b and 9.2b, c) (Aoki 2004; Aoki et al. 2008). It has been demonstrated that specific GPCRs mediate the cellular effects of LPA (Figs. 9.1b and 9.2b, c) (Aoki et al. 2008). Three of seven identified LPA-specific receptors, Edg-2/LPA1 (Hecht et al. 1996), Edg-4/LPA2 (An et al. 1998), and Edg-7/LPA3 (Bandoh et al. 1999), belong to the EDG (endothelial cell differentiation gene) family. The P2Y subgroup comprises of four other LPA-specific receptors, GPR23/P2Y9/LPA4, GPR92/LPA5, GPR87, and P2Y5 (Aoki et al. 2008). LPA is an intercellular lipid mediator with multiple actions, particularly known as an inducer of cell proliferation, migration, and survival (Lange et al. 2008). The physiologic response to LPA signaling depends on cellular context and initiates biological processes such as neurogenesis and tumor progression (Lange et al. 2008). Numerous studies have shown that LPA participates in cancer progression (Mills and Moolenaar 2003). Aberrant expression of LPA receptors is detected in various human malignancies, including ovarian, colorectal, prostate, lung and gastric cancers, and malignant gliomas (Kishi et al. 2006). Studies using mouse models demonstrate that inducible overexpression of the LPA receptor in MDA-MB-231 breast carcinoma cells enhanced the growth of subcutaneous xenografts and promoted osteolytic bone metastasis (Boucharaba et al. 2004; Boucharaba et al. 2006), whereas knockdown or pharmacologic inhibition of LPA receptor in these carcinoma cells significantly reduced the progression of metastases. Moreover, LPA promotes the proliferation and migration of vascular endothelial cells in culture. In brain microvascular endothelial cells, LPA enhances monolayer permeability (Schulze et al. 1997). Mice deficient in one of the LPA-producing enzymes, LPLD/ ATX, suffer from severe vascular defects (Tanaka et al. 2006). Expression analysis reveals that many different cell types produce LPLD/ATX with the malignant glioma, lung cancer, and melanoma cells showing the highest levels in conditioning medium (Moolenaar et al. 2004; Kishi et al. 2006). The major physiological substrate for LPLD/ATX is LPC (Croset et al. 2000). We have shown that LPC produced abundantly and immediately after irradiation with 2–3 Gy (Yazlovitskaya et al. 2008) and, therefore, could be converted to LPA by LPLD/ATX. This suggests that LPA and LPLD/ATX are vital targets for radiosensitization of tumor vasculature and tumor itself.

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9.4 Summary Figure 9.2 illustrates IR-induced cPLA2-regulated prosurvival responses in vascular endothelial and tumor cells. Clinically relevant doses of radiation trigger prosurvival signals in tumor vascular endothelium thus affecting the efficacy of radiation therapy. Specifically, 3 Gy induces activation of cPLA2 within 3 min of irradiation of vascular endothelial cells (Yazlovitskaya et al. 2008; Linkous et al. 2009). We found that the second messenger LPC is the main product of radiation-induced cPLA2 enzymatic cleavage of PC (Fig. 9.2a). LPC can move through cellular membrane and trigger activation of prosurvival signal transduction pathways in both vascular endothelial and tumor cells (Fig. 9.2b, c). LPC can directly bind to and activate RTKs such as Flk-1 (Yazlovitskaya et al. 2008) or GPCRs such as LPC-specific GPCR, GPR4 (Fig. 9.2b). Alternatively, LPC could be further metabolized to LPA, another biologically active lysophospholipid, by LPLD/ATX (Fig. 9.2b). LPA, in turn, activates LPA-specific GPCRs, LPA1 through 7 (Fig. 9.2c). The receptors activated by LPC (Flk-1, GPR4) or LPA (LPA1-7) are expressed both in vascular endothelial and in tumor cells. Moreover, expression of LPA receptors was found to determine tumorigenicity and aggressiveness of cancer cells. Activation of these receptors initiates prosurvival signal transduction pathways such as MAPK/ERK and PI3K/Akt (Fig. 9.2b, c). In summary, along with activation of DNA damage response, therapeutic doses of ionizing radiation trigger cPLA2-dependent prosurvival cell signaling both in tumor and in tumor vasculature (Fig. 9.2). Most importantly, at various steps, this signaling presents molecular targets for sensitization of tumor to radiation therapy. As discussed in this chapter, these targets include RTK inhibitors; inhibitors of PI3K/Akt and their down-stream targets, such as mTOR; MAPK/ERK inhibitors. The most attractive is the strategy targeting cPLA2 and lysophospholipid products of its activation, LPC and LPA, since these molecules have been identified as the initial signaling molecules in irradiated vascular endothelium.

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Chapter 10

EGFR Signaling and Radiation Emily F. Dunn, Shyhmin Huang, and Paul M. Harari

Abstract The epidermal growth factor receptor (EGFR) is an ubiquitously expressed receptor tyrosine kinase that is over expressed or mutated in a broad spectrum of epithelial cancers and serves as an important regulator of oncogenesis. Aberrant EGFR expression and activity have been associated with uncontrolled cancer cell proliferation and survival. Therefore, EGFR has been considered an attractive target in cancer therapy, including studies investigating the ability of EGFR signaling to modulate radiation response. Two primary classes of EGFR inhibitors have been developed in recent years: monoclonal antibodies (mAbs) and tyrosine kinase inhibitors. The most mature clinical data to date derives from studies with the anti-EGFR mAb inhibitor cetuximab combined with radiation in head and neck squamous cell carcinoma (HNSCC) that has provided a new therapeutic option for this disease. Postulated mechanisms of interaction between these two modalities include effects on cell cycle distribution, apoptosis, tumor cell repopulation, DNA damage/repair, and impact on tumor vasculature. The improvement in tumor control rates and overall survival in HNSCC has prompted investigation of this approach in other tumor types. Here we review pertinent literature regarding the interaction of anti-EGFR therapies with radiation, including preclinical and clinical trial data reflecting promising future directions for this approach. Keywords Radiation • EGFR • Head and neck cancer • Resistance • Radiotherapy Disclosure: PMH has held active laboratory research agreements with industry sponsors developing EGFR inhibitors including Amgen, AstraZeneca, Genentech, and ImClone within the last 5 years.

E.F. Dunn (*) Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, K4/332, 600 Highland Avenue, Madison, WI 53792, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_10, © Springer Science+Business Media, LLC 2011

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10.1 EGFR Biology The epidermal growth factor receptor (EGFR) is an ubiquitously expressed receptor tyrosine kinase (RTK) that is important in oncogenesis. The EGFR is a member of the HER family of receptors that consists of four members: EGFR (ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Ligand binding induces receptor activation leading to homodimerization with EGFR or heterodimerization with other HER family members. This activates the RTK and thereby induces autophosphorylation of tyrosine residues in the cytoplasmic tail. These phosphorylated tyrosines serve as docking sites for various proteins that contain Src Homology domains (SH2) and phosphotyrosine binding domains (PB). These events lead to the activation of several signaling cascades most notably the Ras/RAF/MEK/ERK, phosphotidylinositol-3-kinase (PI3K)-Akt, Jak-Stat, and phospholipase C gamma pathways. The activation of these pathways ultimately promotes tumor cell proliferation, survival, invasion, and angiogenesis. Overexpression or increased activity of the EGFR is found in many human cancers including head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and brain cancer. Recent studies have suggested tumor EGFR expression as a predictor of outcome following treatment with radiation (Sheridan et al. 1997; Mendelsohn and Baselga 2003; Ang et al. 2002, 2004; Harari and Huang 2004a). Overexpression of the EGFR is also linked with poor survival and increased locoregional failure in HNSCC (Dassonville et al. 1993; Grandis et al. 1998; Ang et al. 2002). The relationship between high EGFR level and poor clinical outcome following radiation has also been observed in patients with other cancers including gliomas, cervical, and rectal cancers (Chakravarti et al. 2004; Giralt et al. 2005). Targeting the EGFR with molecular inhibitors has been intensely pursued during the last decade as a cancer treatment strategy. Two primary approaches have been developed to target the EGFR, including anti-EGFR monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs) (see Sect. 10.3). In the following sections, we explore the EGFR inhibitors, the combination of EGFR inhibitors with radiation (both preclinical and clinical data), and consider future directions for the use of EGFR inhibitors with radiation.

10.2 EGFR Signaling and Effect on Radiation Response Radiotherapy is an effective treatment modality delivered to approximately one-half of all cancer patients during the course of their disease. Radiation may be given alone or in combination with surgery and/or chemotherapy. Several intensive chemoradiation treatment approaches have produced important advances in cancer patient survival; however, it appears that further advances may be limited by the overall toxicity of treatment. Normal tissue toxicity derives primarily from the lack of specificity of radiation or conventional chemotherapy for cancer cells as opposed to surrounding normal cells. The stepwise advancement of molecular targeted therapies that specifically target

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a berrant growth properties of cancer cells offers the potential to improve outcome with decreased toxicity (Baumann et al. 2004). Aberrant EGFR expression and activity have been associated with uncontrolled cancer cell proliferation and survival. Therefore, EGFR has emerged as an attractive target in cancer therapy, including a role to enhance the efficacy of radiotherapy (Harari 2004; Thariat et al. 2007).

10.2.1 EGFR and Tumor Cell Repopulation Following Radiation Several key findings underlie the rationale to explore the combination of EGFR targeting with radiotherapy. Early studies by Milas and colleagues identified that high levels of EGFR expression correlated with radioresistance of tumor cells (Akimoto et al. 1999; Milas et al. 2004). The EGFR inhibitors show antiproliferative impact on many tumor cell populations and are known to induce cell cycle delay, most notably in G1 phase. Blockade of EGFR signaling can be achieved using at least four types of inhibitors: anti-EGFR mAbs, TKIs, dominant negative EGFR-CD533, and antisense oligonucleotides. Schmidt-Ullrich and colleagues found that all four of these approaches reduced radioresistance of EGFR-overexpressing tumors in vitro and in vivo (Schmidt-Ullrich et al. 2003; Lammering et al. 2004). Extrapolating to the clinical setting, evidence suggests that poor survival of patients with EGFRoverexpressing HNSCC tumors is largely due to a failure of locoregional tumor control as opposed to distant metastasis (Ang et al. 2002). These observations suggest that EGFR may play an important role in tumor cell repopulation, which is a significant contributor to treatment failure in patients treated with radiotherapy. Substantial experimental and clinical data demonstrates that increased overall treatment time (with constant total dose) reduces the probability of locoregional tumor control, particularly for rapidly proliferating tumors such as HNSCC (Baumann et al. 2003; Harari 2005). This appears to result from surviving tumor cells, which are able to maintain or increase their proliferation and repopulation potential during the fractionated course of radiotherapy. Interestingly, EGFR expression during fractionated radiation is found to correlate with repopulation kinetics in human FaDu tumor xenografts (Petersen et al. 2003). In addition, various studies have shown that low dose radiation can activate EGFR signaling which results in the accelerated proliferation or repopulation of cancer cells in a variety of tumor model systems (Kavanagh et al. 1995; Schmidt-Ullrich et al. 1997; Dent et al. 1999). Taken together, these results imply that inhibition of EGFR signaling may serve as a valuable strategy to enhance the efficacy of radiotherapy.

10.2.2 EGFR and DNA Damage Repair Additional interest in combining EGFR inhibitors with radiation is prompted by considering the involvement of EGFR systems in DNA damage repair (Meyn et al. 2009). Ionizing radiation is believed to kill tumor cells primarily by inducing DNA double strand breaks (DSBs). Cell survival following radiation is largely a function

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of successful repair of damaged DNA. There are several ways in which EGFR may affect the DNA repair process. These include the ability of EGFR itself or downstream effectors to modulate the intracellular translocation, transcription, and phosphorylation of key proteins/genes involved in the DNA repair process. Data regarding a potential interaction between EGFR and DNA repair includes the identification of a physical interaction between EGFR itself and DNA-dependent kinase (DNA-PK), a critical molecule in the repair of DNA DSBs. Dittmann et al. reported that ionizing radiation triggers a translocation of EGFR into the nucleus where it forms a physical interaction with the DNA-PK-Ku70/Ku80 complex, which subsequently carries out DNA damage repair (Dittmann et al. 2005a, b). In addition, Toulany et al. showed that inhibition of either EGFR or Akt could suppress radiation-induced phosphory lation of DNA-PK at Tyr-2609, which is an important site for functional DSB repair (Toulany et al. 2006). Recent studies further demonstrate that radiation-induced Src activation and caveolin-1 associated EGFR internalization are linked with nuclear EGFR transport and activation of DNA-PK (Dittmann et al. 2008, 2009). Interestingly, Das et al. reported that lung cancer cells bearing activating mutations in the tyrosine kinase domain of EGFR are more radiosensitive than cells with wild type EGFR. These mutant EGFRs fail to translocate to the nucleus and bind to DNA-PK following radiation (Das et al. 2006, 2007). These results suggest the potential importance of the EGFR–DNA-PK interaction in regulating the radiosensitivity of tumor cells. Beyond EGFR itself, various studies demonstrate that EGFR downstream effectors, such as Akt and MAPK may govern radiosensitivity by affecting certain DNA repair genes/proteins. For example, Toulany et al. discovered a physical interaction between Akt and DNA-PK. Following radiation and Akt knockdown, a strong inhibition to radiation-induced phosphorylation of histone 2AX (g-H2AX) and DNA-PK resulted (Toulany et al. 2008a). Yacoub et al. demonstrated that ionizing radiation could induce upregulation of DNA repair genes XRCC1 and ERCC1 in prostate carcinoma through activating MAPK signaling (Yacoub et al. 2003; Valerie et al. 2007). Since XRCC1 is another crucial molecule involved in the DNA-PK complex, treatment with MAPK inhibitors is found to suppress XRCC1 gene expression and results in enhanced radiosensitivity. A recent study further identified that Akt regulates basal, but not MAPK-regulated radiation-induced XRCC1 expression (Toulany et al. 2008b). Other than XRCC1, Ko et al. showed that treatment of NSCLC cells with either a MAPK inhibitor or an EGFR inhibitor results in decreased expression of another key repair protein, Rad51 (Ko et al. 2008). Taken together, these results suggest that EGFR itself and/or its downstream signaling effectors can regulate DNA damage repair (Fig. 10.1). These findings highlight the potential value of targeting EGFR to improve tumor treatment outcome with radiation.

10.2.3 EGFR Inhibitors Investigators in the EGFR cancer therapeutics field have undertaken several innovative approaches to develop antitumor agents. Following the early notable work by

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a Translocation

EGFR

XRT

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BRCA1

BRCA1

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HRR

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EGFR

b Transcription

Ras PI3K

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c Phosphorylation

Ras PI3K

XRT Raf MEK

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P AKT H2AX

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P

DNA Repair

Fig. 10.1 Schematic illustration highlighting EGFR’s impact on DNA repair. EGFR may influence: (a) translocation of DNA repair proteins to the nucleus. (b) Transcription of DNA repair genes. (c) Phosphorylation and activity of key DNA repair proteins

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Mendelsohn (2003) who demonstrated the antitumor effect of a mAb to EGFR, a broad series of EGFR inhibitors have been designed and explored during the last two decades. Among them, two distinct classes of EGFR inhibitors have demonstrated preclinical and clinical promise and gained U.S. Federal Drug Administration (FDA) approval in recent years. These include mAbs directed against extracellular ligand-binding domain of the EGFR and small molecule TKIs directed against the cytosolic catalytic domain of the EGFR (Harari and Huang 2004b; Harari et al. 2007; Dassonville et al. 2007). Although both can block EGFR activation, the pharmacokinetic profiles of mAbs and TKIs vary considerably. mAbs are delivered intravenously for two main reasons: they are large molecules that are susceptible to degradation in GI tract, and have a long half-life. On the contrary, small molecule TKIs are administered orally (daily most common) due to their short half-life and effective absorption across the GI tract.

10.2.4 Anti-EGFR mAbs A series of anti-EGFR mAbs have been developed for use in the treatment of a variety of cancers as shown in Table 10.1. Among them, cetuximab and panitumumab are the most well studied anti-EGFR mAbs with regard to mature clinical data. Cetuximab (Erbitux®: ImClone Systems/Bristol–Meyers Squibb, New York, NY), formerly known as C225, is a human–mouse chimeric mAb that received FDA approval in 2004 for the treatment of irinotecan-refractory colorectal cancer. Cetuximab is the most comprehensively studied anti-EGFR mAb and is the first EGFR targeting agent approved to combine with radiotherapy in the treatment of HNSCC patients based on promising results from preclinical studies and early clinical trials (Harari and Huang 2006; Baumann et al. 2007; Nyati et al. 2006). Early studies demonstrated that cetuximab can bind to the EGFR with a 2-log greater affinity than that of EGFR ligands and thereby block ligand-induced EGFR activation. Following binding to the EGFR, cetuximab prompts receptor internalization and ultimately results in receptor degradation (Hubbard 2005; Jaramillo et al. 2006). Preclinical studies demonstrated that cetuximab can inhibit the proliferation of a range of human tumor cells and xenografts. This inhibition of proliferation reflects cell-cycle arrest in G1 phase and/or an increase in apoptosis (Prewett et al. 1996; Huang et al. 1999). In addition, cetuximab is found to inhibit the expression of several angiogenic factors in cancer cells, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interleukin-8 (IL-8) (Perrotte et al. 1999; Karashima et al. 2002). This antiangiogenic effect of cetuximab provides a explanation for the finding that the growth inhibitory impact of cetuximab in tumor xenograft models (in vivo) is generally much more pronounced than that observed in cell culture (in vitro) (Huang et al. 2002b). Panitumumab (Vectibix®; Amgen, Thousand Oaks, CA), formerly known as ABX-EGF, is a fully humanized anti-EGFR mAb that gained FDA approval in the treatment of mCRC in 2006 (Van Cutsem et al. 2007). Owing to its fully human

The Institute of Cancer Research, UK

MerckMerdarex

YM BioScience

EMD Pharma

Mouse–human Chimeric Ab Rat Ab EGFR & EGFR vIII

EGFR & CD64

EGFR

EGFR

EGFR

Human Ab

Mouse–human Chimeric Ab Human Ab

Specificity EGFR

Molecule Mouse–human Chimeric Ab

Not known

Not known

62–304 h

4 days

7.5 days

Half life 114 h

Phase I for NSCLC

Phase I for refractory glioma

Clinical uses FDA approved: • mCRC • HNSCC FDA approved: • mCRC Phase I/II clinical trials in NSCLC, CRC, renal and esophageal cancer Phase II trials in NSCLC, colorectal, pancreatic and stomach cancer Currently in late stage development and testing in US for FDA review

Ab antibody, mCRC metastatic colorectal cancer, EGFR epidermal growth factor receptor, FDA Food and Drug Administration, HNSCC head and neck squamous cell carcinoma, NSCLC non-small cell lung cancer

ICR62

Matuzumab EMD72000 Nimotuzumab h-R3 TheraCIM MDX-447

Table 10.1 Anti-EGFR antibodies Name Manufacturer ImClone Systems Inc. Cetuximab Bristol–Myers Squibb Erbitux® C225 Panitumumab AmgenAbgenix Vectibix® ABX-EGF

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sequence, a potential advantage of panitumumab is the low risk of immunogenicity and allergic reactions in cancer patients. However, it has been reported that the chimeric mAb cetuximab may provoke valuable immunologic effects, such as antibody-dependent cellular cytotoxicity (ADCC), which may be important for some of the antitumor effects of cetuximab (Kurai et al. 2007). Ultimately, the comparative therapeutic efficacies of these two distinct anti-EGFR mAbs will be best evaluated in the context of controlled clinical trials.

10.2.5 Anti-EGFR TKIs A second approach to disrupt EGFR function involves the use of synthetic quinazoline-derived TKIs that bind to the ATP-binding pocket of the EGFR tyrosine kinase domain and subsequently inhibit receptor phosphorylation (Table 10.2). Gefitinib (Iressa®; AstraZeneca, London, UK), formerly known as ZD1839, was the first EGFR TKI to gain FDA approval for use in the treatment of chemotherapy-refractory NSCLC. Gefitinib is an effective inhibitor of EGFR kinase activity with an IC-50 in the nanomolar range in vitro. Similar to cetuximab, studies indicated that gefitinib inhibits the growth of a range of human cancer cells in vitro and in vivo via cell cycle arrest and/or apoptosis (Sirotnak 2003; Campiglio et al. 2004). In addition, gefitinib also exhibits antiangiogenic effects via inhibition of VEGF, bFGF, and IL-8 expression (Hirata et al. 2002; Iivanainen et al. 2009). Erlotinib (Tarceva®; OSI/Genentech, San Francisco, CA), formerly known as OSI-774, is the second EGFR TKI approved by FDA for the treatment of chemotherapy-refractory NSCLC patients and advanced pancreatic cancer patients who have not received previous chemotherapy. Like gefitinib, erlotinib is a potent inhibitor of EGFR phosphorylation and has activity against human colorectal, HNSCC, NSCLC, and pancreatic tumor cells in vitro (Akita and Sliwkowski 2003). The combination of an anti-EGFR mAb and a TKI has been explored as a potential cancer treatment strategy to maximize effective EGFR inhibition. Although these two classes of agents have the same ultimate target, namely the EGFR, they do differ in their subcellular site of action and carry distinct toxicity profiles (Dassonville et al. 2007). Anti-EGFR mAbs, but not TKIs, can elicit ADCC and have the capacity to form receptor-containing complexes that result in receptor internalization, a crucial step for attenuating receptor signaling. On the other hand, TKIs show activity against other receptors such as ErbB2/Her2 at high concentrations. By microarray analysis, Baselga and colleagues identified 45 genes that are differentially expressed after treatment with cetuximab or gefitinib, including genes related to cellular proliferation and differentiation, DNA synthesis and repair, angiogenesis, and metastasis (Matar et al. 2004). Although the clinical significance of the differences in activity between anti-EGFR mAbs and EGFR TKIs are not yet clear, preclinical studies have demonstrated a synergistic effect in several xenograft model systems when cetuximab is administrated in combination with gefitinib or erlotinib compared with single agent treatment

Phase II for patients with gefitinibresistant NSCLC Phase III for various neoplasms

36 h

12 h 5 h

20–23 h

EGFR

EGFR EGFR ErbB2 ErbB4 EGFR ErbB2 EGFR ErbB2 24 h

Clinical uses FDA approved for patients who are benefiting or have benefited from gefitiniba FDA approved for: • NSCLC • Metastatic pancreatic cancer Phase I trials for advanced solid malignancies are underway Phase II completed

Half life 48 h

Specificity EGFR

a

EGFR epidermal growth factor receptor, FDA Federal Drug Administration, NSCLC non-small cell lung cancer New patients will be allowed access to gefitinib only if being enrolled into a qualifying clinical trial approved prior to June, 2005

Table 10.2 Anti-EGFR tyrosine kinase inhibitors Name Manufacturer Molecule AstraZeneca Anilinoquinazoline reversible Gefitinib Iressa® ZD1839 Genentech Inc. Anilinoquinazoline reversible Erlotinib Tarceva® OSI-774 PKI166 Novartis Pharmaceuticals Pyrrolopyrimidine CGP 75166 Pfizer Anilinoquinazoline irreversible Canertinib CI-1033 PD-183805 Pelitinib Wyeth Research 3-Cyanoquinoline irreversible EKB-569 Lapatinib GlaxoSmithKline Thiazolylquinazoline reversible Tykerb® GW572016

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(Huang et al. 2004; Jimeno et al. 2005; Matar et al. 2004). These two agents given together are able to exert a superior induction in apoptosis and blockade of EGFR activation and downstream signaling. Interestingly, we and other investigators further find that dual targeting of EGFR can overcome acquired resistance to EGFR mAb inhibitors in cell culture and animal models (Huang et al. 2004; Regales et al. 2009). A recent report from a phase I clinical trial confirms that treatment with cetuximab and gefitinib is feasible in patients with recurrent NSCLC and warrants further clinical investigation (Ramalingam et al. 2008).

10.3 Combination of EGFR Targeting Agents with Radiation A series of preclinical studies have provided proof-of-principle that blockade of EGFR can enhance radiation toxicity in a variety of tumor model systems. Early results showing modulation of radiation response by EGFR inhibitors are predominately achieved with cetuximab. Several studies identified the capacity of cetuximab to enhance the effect of single-dose and fractionated radiations (Huang et al. 1999; Milas et al. 2000). Improved local tumor control, a more clinically relevant endpoint after single-dose irradiation, is also reported in a xenograft model (Nasu et al. 2001). Thereafter, other anti-EGFR mAbs and TKIs have been shown to enhance radiation response in a variety of tumor models (Huang et al. 2002a, b; Bianco et al. 2002; Chinnaiyan et al. 2005; Kruser et al. 2008). The potential mechanisms of action of EGFR inhibitors include specific effects on proliferation, cell cycle progression, apoptosis, DNA damage repair, angiogenesis, and metastatic capacity (Fig. 10.2). EGFR inhibitors may improve radiation response by blocking radiation-induced proliferation and accelerated repopulation. Antiproliferative effects of anti-EGFR mAbs and TKIs have been shown consistently in various preclinical studies (Baumann et al. 2007). A study further demonstrated that simultaneous application of cetuximab during radiation with 30 fractions over 6 weeks in FaDu cancer cells improved local tumor control compared to radiation alone (Krause et al. 2005). This effect in part is caused by reduced tumor cell repopulation by cetuximab. Perturbations in cell cycle progression have also been considered an underlying mechanism for the antiproliferation and radiosensitization effects of EGFR inhibitors since cells exhibit different degrees of radiation sensitivity in different phases of the cell cycle (Ahsan et al. 2009). Both anti-EGFR mAbs and TKIs have been shown to alter cell cycle distribution, with a 10–20% shift from S phase to G0/G1, which would be expected to increase radiation sensitivity (Harari and Huang 2001; Huang et al. 2002a, b; Chinnaiyan et al. 2005). Further studies indicated that EGFR inhibitors cause G1 arrest through upregulation of the cyclin dependent kinase inhibitors p27 and p21 (Busse et al. 2000; Di Gennaro et al. 2003). Therefore, the radiosensitization effect of EGFR inhibitors may result from a decrease of the percentage of cells in S-phase, a relatively radioresistant phase, with concomitant increase in the more radiosensitive G1 phase of the cell cycle. Furthermore, the

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EGF

EGF

mAb EGFR TKI

Cell Membrane

Translocation

Signaling Cascades

Cell Cycle Progression Cytoplasm

G1

DNA Repai r

S

Radiation

DNA Damage Nucleus

M

Growth Arrest Apoptosis

ã G2

Metastasis

Angiogenesis Growth & Repopulation

Invasion Migration Proliferation Differentiation

Endothelial Cell Proliferation Tube formation Vessel recruitment

Fig. 10.2 Mechanisms of action for combined anti-EGFR therapy with radiation. Simplified schematic illustration of the EGFR pathway and potential downstream effects of EGFR signaling inhibition combined with radiation

combined effects of G1 arrest induced by EGFR inhibitors, together with G2/M arrest induced by ionizing radiation, ultimately results in cell cycle checkpoint deregulation and subsequent apoptosis. Induction of apoptosis has been widely observed in a variety of tumors treated with EGFR inhibitors and radiation (Huang et al. 1999; Chinnaiyan et al. 2005). Therefore, promotion of apoptosis has also been proposed as a potential mechanism for EGFR inhibitor-mediated radiosensitization since apoptosis plays a crucial role in regulating response to radiation. Another mechanism of synergy between EGFR inhibition and radiation is through the inhibition of DNA damage repair (Szumiel 2006). In mammalian cells, repair of DNA DSBs is considered to be the critical step for cells to survive following radiation injury. DSBs are mainly repaired in the nucleus via nonhomologous end-joining (NHEJ), which reanneals the separated DNA using several key proteins including Ku70/80 and DNA-PK. There is increasing evidence that EGFR can translocate into the nucleus to act either as a transcription factor or as a cofactor for the DNA-PK–Ku78/80 repair complex (Szumiel 2006). However, when EGFR is blocked by cetuximab, a substantial amount of DNA-PK is retained in the cytosol due to a stalled nuclear import of the EGFR complex (Bandyopadhyay et al. 1998; Dittmann et al. 2005b). As a consequence, radiation-induced DNA-PK activation is abolished and leads to a depressed repair as illustrated by increased numbers of

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repairing foci containing g-H2AX (Dittmann et al. 2005b). Interestingly, the inhibition on DNA-PK translocation does not appear to be restricted to anti-EGFR mAbs. Treatment with gefitinib also resulted in the inhibition of DNA damage repair and a reduction of the level and activity of DNA-PK in the nucleus (Shintani et al. 2003; Friedmann et al. 2006). However, a recent report indicated that gefitinib at low doses in NSCLC cells suppresses DNA repair capacity via another DNA repair protein NBS1, not DNA-PK (Tanaka et al. 2008). The relationship between EGFR signaling and DNA-repair process has not been fully clarified, but these observations raise the possibility that impairment of DNA repair processes, especially for DSB repair, may be involved in the modulation of radiosensitivity by EGFR inhibitors. Finally, it is becoming clear that EGFR inhibitors can interfere with tumor– stromal interactions, such as angiogenesis, and this antiangiogenic effect of EGFR inhibitors has been considered as a potential underlying mechanism for EGFR inhibitor-mediated radiosensitization (Baumann et al. 2007; Guillamo et al. 2009). Cetuximab or gefitinib have been reported to suppress neovascularization in a variety of tumor xenografts (Huang and Harari 2000; Huang et al. 2002; She et al. 2003). The antiangiogenic mechanism of EGFR inhibitors involves not only suppression of several angiogenic factors that are produced by tumor cells but also direct inhibition of the proliferation and migration of endothelial cells, endothelial associated pericytes, and perivascular cells (Iivanainen et al. 2009; Al-Nedawi et al. 2009). Therefore, radiosensitization effects of EGFR inhibitors have been suggested to be due to improved tumor oxygenation, decreased vessel density, and radiosensitization of endothelial cells (Baumann et al. 2007). Among them, the effects of EGFR inhibitors on tumor oxygenation have been widely discussed since this represents a paradoxical issue for most antiangiogenic therapies. Theoretically, one would expect that impairment of tumor vasculature would hinder oxygen delivery, and the resultant hypoxia could increase radioresistance of tumor cells. Such a negative effect of antiangiogenic agents on the combined treatment with radiation has been observed in some studies (Murata et al. 1997; Fenton et al. 2004). However, emerging preclinical and clinical evidence supports an alternative hypothesis suggesting that antiangiogenic agents may serve to transiently “normalize” the abnormal structure and function of tumor vasculature to make it more efficient for oxygen and drug delivery (Jain 2005; Ma and Waxman 2007, 2008). A “vascular normalization index” has been proposed and shown to be a potential biomarker to predict survival after antiangiogenesis treatment in glioma patients (Sorensen et al. 2009). In addition, by determining the optimal scheduling and dose of antiangiogenic agents, the combination of radiation with antiangiogenic agents can improve therapeutic outcomes via vessel normalization (Dings et al. 2007). Therefore, detailed investigations regarding the temporal changes of the tumor microenvironment appear important for our understanding of EGFR/ radiation interactions. In several studies, simultaneous application of cetuximab during fractionated irradiation is shown to improve reoxygenation of tumor xenografts, which contributes to improved local control of FaDu tumors (Petersen et al. 2003;

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Krause et al. 2005). Further studies indicate that treatments with EGFR inhibitors results in a reduced expression of the hypoxia related protein, hypoxia-inducible factor alpha (HIF-1 alpha) (Pore et al. 2006; Riesterer et al. 2009). Importantly, several studies recently confirm that EGFR inhibitors can modulate the microenvironment by vascular normalization to improve radiotherapy efficacy (Gan et al. 2009; Cerniglia et al. 2009; Qayum et al. 2009). Maity and colleagues demonstrate that treatment with erlotinib inhibits tumor-expressed HIF-1 alpha and VEGF in vitro and in vivo (Cerniglia et al. 2009). Imaging studies further identify changes in vessel morphology in erlotinib-treated tumor xenografts accompanied by increased tumor blood flow, decreased vessel permeability, and reduced hypoxia that are typical characteristics of tumor vascular normalization. Those changes correlate with improved drug delivery and increased response to radiation (Cerniglia et al. 2009). These findings suggest that improvement in blood flow leads to better drug delivery or increased tumor oxygenation and offers a rationale for why EGFR inhibitors can enhance the radiation response of tumors. They also highlight the importance of careful selection of dose and administration schedules for EGFR inhibitors when combined with radiotherapy.

10.4 Clinical Trials Combining EGFR Inhibitors and Radiation Several trials have been completed that combine EGFR inhibitors with radiation in HNSCC, NSCLC, esophageal cancer, and glioblastoma multiforme (GBM). Clinical data in head and neck cancer patients confirms an inverse correlation between EGFR expression level and tumor control following a course of fractioned radiation treatment (Ang et al. 2002; Bentzen et al. 2005). To date, one phase III clinical trial has been reported with long-term followup that assessed the use of the anti-EGFR mAb cetuximab with radiation in HNSCC. Bonner et al. studied cetuximab in locoregionally advanced HNSCC, and compared the efficacy of radiotherapy alone to radiotherapy plus weekly cetuximab (Bonner et al. 2006, 2010). The results of this trial confirmed an increase in locoregional control, progressionfree survival, and overall survival with the addition of cetuximab and led to the FDA approval of cetuximab for use in HNSCC in combination with radiation in 2006. The 5-year update report confirms stability of the advantage for the cetuximab arm with a 10% improvement in overall survival compared to the radiation alone arm (Bonner et al. 2010) (Fig. 10.3). A second phase III trial, RTOG 0522, has recently completed enrollment in March 2009. This study is evaluating the addition of cetuximab to a standard-of-care concurrent radiation/cisplatin regimen in over 900 patients. Patients were randomized to one of two treatment arms: (1) accelerated fractionation plus cisplatin versus (2) accelerated fractionation plus cisplatin and cetuximab. Followup data is currently maturing for this study. This study will help define the potential benefit of adding cetuximab to concurrent chemoradiation in locoregionally advanced HNSCC.

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1·0

Overall survival

0·8

Stratified log-rank p=0·018

0·6 0·4 0·2 0 0

Number at risk Radiotherapy+ cetuximab Radiotherapy alone

10

20

30

40

50

60

70

Months 211

177

136

117

105

90

49

··

213

162

122

98

85

77

49

··

Adapted with permission by Bonner et al.(97)

Fig. 10.3 Survival advantage of cetuximab combined with radiation in HNSCC. Overall survival advantage of 10% with addition of weekly cetuximab to radiation for locoregionally advanced HNSCC patients, 5-year update (median follow-up 60 months)

Several other trials investigating the role of cetuximab are ongoing or have been reported in HNSCC (Table 10.3). For example, Argiris et al. presented at ASCO 2008, a study of 39 patients with stage III/IV HNSCC treated with induction docetaxel, cisplatin, and cetuximab followed by concurrent cisplatin and cetuximab with 70 Gy radiation. Preliminary reports from this study demonstrate high response rates (Argiris et al. 2008). Toxicities were deemed acceptable with this regimen, but survival data has yet to be reported. Similar trials are ongoing using the small molecule tyrosine-kinase inhibitors combined with radiation in HNSCC. Table 10.3 provides a listing of clinical trials that will help further define the role of EGFR inhibitors combined with radiation or chemoradiation in HNSCC in the years to come. Several trials in NSCLC combining EGFR inhibitors with radiation have been undertaken. One such phase II study has been completed in NSCLC. This study compares the regimen of carboplatin, pemetrexed (500 mg/m2), and thoracic radiation (70 Gy) alone and in combination with weekly cetuximab for 6 weeks (Govindan et al. 2009). Preliminary data identifies that 73% of patients on both arms had a complete or partial response. As reported at ASCO 2009, it does not appear that the addition of cetuximab confers survival benefit in this study although further maturation of followup is required (Govindan et al. 2009). A second phase II trial (the NEAR protocol) is ongoing in stage III NSCLC patients that combines cetuximab with IMRT (Jensen et al. 2006). The study has been open since 2006 with plans to accrue 30 patients still in progress. Another disease site under active investigation regarding the combination of EGFR inhibitors and radiation is esophageal cancer. Currently, one phase II trial has

NA 63

NA 85 98

II II III II I/II II

IMRT plus cisplatin vs. IMRT plus cisplatin and radiation

Radiotherapy, cisplatin/5-FU and gefitinib vs. chemoradiation alone Docetaxel, carboplatin, and 5-FU followed by radiotherapy and docetaxel, all combined with gefitinib Carboplatin and paclitaxel followed by radiotherapy, hydroxyurea, and 5-FU, all combined with gefitinib 91

32

NA

NA

87.5 52

84 89

33 73

86

CRR (%) 87 59 NA NA 68

OS at 2 years: 82% DFS at 2 years: 74% OS at 2 year: 86% PFS at 1 year: 68% OS at 3 years: 73% PFS at 3 years: 64%

NA OS at 2 years: 56% PFS at 2 years: 47% NA

NA PFS at 1 year: 78% OS at 1 year: 89% NA PFS at 1 year: 85%

NA

Survival index NA NA 62a versus 55% PFS at 3 years: 59% NA

NA

NA

NA

NA

NA NA

NA NA

NA NA

NA

Median overall survival (months) NA NA 54a versus 28 NA NA

Ahmed et al. (2007)

Doss et al. (2006)

Ongoing trial, patient accrual recently completed Adelstein et al. (2008)

Chen et al. (2007) Rueda et al. (2007)

Herchenhorn et al. (2007) Cohen et al. (2005)

Kuhnt et al. (2008) Savvides et al. (2008)

Argiris et al. (2008)

Reference Robert et al. (2001) Harrington et al. (2006) Bonner et al. (2006) Su et al. (2005) Merlano et al. (2007)

Adapted with permission from Bernier et al. (2009) CRR complete response rate, DFS disease free survival, 5-FU 5-fluorouracil, NA not available, ORR overall response rate, OS overall survival, PFS progression-free survival, HNSCC head and neck squamous cell carcinoma, IMRT intensity modulated radiation therapy a In cetuximab-containing arm

NA

NA 98

91 NA

I II I/II II

100

II

Radiotherapy, cisplatin, and erlotinib Radiotherapy, carboplatin paclitaxel induction 5-FU, hydroxyurea, and gefitinib Radiotherapy, cisplatin, and gefitinib Radiotherapy, cisplatin, and gefitinib

Drug and treatment regimen Radiotherapy and cetuximab Radiotherapy, cisplatin, and lapatinib Radiotherapy and cetuximab vs. radiotherapy alone Radiotherapy, cisplatin, and cetuximab Radiotherapy, cisplatin, 5-FU, and cetuximab (rapid alternating treatment) Induction chemotherapy followed by chemoradiation and cetuximab during sequential treatment Accelerated radiotherapy, cisplatin, and cetuximab Radiotherapy, docetaxel, and bevacizumab

ORR (%) NA 89 NA 76 100

Study phase I I III II II

Table 10.3 Clinical trials combining EGFR inhibitors with radiation in HNSCC 10 EGFR Signaling and Radiation 215

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completed enrollment with a total of 60 patients with esophageal (57 patients) or gastric cancer (three patients). The primary aim of the study was to examine the safety of the addition of cetuximab to paclitaxel, carboplatin, and radiation. Patients received this chemotherapy regimen for 6 weeks along with 50.4 Gy of radiation (Safran et al. 2008). A clinical response rate of 70% was demonstrated within this group with 16% of the group displaying grade 3 or 4 esophagitis (Safran et al. 2008). The summary impression from this trial considered the regimen safe, but further clinical trials are necessary to evaluate the ultimate effectiveness in esophageal cancer. GBM is another challenging disease site in which clinical trials are evaluating the combination of EGFR inhibitors with radiation. Notable among these is a phase II study evaluating erlotinib plus temozolomide during and after radiation therapy (Prados et al. 2009). A total of 65 patients were enrolled in the study. Patients were given 100 mg/day of erlotinib during radiation and were escalated after radiation therapy to 150 mg/day or until the development of an erlotinib-associated grade-2 rash. Patients receiving antiepileptic medications were given a higher dose of erlotinib (200 mg/day during radiation and 300 mg/day after radiation therapy). The median survival was 5.2 months longer than that projected in comparison with historical controls patients. Though this is a small study, some interesting conclusions were noted. Patients with MGMT methylation had a better overall survival of 25.5 months compared to patients without MGMT methylation, which demonstrated a survival of 14.6 months (hazard ratio = 0.31; 95% CI, 0.14–0.71; p = 0.006) (Prados et al. 2009). These conclusions may provide information regarding patient selection for future clinical trials, though further studies are necessary to determine the importance of MGMT methylation in this patient cohort. In addition to this study, there are several other phase I and II studies that have been completed combining EGFR inhibitors with radiation in GBM, including studies in pediatric GBM that requires further investigation.

10.4.1 Toxicities of Anti-EGFR mAbs and TKIs Although EGFR inhibitors generally carry a modest toxicity profile compared with conventional cytotoxic agents used in the treatment of HNSCC and NSCLC, there are certainly toxicities that warrant further understanding. As noted in the cetuximab prescribing information, serious grade-3 or 4 hypersensitivity reactions to cetuximab can occur, which may present as acute airway obstruction with bronchospasm and stridor, hypotension, and anaphylaxis. These reactions have been observed in 2–5% of patients. However, incidence reports from different geographic regions appear to vary significantly, with lower rates (<1%) seen in the Northeast US, and higher rates reported in North Carolina, Arkansas, Missouri, Virginia, and Tennessee (O’Neil et al. 2007; Chung et al. 2008). Serious cetuximab-related infusion reactions were reported as high as 22% in 88 patients treated in trials at two academic institutions in North Carolina and Tennessee (O’Neil et al. 2007). Recently, a mechanism of hypersensitivity reaction to cetuximab was identified as the presence of preexisting IgE antibodies against galactose-a-1,3-galactose, an oligosaccharide present on the

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Fab portion of the recombinant antibody molecule (Chung et al. 2008). In clinical practice, it is recommended to premedicate patients with an H1 antagonist such as diphenhydramine prior to cetuximab infusion. In addition, patients should be monitored for at least 1 h after infusion, particularly with the first infusion as the majority of severe reactions occur at this time even with pretreatment. A significant proportion of patients (45–100%) experience some form of cutaneous toxicity with the administration of EGFR inhibitors. These can include a papulopustular rash, dry skin, fissuring, hair and nail alterations, and periungual inflammation as the most common side effects seen with anti-EGFR mAbs and TKIs (Lacouture 2006). These cutaneous effects are believed to result from EGFR inhibition in keratinocytes in the basal layer of the epidermis and outer layers of the hair follicle, leading to apoptosis of keratinocytes, inflammatory reactions, and pathological skin manifestations. The characteristic rash generally develops within the first 2 weeks of therapy, primarily involving the face, scalp, neck, and upper torso. Although these EGFR-related cutaneous toxicities are problematic for some patients, they are also commonly associated with an increased likelihood of favorable response to the EGFR inhibitor. As reported in several clinical trials, a strong correlation has been observed between the development of skin toxicities to cetuximab therapy and favorable tumor response (Cohen et al. 2003; Saltz et al. 2004). The development of a cetuximab-related skin reaction was correlated with an improvement in overall survival in a phase III ECOG trial evaluating cisplatin plus placebo vs. cisplatin plus cetuximab in metastatic/recurrent HNSCC (HR 0.42, p = 0.01) (Burtness et al. 2005). A similar correlation between severity of skin reaction and survival outcome was identified in the phase III radiation-cetuximab trial (Bentzen et al. 2005). Fortunately, cetuximab-related skin toxicities are generally self-limited and not severe for the majority of patients. Furthermore, it appears that cetuximab does not increase the incidence and severity of overall radiation-induced reactions (Bonner et al. 2006), although as noted above there appear to be some geographic variation in these response profiles.

10.5 Resistance to EGFR Inhibitors As increasing use of EGFR inhibitors is maturing worldwide, we are beginning to identify issues of resistance to EGFR therapy that compromise our ability to maximize the impact of this promising class of molecular agent.

10.5.1 Genetic Mutations and Resistance The problem of resistance to EGFR inhibitors is becoming well established. Not all patients respond favorably to an initial challenge with EGFR inhibitors. This represents intrinsic resistance. Several biomarkers of resistance have been identified within the categories of colorectal cancer and lung cancer. Mutations have been

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established within the first four exons (exons 18–21) of the tyrosine kinase domain of the EGFR that predict response to gefitinib or erlotinib in lung cancer patients, representing a landmark development in the EGFR field (Lynch et al. 2004; Paez et al. 2004; Pao et al. 2004). For example, mutation in exon 21 of L858R may predict increased sensitivity to TKIs, whereas the T790M mutation in exon 20 is associated with acquired resistance to TKI therapy (Pao et al. 2005; Inoue et al. 2009). Beyond the EGFR itself, mutations in several EGFR downstream signaling molecules are also found to affect response of EGFR therapy. Colorectal cancers with Kras and Braf mutations have been strongly correlated with resistance to cetuximab therapy in clinical studies (Normanno et al. 2009). It has recently been recommended by ASCO in 2009 that patients with colon cancer and a Kras mutation should not receive cetuximab therapy (Allegra et al. 2009). PIK3CA mutations and PTEN overexpression was also linked with response to cetuximab in colon cancer (Jhawer et al. 2008). These mutations all provide important clinical tools for rational identification of patients more likely to respond to EGFR inhibitors.

10.5.2 Tyrosine Kinase Receptors and Resistance Despite the efficacy of EGFR TKIs in lung cancer patients with EGFR activating mutations, some patients ultimately develop resistance (i.e., acquired resistance) to these drugs. Acquired resistance to EGFR inhibitors may result from the activation of alternative membrane-bound RTKs that bypass the EGFR pathway and/or activate signaling pathways downstream of EGFR (Camp et al. 2005). Engelman et al. reported that tumors with EGFR activating mutations eventually acquire resistance to gefitinib following long-term exposure. Interestingly, it was demonstrated that amplification of the RTK, c-Met (hepatocyte growth factor receptor), caused resistance to gefitinib because of activation of an ErbB3-dependent PI3K/Akt pathway (Engelman et al. 2007; Engelman and Janne 2008). This activation of ErbB3 permits the cells to transmit the same downstream signaling in the presence of EGFR inhibitors. Similar to this finding, we and other investigators identified the activation of HER2, ErbB3, and c-Met in EGFR inhibitor-resistant tumor cells (Frolov et al. 2007; Wheeler et al. 2008). However, distinct from previous findings, c-Met was not amplified in these cells. Instead, specific RTKs could be activated in an EGFR-dependent manner since an increased association of EGFR with HER2, ErbB3, or c-Met was observed in the resistant cells. The blockade of EGFR and HER2 led to the loss of ErbB3 and PI3K/ Akt signaling. Other than these RTKs, IGFR and VEGFR have also been identified as important molecules to regulate resistance to EGFR inhibitors (Viloria-Petit et al. 2001; Jones et al. 2004; Morgillo et al. 2007; Benavente et al. 2009). Taken together, these studies suggest that if tumor cells can maintain activity of downstream signaling, especially PI3K/Akt, in the presence of EGFR inhibitors via the activation of alternative RTKs, this may contribute to resistance to EGFR inhibitors. More importantly, these findings suggest that combinations of molecular targeted therapies blocking EGFR and other selected RTKs may offer a promising strategy to overcome acquired resistance and enhance the effectiveness of EGFR therapy.

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10.5.3 Nuclear EGFR and Resistance Another avenue of active investigation regarding acquired resistance to EGFR inhibitors involves the function and purpose of EGFR within the nucleus. The expression of nuclear EGFR has been associated with decreased overall survival in both breast and HNSCC (Lo et al. 2005; Psyrri et al. 2005). Lo et al. analyzed 130 breast carcinomas for nuclear EGFR and found an inverse correlation between nuclear EGFR expression and overall survival (p = 0.009) (Lo et al. 2005). Psyrri et al. analyzed total EGFR and nuclear EGFR in 95 patients with HNSCC. Tissue microarray demonstrated that nuclear EGFR was inversely correlated with survival as well as radiation response (Psyrri et al. 2008). Further studies suggest that the role of nuclear EGFR is linked with other pathways that are important in tumor biology. Nuclear EGFR has also been associated with increased expression of cyclin D1 and Ki67 in breast carcinoma cells, suggesting increased proliferation in cells expressing nuclear EGFR (Lo et al. 2005). Recent studies have shown that nuclear EGFR phosphorylates and stabilizes proliferating cell nuclear antigen (PCNA), promoting cell cycle progression (Psyrri et al. 2005). These preliminary data suggest that nuclear EGFR is associated with characteristics of more aggressive tumors that manifest increased proliferation and accelerated cell cycle progression. Several studies have demonstrated that radiation can induce EGFR translocation to the nucleus (Dittmann et al. 2005a, 2008, 2009). Nuclear EGFR has been linked to acquired resistance to cetuximab (Wheeler et al. 2008; Li et al. 2009). Dasatinib in combination with cetuximab can prevent the translocation of EGFR to the nucleus and resensitize cells to cetuximab in preclinical models (Li et al. 2009). From this data, it is theorized that nuclear EGFR may offer an escape mechanism from anti-EGFR therapy and suggests a new kinase-independent role for EGFR. Further studies are required to clarify this mechanism of resistance as well as potential strategies to overcome resistance in an effort to further enhance EGFR treatment approaches.

10.6 Conclusions A beneficial impact of combining EGFR signaling inhibition with radiation in cancer therapy has been established in recent years. This is particularly evident in HNSCC where mature phase III trials have been completed. Strong preclinical data highlighting a favorable interaction of anti-EGFR therapies with radiation has provided a logical framework for the ongoing clinical trials program. There is growing evidence that these promising EGFR inhibitor-radiation combinations may benefit patients with a variety of epithelial tumor types as briefly described within this review. Our improved understanding of the molecular and cellular mechanisms underlying the enhancement of radiation response with EGFR signaling inhibition is central to further refinement of this cancer treatment strategy. Sharpening our ability to identify those patients (tumors) most likely to benefit from EGFR/ radiation approaches is also a primary objective for the future. With well over 50%

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of cancer patients worldwide receiving radiation as an integral component of their treatment, this approach may provide significant new leads to guide not only the combination of radiation with EGFR inhibitors, but also with other molecular- targeting drugs for the improvement of cancer therapy. Acknowledgements Supported by NIH/NCI Grant R01 CA 113448 to PMH; EFD supported by NIH Physician Scientist Training in Cancer Medicine.

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Chapter 11

Thermal Modulation of Radiation-Induced DNA Damage Responses Joseph L. Roti Roti, Robert P. VanderWaal, and Andrei Laszlo

Abstract The goal of this review is to delineate the interaction between the DNA damage response and the proteotoxicity induced by hyperthermia which leads to increased sensitivity to ionizing radiation. The radiosensitization must come from an interaction between the proteotoxicity induced by hyperthermia and DNA damage responses occurring after ionizing radiation. Recently, the cellular response to DNA DSB has been described as a combination of classical signal transduction cascades triggered by a series of phosphorylation-dependent events in which a “signal” (DNA damage) is detected by “sensors” (DNA-damage binding proteins), which then triggers the activation of a “mediator” system (protein kinase cascade) which, in turn, amplifies and diversifies the signal targeting of a series of downstream “effectors” of the DNA damage response, including DNA repair, cell cycle checkpoints, and apoptosis. Proteins that perform “sensor” or “transducing/mediating” functions are likely to be targets for heat radiosensitization through protein aggregation, possibly in concert with other mechanisms secondary to, or triggered by, the proteotoxicity associated with protein aggregation. Recent studies (reviewed herein) show three types of interaction between proteotoxic pathways and DNA DSB response pathways; (1) blocking of ability to sense DSBs via alterations in the proteins that bind DNA-nuclear matrix attachment points, (2) modulation of chromatin remodeling and (3) perturbations of the ability to assemble DNA repair complexes. Specifically, heat effects on Mre11, gH2AX, and 53BP1 will be described. In addition, it has been found that the nucleolar protein, nucleophosmin, contributes to radiosensitization only in cells that have been heat shocked. Keywords Radiation sensitivity • DNA repair • Hyperthermia • Heat shock proteins • MRE11 • gH2AX • 53BP1 • Nucleophosmin

J.L. Roti Roti (*) Radiation and Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_11, © Springer Science+Business Media, LLC 2011

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11.1 Introduction This paper will review the state of knowledge of the interaction between the DNA damage response and the proteotoxicity induced by hyperthermia, which leads to increased sensitivity to ionizing radiation. The challenge in this area of study is that heat does not directly produce DNA damage, which is the cause of cell death due to ionizing radiation, while low LET radiation does not induce significant proteotoxicity at clinically relevant doses. Further, it has long been known that the enhanced cell killing from the combination of hyperthermia and radiation (compared to that induced by each alone) is due to an enhancement of radiation-induced cell death. Thus, the radiosensitization must come from an interaction between the proteotoxicity induced by hyperthermia and DNA damage responses occurring after ionizing radiation. However, the actual DNA damages [~40–60 double stand breaks (DSBs) per cell per mean lethal event] and fraction of proteins, undergoing an endothermic change needed to produce radiosensitization are relatively small. To illustrate the latter point, a thermal enhancement ratio (TER) of 3 is produced by a time temperature combination that induces less than 10% of the measurable endothermic changes [extrapolated from Lepock (2004)]. Note that TER is a measure of the extent of radiosensitization (Robinson et al. 1974). Therefore, delineating the protein change, that critically impacts DNA damage response pathways, remains a challenge. Because the radiosensitizing effects of hyperthermia have been shown to improve clinical radiation therapy (Horsman and Overgaard 2007), its study has significant clinical relevance for the potential improvement of radiotherapy. The purpose of this review is to provide the reader with an up to date picture of the knowledge of the mechanisms of heat-induced radiosensitization and delineate some of the intriguing new concepts that are emerging. Both the DNA damage response and the response to proteotoxicity (also known as the “heat shock response”) are stress responses that are well conserved throughout evolution. Recently it has been suggested that the survival of malignant tumor cells is dependent on one or more stress response pathways (Luo et al. 2009). The review by Luo et al. (2009) concludes that tumors are characterized by both oncogene and non-oncogene addictions, which could “be exploited through stress sensitization and stress overload to kill …cancer cells.” These authors (Luo et al. 2009) propose that effective tumor cell killing can be accomplished by developing orthogonal cancer therapies. Two of the stress support pathways are the DNA damage response and the proteotoxic stress response. Radiotherapy and certain chemotherapeutic agents overload the DNA damage response pathways. Heat shock has long been recognized as a major source of proteotoxic stress. Thus hyperthermia combined with radiation therapy is an excellent example of an orthogonal therapy. In fact, since the effectiveness of hyperthermia as an agent to improve radiation therapy has been established by numerous preclinical studies and is supported by numerous clinical trials

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(see below), the combination of proteotoxic stress from hyperthermia and the DNA damage response induced by radiotherapy is a classic example of an orthogonal cancer therapy. Therefore, delineating the intersecting pathways of these orthogonal therapies should delineate effective strategies and molecular targets to improve cancer treatment. Recent meta-analysis of all completed clinical trials of hyperthermia combined with radiation therapy shows that hyperthermia significantly improves radiation therapy (Horsman and Overgaard 2007). Further, hyperthermia is one of the most potent known radiosensitizers, since acute hyperthermia (see below) produces TERs from 3 to 5 for both oxygenated and hypoxic cells (Kampinga and Dikomey 2001; Horsman and Overgaard 2007). However, despite clear evidence that hyperthermia improves the effectiveness of radiation therapy; it has made little impact on the clinical practice of clinical radiation oncology. One explanation is that, clinically, the acute hyperthermia target thermal dose, 1 h at 43°C, proved to be unachievable, while the achievable moderate hyperthermia (41– 42°C) produced TERs on the order of 1.5 or less. By better understanding the mechanisms of heat induced radiosensitization, we anticipate that strategies for mimicking or enhancing the TERs produced by clinically achievable temperatures will be elucidated. Thus, this review will focus on the molecular changes induced by hyperthermia in proteins that are critical for the cell’s responses to DNA damage. At the outset, it is important to note that temperature and/or thermal dose (i.e., the combination of time and temperature) is key factor in whether any given effect is triggered to a consequential level. Thus, it may be useful to present a terminology to facilitate any discussion of temperature dependence. Just above the normal temperature range, 37 ± 0.5, is fever range 38–40°C, which will have little effect on cell viability or radiation sensitivity. Thus, fever range hyperthermia will not be considered in this review. The next interval, 41–42.5°C, will be referred to as moderate hyperthermia. Heating times of 1–2 h at moderate hyperthermia will cause little cell killing, but will have modest to significant effects on radiation sensitivity. In this temperature interval the radiosensitizing effects are often cell line dependent. Most clinical hyperthermia achieves tumor heating to temperatures in this range. The next interval is 42.5–46°C, which we refer to as acute hyperthermia. In the acute temperature range, cell killing and radiosensitization are achieved in 5–60 min depending on temperature. While radiosensitization will affect all of the heated cells, a significant fraction will be clonogenically killed by the direct effects of heat. In particular, S-phase and mitotic cells are preferentially killed at these temperatures. The next temperature range is 47–57°C, which we refer to as severe hyperthermia. In the severe temperature range radiosensitization can occur in as little as 15 s and massive cell death occurs in 5–10 min. Above 58°C the temperatures are used for thermal ablation referred to as the ablative range. Cell death is so rapid at these temperatures that radiosensitization is not relevant. Thus, the ablative range is not relevant to this review.

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11.2 Evidence that Heat Effects on Proteins Involved or Impacting DNA Repair Pathways are Responsible for Radiosensitization The lethal effects of ionizing radiation are thought to be largely due to incomplete or misrepair of DSBs even though DSBs are only one of several types of DNA damage induced by ionizing radiation. In contrast, it is clear that hyperthermia does not directly induce DNA damage, but does indirectly through effects on DNA replication and DNA repair (Wong et al. 1995). Therefore, in order for hyperthermia to increase the lethal effects of radiation, it must modulate the radiation response via alterations in key protein–protein and protein–DNA interactions in critical steps in DNA repair pathways. The principal molecular effect of hyperthermia (heat shock in the 40–46°C) is the unfolding and aggregation of a significant number of proteins, which is, in fact, a relatively small fraction of the total proteins. For example, TERs (which is the ratio of the radiation dose alone to that in combination with heat to achieve isosurvival) >3.0 can be obtained by thermal exposures that induce conformational changes in less than 10% of the cellular protein (calculated from Lepock 2004). It has long been recognized that hyperthermia induces a wide variety of structural and functional effects on cells. These effects fall into two broad categories: the effects of heat on cells and the responses of cells to such effects. Among the latter are the much studied heat shock proteins that are synthesized in response to protein unfolding (Kampinga 1993). An example of the former category is protein aggregation that occurs in both the cytoplasm and the nucleus [reviewed in Lepock (2004)]. Such aggregates are known to contain both unfolded proteins and normally folded proteins that are trapped in the aggregates (Stege et al. 1995). Heat induced protein aggregation is associated with both cell killing and radiosensitization and the induction of heat shock proteins, which act to disperse the aggregates and to restore the proper folding of unfolded proteins (Kampinga 1993), as illustrated in Fig. 11.1. While little is known regarding the particular proteins involved in these aggregates and the specifics of the loss (and even possible gain) of function of the thermally affected and putatively unfolded proteins, recent work is beginning to delineate some of these changes as they relate to DNA repair inhibition and heat radiosensitization. Classic and several recent studies have shown that heat-induced radiosensitization is due to the inhibition of the repair of X or g-ray induced DNA damage (Dewey et al. 1978; Kampinga and Dikomey 2001; Kampinga et al. 1997), especially DSBs (Kampinga and Dikomey 2001; Illiakis et al. 2008). DNA DSBs pose considerable threat to the cell, since DNA loses integrity and information on both strands, in addition to its physical connection to the centromere (Shiloh 2004). Recently, the cellular response to DNA DSB has been described as a combination of classical signal transduction cascades triggered by a series of phosphorylationdependent events in which a “signal” (DNA damage) is detected by “sensors” (DNA-damage binding proteins), which then triggers the activation of a “mediator” system (protein kinase cascade) which, in turn, amplifies and diversifies the signal

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targeting of a series of downstream “effectors” of the DNA damage response, including DNA repair, cell cycle checkpoints, and apoptosis (see article by Jackson 2002). In this model, the nonhomologous end joining, NHEJ, and the homologous recombination, HR, repair pathways for DNA DSBs are viewed as “effectors.” The foregoing concept is important for understanding the observations that the magnitude of heat radiosensitization is not diminished in cells that are mutated in, or have deletions of, several different key components of the either the NHEJ and HR repair pathways (Kampinga et al. 2004). Recently, it has been demonstrated that the base excision pathway is also not a target for heat radiosensitization (Batuello et al. 2009). These results suggest that the target(s) of heat radiosensitization are upstream of the “effectors” in the cellular response to DNA DSB. Thus, proteins that perform “sensor” or “transducing/mediating” functions are likely to be targets for heat radiosensitization through protein aggregation, possibly in concert with other mechanisms secondary to, or triggered by the proteotoxicity associate with such aggregation (Fig. 11.1). Recent studies (reviewed herein) show three types of interactions between the proteotoxic pathway and DNA DSB response pathway, Proteotoxicity and the DNA Damage Response Hyperthermia does not cause DNA damage, but affects intracellular processes via changes in protein folding

Aggregated Unfolded Proteins

Hyperthermia

= targets for radiosensitization?

Blocks access to site of DSB Alters the availability of repair proteins

Sensors:

γ-H2AX

HSP70 & HSPs

Unfolding & exposure of hydrophobic domains Hyperthermia

Proteins involved in the DNA damage response can be affected by heat shock:

MDC1 Inhibits assembly of transducing proteins

HSP70 & HSPs

53BP1

Signal Amplification Repair

Correctly folded and functional proteins

MRN

Cell Cycle Control

ATM

Transducers: Kinase Cascades Apoptosis

Other Effectors

Enhanced cell killing by radiation

Fig. 11.1 Illustration of the potential impact of heat-induced proteotoxicity on the DNA damage response. The pathway on the left shows the effects of moderate or acute hyperthermia on protein folding, which in effect, makes hyperthermia a proteotoxic stress. Beginning with the correctly folded protein at the lower left, the application of thermal energy increases the exposure of hydrophobic domains that can aggregate to render the affected proteins nonfunctional. The pathways on the right illustrate the steps in the response of cells to DNA DSBs. Some of the steps known to be disrupted by heat shock, leading to radiosensitization are illustrated by red arrows and red text

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which are illustrated by the red arrows (Fig. 11.1); (1) blocking of ability to sense DSBs via alterations in the proteins that bind DNA-nuclear matrix attachment points, (2) modulation of chromatin remodeling, and (3) perturbations of the ability to assemble DNA repair complexes.

11.3 Perturbation of Proteins Known to Participate in DNA Repair Signaling Pathways Several of the “sensor” and “modulator” proteins become rapidly associated with the region around newly generated DNA DSB. Within a few minutes after irradiation, cytologically observable foci, ionizing radiation induced foci (IRIF) are visible (Ward and Chen 2004; Stucki and Jackson 2006), which contain several “sensors” of DNA DSBs, including the Mre11/Rad50/Nbs1, (MRN), complex, g-H2AX, MDC1, BRCA1 and p53 binding protein 1, and 53BP1 (Ward and Chen 2004; Stucki and Jackson 2006). One of the earliest responses to radiation-induced DSBs is a chromatin region containing g-H2AX, the phosphorylated form of histone H2AX, which has been suggested to play a direct role the organization of multiprotein complexes involved in the repair of DNA DSB (Paull et al. 2000; Ward and Chen 2004; Stucki and Jackson 2006). Several recent studies show that heat interferes with the cellular DNA DSB response elicited by radiation by altering some of these early events in this signaling cascade, including the MRN complex, g-H2AX, and 53BP1.

11.3.1 Hyperthermia Effects on the MRN Complex The MRN complex (Mre11–Rad50–NBS) is composed of two highly conserved subunits that have enzymatic activity, Mre11 and Rad50, and a catalytic component, Nbs1. Mre11 has a 3¢–5¢ double strand exonuclease and endonuclease activity that efficiently degrades DNA DSB substrates with blunt or 3¢ recesses ends, both in vivo and in vitro, while Rad50 has been shown to have an ATP dependent DNA double strand binding activity. These enzymatic activities are increased in the MRN complex, indicating that the complex is important in carrying out the activity of Mre11 (D’Amours and Jackson 2002; van den Bosch et al. 2003). Although no known enzymatic activity is associated with Nbs1, it may have a catalytic activity in forming the MRN complex, since no foci containing Mre11 are observed in cells that lack Nbs1 (van den Bosch et al. 2003). The formation of foci containing the MRN complex is one of the most rapid events following irradiation, which has been observed within 45 s (van den Bosch et al. 2003). The foci containing MRN have been shown to be associated with the nuclear matrix fraction that is insoluble in CSK buffer (Mirzoeva and Petrini 2001; Zhu et al. 2001). Most, if not all, of Mre11

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is found in the nucleus (Zhu et al. 2001). Heat shock has been shown to increase the protein content of the nuclear matrix (Roti Roti et al. 1998). Thus, it was surprising that in U-1 human melanoma cells and HeLa cells after a 15 min heat shock at 45.5°C, there was an increased association of Mre11 with the cytoplasm, concomitant with a decrease in its nuclear localization, within 30 min following heat shock. This altered solubility, inferring altered subcellular localization, peaked at 4 h following the treatment at 45.5°C for 15 min, and returned to nearly control levels by 7 h of recovery at 37°C (Zhu et al. 2001). Irradiation with 12 Gy did not lead to observable changes in the nuclear Mre11 content, but displayed a greater relative increase in its distribution in the cytoplasm after the combined ionizing radiation and heat treatment. Such heat-induced alterations in the localization of Mre11 were also observed in immunofluorescence experiments (Zhu et al. 2001). Heat effects on the solubility and localization of Mre11 by acute heat shock were further studied (Dynlacht et al. 2004). Increased cytoplasmic association after heat shock at 45.5°C with or without radiation was observed for the other two components of the MRN complex, Rad50 and Nbs1. Further, immunoprecipitates with an Nbs1 antibody were shown to contain both Mre11 and Rad50, indicating that at least some of the MRN components in the cytoplasm are part of a MRN complex (Seno and Dynlacht 2004). Increased cytoplasmic MRN components were observed immediately after the heat shock, with the maximal cytoplasmic fraction occurring 30–60 min after heat shock and a return to control levels after 7 h. These effects of heat on the MRN components were observed in cells that were heated and irradiated, independent of the sequence of heat shock and irradiation. It should be noted that the increased cytoplasmic localization of the members of the MRN complex in heated and heated irradiated cells was not due to ongoing protein synthesis as it was unaffected by cycloheximide (Seno and Dynlacht 2004). The effect of heat shocks at a lower temperature was also determined. A heat shock of 2 h at 42.5°C induces the same cell killing as the 15 min heat shock at 45.5°C. However, the relative increase in the amount of the cytoplasmic MRN was higher in the cells heated at 42.5°C. In contrast with the cells heated at 45.5°C, the maximal increase in cytoplasmic NRM in cells heated at 42.5°C was observed immediately after heating. The return to pretreatment MRN distribution was observed to be 7 h similar to that found in cells heated at 45.5°C. All three components of the MRN complex are known to be phosphorylated after radiation (van den Bosch et al. 2003). The phosphorylated form of Nbs1 was found in the nucleus of the irradiated cells, but not in the cytoplasm of cells that had been both heated and irradiated. Six hours after heat shock, the phosphorylated form of Nbs1 was not observed in either the nucleus or the cytoplasm. Interestingly, heat inhibited the normal phosphorylation of Nbs1 that is induced in the nucleus as a consequence of irradiation. The decrease in the nuclear fraction of the MRN complex was not observed in cells that had been treated with leptomycin B prior to heating, suggesting that the heat induced reduction in the nuclear association of MRN is an active process facilitated by the CRM1 nuclear export pathway (Seno and Dynlacht 2004). In cells heated at a clinically achievable temperature of 41.1°C a significant increase in the cytoplasmic localization of Mre11 was observed after 1 h,

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and reached a maximum after 2 h (Xu et al. 2002). However, this effect was highly cell line dependent and was dependent on the initial Mre11 levels in various cell lines (Xu et al. 2007). The fraction of nuclear Mre11 decreased with a concomitant increase in the cytoplasmic Mre11 in cells that were heated at 41.1°C (Xu et al. 2002). Meanwhile, the total levels of Mre11 and Rad50 did not change in the course of the various treatments. The magnitude of the increase of cytoplasmic Mre11 correlated with heat radiosensitization across four human tumor cell lines (Xu et al. 2007). Immunoprecipitation experiments with anti Mre11 antibody indicated an increase in the interaction of Rad50 and Mre11 in irradiated cells, but not in heated cells, where a decrease in this interaction was observed after 2 h of heating at 41.1°C. Significantly, the treatments that led to the largest radiosensitization were associated with the maximal decrease of Rad50 that co-immunoprecipitated with Mre11 and maximal increase in HSP70 that co-immunoprecipitated with either Rad50 or Mre11 (Dynlacht et al. 2004). These changes are consistent with the observation that the interaction of Mre11 with Rad50 and Nbs1 is decreased in cells heated at 45.5°C and then irradiated (Seno and Dynlacht 2004). Heat induced changes in the interactions between the members of the MRN complex appear to be due to denaturation of one or more of its component proteins. The heat induced denaturation of cellular proteins which leads to proteotoxic stress is accompanied by an increase in the fraction of such proteins that are insoluble in buffers containing the nonionic detergent, TX-100 (Laszlo 1992a; Laszlo et al. 1993). Many, if not all, of the heat denatured proteins in the nuclear fraction are associated with the nuclear matrix, (Lepock 2004). To gain more insight regarding the effects of heat on the residual nuclear matrix associated Mre11, it was visualized in cells that had been extracted with CSK buffer extracts the soluble cellular components, leaving the nuclear matrix and the cytoskeleton (Fey et al. 1984; He et al. 1990) after exposure to 41.1°C with and without irradiation with 12 Gy (Dynlacht et al. 2004). After heat alone, the residual Mre11 in the nucleus is localized at the nuclear periphery (Dynlacht et al. 2004). This result is consistent with earlier data which shows localization of residual, nuclear matrix-associated Mre11 around the nuclear periphery after 1 h at 41.1°C (Xu et al. 2002, 2007). When these preparations are made from cells irradiated with 12 Gy and allowed to recover for 15 min–1 h after irradiation, repair foci containing Mre11 are seen as bright single specks. However, if cells are irradiated during a heat exposure, the Mre11 repair foci appear to be clumped or aggregated and are seen as large bright blobs. When cells were irradiated first and then maintained at 41.1°C for 15 min, Mre11 appeared to form aggregates throughout the nucleus, seen as bright blobs or clumps of the smaller specks (Dynlacht et al. 2004). This observation may explain, in part, the greater radiosensitization that has been observed when cells are heated during irradiation (Xu et al. 2002). The heat shock protein hsp70 has been shown to interact with proteins that become insoluble in CSK buffer in heated cells (Beckmann et al. 1992; Laszlo 1992a). Immunoprecipitation experiments have shown that Mre11 and Rad50 interact with Hsp70 during heating at 41.1°C. When HeLa cells were heated for 6 h at 41.1°C (a radiosensitizing heat dose for HeLa cells), both Rad50 and Mre11 co-immunoprecipitated with hsp70 as was seen earlier for a 2 h heating of NSY

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cells (Xu et al. 2002). Reciprocal immunoprecipitation experiments found hsp70 in immunoprecipitates of Mre11 and Rad50, and Mre11 and Rad50 in immunoprecipitates of hsp70. The amount of Hsp70 immunoprecipitating with both Rad50 and Mre11 increases after 4 h at 41°C and reaches a maximum at 6 h leading to a plateau for up to 8 h. The association between Mre11 and hsp70 increases 10–100fold and that between Rad50 about tenfold, while the amount of hsp70 increases two- to threefold during heating times (Dynlacht et al. 2004). Thus, these interactions are probably associated with heat effects on Rad50 and/or Mre11. One possibility would be that heat causes some unfolding of these molecules and that hsp70 interacts with the unfolded regions. The changes in hsp70 association with Mre11 and Rad50 correlate reasonably well with radiosensitization of HeLa cells by 41°C. The most compelling data to support the notion that Mre11 might be an important target for radiosensitization by moderate hyperthermia comes from recent Mre11 small interfering RNA (siRNA) experiments (Xu et al. 2004). Cells in which Mre11 was knocked down to 30–40% of control levels were significantly radiosensitized. However, when heat radiosensitization was compared between cells expressing normal levels of Mre11 and cells in which Mre11 expression was suppressed, the degree of radiosensitization was not increased further in the Mre11 knockdown cells. This result suggested that reduced availability of Mre11 is a consequence of the heat-induced Mre11 delocalization from the nucleus and its aggregation or clumping, contributes to heat radiosensitization (Xu et al 2004).

11.3.2 Hyperthermia Effects Involving g-H2AX Phosphorylation of the carboxyterminal SQE motif of histone H2AX and its appearance in cytologically observable foci, called g-H2AX foci, is one of the earliest events in the orchestrated cellular response to DNA DSBs (Fernandez-Capetillo et al. 2004; Takahashi and Ohnishi 2005). For ionizing radiation, there is a good correlation between the number of g-H2AX foci and the number of DSBs (Sedelnikova et al. 2002). Thus the formation of g-H2AX foci has been used as a measure of DNA DSBs (Takahashi and Ohnishi 2005). The rapid phosphorylation of histone H2AX at serine 139 has been postulated to form the type of scaffold mentioned above for the docking of other repair proteins (Paull et al. 2000; Fernandez-Capetillo et al. 2004), since in mammalian cells H2AX is phosphorylated in regions as large as one megabase on either side of a DSB (Rogakou et al. 1999). Thus, it was of interest to determine the effect of heat on the formation of g-H2AX foci. Surprisingly, heat shock alone induced the formation of g-H2AX containing foci from temperatures of 41–45°C (Takahashi et al. 2004; Kaneko et al. 2005; Hunt et al. 2007). Interestingly, severe, brief (30–60 s at 50°C) heat shocks did not induced the formation of g-H2AX foci (Roti Roti et al. 2009). Since acute hyperthermia alone induced g-H2AX foci, the effect of heat on the radiation induced g-H2AX response could only be determined after brief high temperature heat shocks. In such cells, the resolution of radiation induced g-H2AX foci was delayed (Roti Roti et al. 2010). Results using immunoblotting techniques demonstrated

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that heat and irradiation were additive with respect to the amount of g-H2AX observable by these techniques in heated (42–45°C) irradiated cells (Laszlo and Mueller, unpublished results). The induction of g-H2AX foci by heat generated a controversy whether or not these foci represented DNA DSB, indicating that heat alone induced direct DNA damage (Takahashi et al. 2004; Kampinga and Laszlo 2005). A 60 min at 43°C treatment led to the formation of 60–70 g-H2AX foci, equivalent to that associated with 2 Gy of irradiation. However, measurements of DNA damage by three different sensitive biochemical and cytological assays in heated cells could not detect DNA DSB and furthermore, there was no increase in DNA DSB in heated-irradiated cells (Hunt et al. 2007). Moreover, heat did not induce foci containing 53BP1, Chk2, and Ser 957 phosphorylated SMC1, all of which are associated with DNA DSB induced by irradiation. Thus, the mechanism of induction of g-H2AX foci in heated cells is not due to the direct induction of DNA DSBs. Heat induced g-H2AX foci are ATM dependent and hsp70 independent. Heat induced the activation of the ATM protein kinase, as monitored by both the phosphorylation of Ser 1981 and increase of kinase activity. Interestingly, heat, but not radiation, induced ATM kinase activation in A-TLD1 cells that are hypomorphic for Mre11 and lack this activity (Hunt et al. 2007). Thus heat is an agent that induces MRN-independent activation of ATM; in contrast to the case after for ionizing radiation, where it has been shown that the activation of ATM kinase activity is an event upstream from MRN complex and focus formation (Lee and Paull 2004, 2005). Therefore, while the heat-induced g-h2AX foci are well characterized, the mechanism of their formation remains to be elucidated. Significantly, an extensive study using cells with different thermal sensitivities indicated that there was no correlation between heat-induced cell killing and the heat induced g-H2AX response (Laszlo and Fleischer 2009b) showing that formation of these foci does not play a role in heat-induced cell death. Treatment with amino acid analogs, agents which induce proteotoxic stress, (Li and Laszlo 1985; Laszlo and Li 1993) did not interfere with the formation of radiation induced g-H2AX foci, but did delay the timely resolution of such foci. In addition, under conditions where heat does not induce g-H2AX foci, when cells are heated at temperatures >50°C, the resolution of the radiation induced g-H2AX foci is delayed, a process analogous to that observed in radiosensitive cells (Paull et al. 2000; Mirzayans et al. 2006). These experimental results show that heat interferes with the radiation induced g-H2AX response, which could contribute to radiation sensitivity. However, due to the fact that acute hyperthermia induces g-H2AX foci, the role of the delay due to heat of radiation-induced g-H2AX in radiosensitization at the survival level has yet to be quantified.

11.3.3 Hyperthermia Effects on 53BP1 Another early event in the cellular DNA DSB response cascade is the formation of foci containing 53BP1, which colocalize and require foci containing g-H2AX and

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MDC1 (Ward and Chen 2004; Mochan et al. 2004; Adams and Carpenter 2006). Radiation induced 53BP1 foci are thought to be at sites of DNA DSBs since they colocalize with other foci known to mark sites of DNA DSBs, such as g-H2AX, MDC1, and the MRN complex (Ward and Chen 2004; Adams and Carpenter 2006). The number of 53BP1 foci increases linearly over time, reaching a maximum at about 15–30 min after irradiation and then steadily decreases to baseline levels within the next 16 h (Schultz et al. 2000; Rappold et al. 2001). The number of 53BP1 foci, closely parallels the number of DNA DSBs and the kinetics of resolution of 53BP1 foci is very similar to the kinetics of DNA DSB repair following ionizing radiation (Mochan et al. 2004; Adams and Carpenter 2006). 53BP1 is thought to bind to long stretches of DNA or chromatin on either side of the DNA DSB (Mochan et al. 2004) as has been shown for g-H2AX (Rogakou et al. 1999; Fernandez-Capetillo et al. 2004; Lou et al. 2006). Since heat does not induce the formation of 53BP1 foci (Hunt et al. 2007), the effects of heat on the formation and resolution of radiation induced 53BP1 foci were investigated (Laszlo and Fleischer 2009a). The heating of cells prior to or after irradiation leads to the delay of the formation of the radiation induced 53BP1containing foci. The length of the delay is heat-dose dependent, but radiation dose independent. The appearance of radiation-induced 53BP1 containing foci is independent of de novo protein synthesis in the heated irradiated cells indicating that the 53BP1 foci formed in a delayed manner contain preexisting 53BP1 molecules. The heat-induced delay of radiation-induced 53BP1 foci formation was observed in heated-irradiated hamster, mouse, and human cells; normal and transformed; and independent of p53 status (Laszlo and Fleischer, unpublished observations). The delay in 53BP1 foci formation in heated-irradiated cells was observed with five different anti-53BP1 antibodies, indicating that such delay was not due to heatinduced epitope masking (Laszlo and Fleischer 2009a). The heat-induced delay in 53BP1 complex formation was reversible, as indicated by irradiating at various times post heating. Cells X-irradiated 6–8 h after heat show no measurable delay in 53BP1 foci formation, indicating that the cells had recovered from the heat effects responsible for the delay. Interestingly, heating at various times post irradiation led to the “dispersal” of the 53BP1 foci induced the irradiation. Heat induced g-H2AX containing foci, also contain MDC1 (Hunt et al. 2007). Foci containing both of these proteins are a prerequisite for formation of foci containing 53BP1 after irradiation (Ward and Chen 2004; Adams and Carpenter 2006). Thus, either the heat-induced g-H2AX/MDC1 complexes are different from their radiation-induced counterparts, and/or heat alters the binding capacity of 53BP1 to this complex, leading to the lack of formation of 53BP1 foci in heated cells. Since transient heat-induced insolubilization and/or aggregation of 53BP1 associated with the heat-induced proteotoxicity would prevent it from being associated with the g-H2AX/MDC1 complex in heated-irradiated cells, we determined if heat altered the solubility of 53BP1. We performed in situ cell extractions with a buffer containing a nonionic detergent, followed by quantitative immunofluorescence (Laszlo and Fleischer 2009a). The following results were obtained. In control cells, ~ 50% of 53BP1 was insoluble; there is an increase in the insolubility of 53BP1 in

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irradiated cells, confirming the results of others that the 53BP1 foci are not soluble (Pryde et al. 2005). On the other hand, heat prevented the radiation-induced insolubilization of 53BP1. This result was consistent with the heat-induced delay in 53BP1 foci formation. Heat alone did not lead to the insolubilization of 53BP1. Thus, the heat-induced delay in the formation of 53BP1 foci in heated-irradiated cells was not associated with heat-induced aggregation of 53BP1 as measured by insolubility. It is conceivable that heat did lead to insolubilization of factors upstream from 53BP1 that are required for the formation of foci containing this protein (Huen et al. 2007; Kolas et al. 2007; Mailand et al. 2007). However, the observation that heat can disperse preexisting 53BP1 foci induced by X-irradiation argues against this possibility. Thus, failure to form 53BP1 foci after irradiation is likely due to an alteration in the protein complex required for its assembly. To ascertain if molecular chaperones can modulate the heat-associated delay in 53BP1 complex formation, we determined the response of thermotolerant (TT) cells. Thermotolerance is the transient ability of cells which have survived a prior heat shock to display increased resistance to subsequent heat shocks, and is associated with elevated expression of several molecular chaperones (Laszlo 1992a, b). Heat radiosensitization was attenuated in TT cells as measured by the TER. Recovery from the heat-induced delay in 53BP1 complex formation was attenuated in irradiated TT cells indicating that in the decrease of the TER in TT cells is associated with the attenuation of this delay. We also determined the delay in 53BP1 complex formation in two other cell systems with elevated expression of Hsc70, heat radiosensitization-resistant (HR-1) cells (Laszlo and Li 1985; Laszlo et al. 2006), and cells infected with Hsc70 adenovirus constructs (Newmyer and Schmid 2001). In HR-1 cells (Laszlo and Li 1985), there is increased expression of Hsc70 due to gene amplification (Chen et al. 1996); the decrease in TER was also associated with attenuation of the delay in 53BP1 complex formation. We found that there was a correlation between the magnitude of the delay in 53BP1 complex formation and TER (in control HA-1 cells, TT cells, and HR1 cells, with a linear correlation with R = 0.9986 and p < 0.001) (Laszlo and Fleischer 2009a). The impact of modulating the levels of functional Hsc70 on the heat-induced delay of 53BP1 complex formation was determined by comparing the effect of overexpressing wild type Hsc70 or a dominant negative mutant of Hsc70. The expression of the Hsc70-WT construct significantly decreased the heat-induced delay of 53BP1 complex formation in heated-irradiated cells, while expression of the dominant negative Hsc70D199S mutant considerably extended this delay. Thus, three different approaches indicated that Hsc70, an important molecular chaperone (Newmyer and Schmid 2001; Bukau et al. 2006), can modulate the heat-induced delay of 53BP1 complex formation in heated-irradiated cells (Laszlo and Fleischer 2009a). These results indicate that the heat-induced delay in the formation of 53BP1 complexes plays a role in heat radiosensitization and that the delay is modulated by molecular chaperones. Heat radiosensitization was determined in 53BP1 knock out MEFs. There was no significant difference in the heat sensitivity of 53BP1+/+ and 53BP1−/− MEFs, while there was a significant difference in their radiation sensitivity as previously

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reported (Ward et al. 2003; Riballo et al. 2004). Heat radiosensitization was significantly attenuated in 53BP1−/− MEFs compared with 53BP1+/+ MEFs (Laszlo and Fleischer 2009a). The attenuation of TER in 53BP1−/−MEFs suggests that 53BP1 plays a role in heat-radiosensitization. Since heat radiosensitization was still observed in the 53BP1−/− MEF albeit at a much lower TER, heat effects on other components of the cellular response to ionizing radiation-induced DNA DSB also play a role in heat radiosensitization. The 53BP1−/− MEFs are the first radiosensitive cell line studied that displays reduced heat radiosensitization, since that reported for ATM cells after acute hyperthermia (Mitchel et al. 1984). Knocking down or knocking out 53BP1 has indicated that several processes involved in the cellular response to ionizing radiation-induced DNA damage, including the phosphorylation of SMC1 and Chk2 and the formation of foci containing them, are events downstream from 53BP1 (DiTullio et al. 2002; Mochan et al. 2004; Adams and Carpenter 2006). Heat alone did not induce foci containing Ser 957 phosphorylated SMC1 (Hunt et al. 2007), while radiation induced a robust response. The formation of such foci was delayed in heated-irradiated cells. Similar results were obtained with the formation of foci containing Thr 68 phosphorylated Chk2. The heat-induced delay in these two processes downstream from 53BP1 in heated-irradiated cells paralleled the heat-induced delay in 53BP1 complex formation (Laszlo and Fleischer 2009a).

11.4 Masking of DNA Damage by Proteins Normally Not Associated with DNA Repair The “masking effect” is a reduction in accessibility to the site of DNA damage based on the observation that after hyperthermia, a fraction of DNA strand breaks is not detected by various assays unless certain protein(s) are removed. It is hypothesized that the masking effect involves the aggregation of proteins to the nuclear matrix or chromatin, thereby stabilizing these structures and masking DNA damage sites in the nuclear matrix associated DNA from DNA repair complexes. It has been reported that hyperthermia induces masking of DNA damage using the neutral comet assay (Vanderwaal et al. 2009), which measures DNA DSBs and the halo assay which measures single strand DNA damage (Kampinga et al. 1988; Wynstra et al. 1990; Laszlo et al. 2006; Vanderwaal et al. 2009). Further, the nucleolar protein, nucleophosmin (NPM) was identified as a major player in the masking of DNA damage (Vanderwaal et al. 2009). As will be discussed below, a link was found between these masking effects and radiosensitization, measured by clonogenic survival assays. The first evidence for the masking effect was that heat-induced changes in nuclear protein binding masked radiation induced DNA base damage (Warters and Roti Roti 1978) and DNA strand breaks (Clark et al. 1981; Mills and Meyn 1981). Further evidence that masking of DNA lesions is critical to radiosensitization is the correlation between unrepaired DNA strand breaks, cell killing, and the remaining

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enhanced nuclear protein binding with time between heating and irradiation (Mills and Meyn 1983), suggesting that enhanced nuclear protein binding could be related to a fraction of irreparable DNA damage. Studies to determine which part of the nucleus was affected by heat-induced enhanced nuclear protein binding and what DNA is being masked from repair pathways showed that the nuclear matrix was the site of binding of many proteins (Kampinga et al. 1989; VanderWaal et al. 1996). Masking of the nuclear matrix associated DNA or MAR DNA is associated with a reduction in DNA repair rate and an increase in residual DNA damage (Kampinga et al. 1988; Wynstra et al. 1990). Furthermore reduced masking of DNA damage was reported for variant cell lines that were resistant to heat radiosensitization, the reduction in masking correlating with a reduction in heat radiosensitization (Laszlo et al. 2006). Two isoforms of NPM were among those identified as proteins that become associated with the nuclear matrix and its associated DNA (VanderWaal and Roti Roti 2004).

11.4.1 Evidence from Neutral Comet Studies The neutral comet assay measures DNA DSBs. Vanderwaal et al. (2009) reported that following heat shock; the initial DNA damage detected was reduced by more than 50%, when the cells were treated with hyperthermia and 8 Gy ionizing radiation relative to cells exposed to radiation alone. However, when cells were treated with proteinase K during the lysis step of the neutral comet assay, the measured DNA damage in cells treated with or without hyperthermia plus ionizing radiation was the same. Thus, the hyperthermia treated cells did not experience an actual reduction in DNA DSBs. This observation is explained as hyperthermia induced a protein dependent “masking” of a fraction of the DNA DSBs. Hyperthermia treated cells also exhibited a reduction in DNA repair rates and 50% of the initial DNA damage remained after 2.5 h of repair, supporting the hypothesis that the heat induced alteration in chromatin structure which masks DNA damage also provides sufficient steric hindrance to limit DNA repair. To determine if NPM participated in this steric hindrance, the effect of NPM knockdown on hyperthermia masking of DNA damage and repair inhibition was measured. In cells treated with a 95% reduction in NPM, heat induced masking of DNA DSB was reduced by 40%, DNA repair rates were greatly enhanced, and the residual DSBs remaining after 2.5 h was 12%. Without hyperthermia, NPM knockdown had no affect on either the DNA DSBs measured by the neutral comet assay, the DNA DSB repair rates, or the residual unrepaired damage, showing that heat effects on NPM are contributing to these heat effects on DNA DSB repair. Previously, VanderWaal and Roti Roti (2004) showed that heat shock induces an increase in NPM associated with the nuclear matrix of HeLaS3 cells and increase its association with DNA. Moreover, VanderWaal et al. (2009) showed that NPM is induced by hyperthermia to relocate from the nucleoli to the nucleoplasm. Results from the chromatin immunoprecipitation (ChIP) assay showed that hyperthermia

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enhances the association of NPM with matrix associated regions (MAR) DNA sequences, i.e., those associated with the nuclear matrix DNA attachment regions. In contrast, there was no detectable association with exon or promoter sequences. These results are consistent with the conclusion that NPM plays a role in the masking effect and explain the results from the halo assay described below.

11.4.2 Evidence from DNA Supercoiling Studies (Halo Assay) The halo assay is a DNA supercoiling assay that depends on DNA-nuclear matrix anchoring (Wright et al. 2001). DNA in mammalian genomes are organized in loop domains which are anchored on either end to the nuclear matrix by MAR DNA. These loop domains are negatively supercoiled in living cells. The halo assay measures DNA supercoiling ability by increasing the concentration of the intercalating dye, propidium iodide (PI) in a lysis buffer. PI intercalating between the DNA base pairs forces an unwinding of the DNA loop domains. As the PI concentration increases, the DNA loops initially become fully relaxed. Under microscopic examination, a halo of DNA loops is seen to surround the nucleoids. At increasing higher PI concentrations, the loop domains are rewound in the opposite orientation, and again become supercoiled. Consequently, the halo of DNA loops diminishes in size. Single-strand DNA breaks induced by ionizing radiation inhibit the ability of the loops to rewind supercoils (Roti Roti and Wright 1987). The inability of DNA loops to be rewound is quantified as the difference in nucleoid diameter between rewinding in the absence versus the presence of DNA damage. This difference is called the excess halo diameter (EHD) (Roti Roti and Wright 1987; Wright et al. 2001). VanderWaal et al. (2009) observed that cells with normal NPM levels had a hyperthermia-induced reduction in the inhibition of DNA supercoil rewinding (i.e., masking of DNA damage) following irradiation. Specifically, after 10 Gy, the EHD was 45 mm in HeLa cell and 35 mm in HCC1806 breast cancer cells. In cells heated at 45°C for 30 min and irradiated with 10 Gy, these values were 10 and 5 mm, respectively. In contrast, in cells treated with a 95% NPM knockdown, hyperthermia had no statistically significant effect on DNA supercoil rewinding after irradiation. Thus, the masking of DNA damage, as detected by the halo assay, was essentially removed in both HeLa and HCC1806 cells with a 95% reduction in NPM protein levels. These data suggest that NPM plays a significant role in masking of DNA damage as detected by the halo assay.

11.4.3 Evidence that Masking of DNA Damage Increases Sensitivity to Ionizing Radiation The hyperthermia induced association of NPM with MAR DNA results in the masking of DNA damage and inhibits DNA repair. Therefore, it was important to

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determine its impact on heat-induced radiosensitization in terms of cell survival. As measured by the clonogenic survival assay, (VanderWaal et al. 2009) found that hyperthermia (30 min at 45°C) plus ionizing radiation greatly reduced clonogenic cell survival after 2, 4, and 6 Gy compared to radiation alone. However, a 95% reduction in NPM levels reduced heat-induced radiosensitization by ~40% in both HeLa and HCC1806 cells. The TER values were calculated as the ratio of radiation dose given without hyperthermia to the radiation dose given with hyperthermia, which produces the same survival value. For HeLaS3 and HCC1806 cells, TER values were calculated at the control survival level after 2, 4, and 6 Gy, which were chosen because these doses are commonly used in clinical radiation therapy and averaged. Hyperthermia significantly increased the cell’s sensitivity to X-rays, TER of 2.22 and 2.08 for HeLaS3 and HCC1806, respectively. A 95% reduction of NPM levels significantly lowered the TER to 1.74 and 1.39 in HeLaS3 and HCC1806 cells, respectively (Table 11.1). Thus, heat-induced radiosensitization is due, in part, to the presence of NPM. Without heat shock, NPM knockdown had no effect on survival after 2, 4, or 6 Gy. Thus NPM is a unique protein in that it plays a significant role in the radiation response and post irradiation cell survival in heated cells, but has no effect on the radiation response or cell survival in unheated cells.

Table 11.1 The impact of various proteins on radiation sensitivity and heat plus radiation sensitivity Target molecule Thermal dose/TER reduction DMF (A) TER reduction (and DMF) after siRNA knock-down of target molecule MRE11, 60–70% 2 h at 41°C/TER = 1.28 1.39 NSY Reduced to 1.00 NPM, 90–95% 30 min at 45°C/TER = 2.22 1.00 HeLa Reduced to 1.72 NPM, 90–95% 30 min at 45°C/TER = 1.89 1.00 NCC1806 Reduced to 1.57 (B) TER reduction (and DMF) in cells lacking target molecule or its function ATM (AT vs. normal fibroblasts) 30 min at 45°C/TER = 2.7 0.32 Reduced to 1.2 (Mitchel et al. 1984) 60 min at 43°C/TER = 2.29 53BP1, 100% MEF 1.91 Reduced to 1.42 30 min at 43°C/TER = 1.37 HSP70, 100% MEF 1.81 Increased to 1.55a Individual proteins are indicated in the left column. The impact of knocking down the protein (A) or its absence through mutation or knock out (B) on heat radiosensitization as measured by changes in the TER is shown in the middle column. The impact of knocking down the protein (A) or its absence through mutation or knock out (B) on radiation sensitivity as measured by the dose modifying factor (DMF) is shown in the right column. DMF is equal to the ratio of the dose with the wild type protein level divided by the dose with the protein reduced or absent to achieve the same level of cell killing with radiation alone a Reversed by HSP70 expression

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11.5 Summary of the Radiosensitization Resulting from Key Interactions Between the Proteotoxic and DNA Damage Stress Responses Throughout this chapter we have described several effects due to proteotoxic stress on different steps of the DNA damage response. The question becomes whether any or all of these effects contribute to the enhanced cell killing induced by the combination of heat and X or g-irradiation. To answer this question it is useful to look at an overview of how the absence of any of the proteins discussed above impacts the TER resulting from a given heat dose. As shown in Table 11.1, all of these proteins have an impact on heat-induced radiosensitization. Of the five proteins considered in Table 11.1, only NPM had no effect on radiation sensitivity in the absence of heat, consistent with the absence of a role for NPM in normal radiation resistance. Most of the proteins that have an impact on heat radiosensitization also play a role in normal radiation resistance. Interestingly, the absence of HSP70 increased the TER consistent with its role as molecular chaperon that reduces the proteotoxicity from a given heat shock. Thus, just as resistance to ionizing radiation depends on many different proteins, several proteins are involved in the resistance to heat plus radiation. It is also worth noting that of all of the proteins affecting the TER appear to be involved in the sensor or mediator parts of the DNA DSB response (Fig. 11.1), which is consistent with previous reports that mutations in the effector parts of the DNA damage response do not alter the TER (Kampinga et al. 2004).

11.6 Nuclear and Chromatin Structure Another underlying theme of these observations is that there appears to be a structural component to the intersection of the proteotoxic stress pathway and the DNA DSB response pathway. For example, g-H2AX has been implicated in tethering together broken chromosome ends through long-range synapsis at the switch regions (Adams and Carpenter 2006; Stavnezer et al. 2008), leading to the proposal that g-H2AX participates in an end rejoining process that is distinct from classical NHEJ, called “anchoring” (Manis et al. 2004; Posey et al. 2004). 53BP1 has been proposed to have a similar anchoring function (Manis et al. 2004; Posey et al. 2004). In this capacity, both g-H2AX and 53BP1 facilitate the repair of DNA DSB at broken chromosomes by anchoring DNA ends in preparation for end processing and ligation by the NHEJ machinery (Bassing and Alt 2004; Manis et al. 2004; Posey et al. 2004; Stavnezer et al. 2008). We hypothesized that heat may interfere with the putative “anchoring” function of 53BP1, perhaps by affecting chromatin mobility due to the increased association of denatured proteins with chromatin in heated cells (Laszlo 1992a, b; Roti Roti et al. 1998), leading to heat-radiosensitization (Laszlo and Fleischer 2009a). Recently, it has been demonstrated that 53BP1 promotes the synapsis of DNA ends that are far from each other by increasing local

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chromatin mobility (Dimitrova et al. 2008; Difilippantonio et al. 2008; Huen and Chen 2008). Increased chromatin mobility could be impaired in heated cells, as implied by classic DNA supercoiling studies (Kampinga et al. 1988) and nuclear matrix rigidity assays (Wright et al. 1989). Key in this consideration is the observation that heat-induced protein (including NPM) binding to the nuclear matrix inhibits the ability of nuclear matrix-MAR DNA binding proteins (including PDI) to relax the tightness of his anchoring. Thus, a common theme with respect to heat radiosensitization is a structural alteration due to aggregation of unfolded proteins resulting in a reduction of chromatin fluidity required for the ability to assemble DNA repair complexes and their ability to manipulate damaged DNA ends.

11.7 Targeting the Intersection of the Proteotoxic and DNA Damage Stress Response Pathways to Improve the TER at Clinically Achievable Temperatures Phase III clinical studies have established that radiation therapy combined with adjuvant therapeutic hyperthermia can produce significant local control in disease refractory to ionizing radiation (Horsman and Overgaard 2007). However, for many cancers, suboptimal thermal doses limit radiosensitization. Thus, agents designed to enhance hyperthermia have the potential to improve radiotherapy outcomes. Consideration of the mechanisms by which heat shock induces radiosensitization offers an opportunity for innovative chemistry-driven drug discovery of small molecules that convert subtherapeutic hyperthermic exposures into efficacious thermal doses that yield robust radiosensitization. The foremost molecular event governing thermal radiosensitization is the unfolding and aggregation of a subset of cellular proteins (Lepock 2005). Heat shock-mediated alterations of the biophysical properties of irradiated chromatin, temperature-dependent inhibition of DNA DSB ligation (Kampinga and Dikomey 2001), and the perturbation of signal transduction pathways are mainly a consequence of protein unfolding and aggregation (Laszlo and Fleischer 2009a). Thus, thermal destabilization of protein conformation represents an underlying biophysical process that can be exploited in the design of agents to enhance thermal radiosensitization. Indomethacin is a nonsteroidal anti-inflammatory drug shown to lower the temperature required for initiation of the heat shock response (Roussou et al. 2000; Locke et al. 2002) and the threshold temperature for hyperthermic radiosensitization (Locke et al. 2002). Thus, the N-benzoyl indole moiety of the indomethacin molecule represented a structural platform upon which a chemistry-driven synthesis approach could be initiated (Sekhar et al. 2007). A series of novel indole-N-substituted IMZO3-ols, their corresponding 3-keto analogs, and other structurally related non-indolic compounds were synthesized and were structurally characterized by 1H and 13C nuclear magnetic resonance spectroscopy, gas chromatography–mass spectroscopy analysis, and elemental combustion analysis. X-ray analysis was used to identify the

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molecular geometry, conformation, stereochemistry, and atom connectivity of two representative analogs (Sekhar et al. 2007; Sonar et al. 2007). HT29 colon carcinoma cells, which are intrinsically resistant to heat shock (Xu et al. 2002), were used as a model in a comparative structure-activity analysis of drug development. Treatment with the representative analogs led to a lowering of the threshold for radiosensitization induced by a given heat exposure. For example, a 41°C heat shock did not induce radiosensitization; however, when treated with the analog, the 41°C heat shock produced a TER of 3. Therefore, we call these analogs as thermal enhancers. As reviewed above, moderate hyperthermia capable of producing radiosensitization induces a fraction of nuclear Mre11 to become associated with the cytosol, which is correlated with the TER, indicating that this parameter can be used as a molecular signature of heat radiosensitization. FRAP experiments with a GFP tagged Mre11 protein, indicated that treatment with the heat enhancer at 41°C led to an immobilization of GFP-Mre11 similar to that achieved by treatment at 43°C alone (Sekhar et al. 2007; Sonar et al. 2007). Proteotoxicity induced by heat leads to protein destabilization which, in turn, leads to the acquisition of DNA binding ability of the heat shock transcription factor (HSF). The minimum temperature for this activation of DNA binding activity was found to be the same as the minimum temperature that caused cellular proteins with intrinsically low conformational stability to unfold and denature (Senisterra et al. 1997). Indeed, treatment with the heat enhancers significantly increased the activation of HSF at 41°C and led to an increased synthesis of hsp70. Treatment with the heat enhancers led to a loss of the mitochondrial membrane potential and also led to cell death by mitotic catastrophe (Sekhar et al. 2007). In addition, these agents enhance heat inhibition of the radiation induced 53BP1 response. For example, treatment with the heat enhancer at 41°C produced a significant delay in 53BP1 focus formation after X-irradiation; whereas 41°C without the enhancing agent had no affect on 53BP1 focus formation (Sekhar et al. 2007). In summary, the synthesis of these small molecules represents a promising tool for the clinical application of therapeutic hyperthermia as a radiation sensitizer for the treatment of recurrent tumors. As the small molecule-thermal enhancing agents increase multiple thermal effects it may be hypothesized that these agents have the potential to enhance additional cancer therapies augmented by hyperthermia, such as chemotherapy and immunotherapy (Coffey et al. 2006).

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Chapter 12

Radiation-Induced Immune Modulation Charles G. Drake

Abstract Ionizing radiation is commonly used to treat cancer, with the explicit goal of eliminating an in situ tumor through reasonably well-understood direct killing mechanisms. However, the tumors that afflict patients do not evolve on plastic dishes or in immune-compromised mice. Instead, tumors develop in hosts with an intact immune system, which may serve to edit and shape the immunological phenotype of a particular lesion. Radiotherapeutic treatment of a mature tumor in an intact host results in immunologically relevant phenotypic changes in the targeted tumor cells themselves, and also has effects on the multitude of other cells that make up the tumor-associated stroma. Although many of these changes are both poorly investigated and poorly understood, recent studies have provided a new level of insight into the immunological effects of radiation, both at a local, molecular level, as well as at a systemic, whole-body level. Many of the “off-target” effects of radiation therapy might be expected to be proinflammatory, but harnessing these effects in a synergistic treatment regimen will undoubtedly require a greater knowledge and appreciation of the manner in which ionizing radiation interacts with the host. Keywords Immunotherapy • Vaccine • Lymphocyte • CD4 • CD8 • Fas • TGF-b • IL-6

12.1 The Abscopal Effect An out-of-field effect for therapeutic radiotherapy was first described by RH Mole, who in 1953 suggested that systemic effects could result from localized treatment (Mole 1953). He termed such effects “abscopal,” i.e., off-target; such C.G. Drake (*) Johns Hopkins Kimmel Cancer Center, 1650 Orleans Street, CRB 410, Baltimore, MD 21231, USA e-mail: [email protected] T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1_12, © Springer Science+Business Media, LLC 2011

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effects are purported to occur when therapeutic irradiation to a target lesion results in a regression of a second out-of-field mass or metastasis. The relevance of such effects to clinical radiation therapy are controversial (Kaminski et al. 2005) – indeed documented instances of clinical responses best explained by an abscopal effect exist primarily at the level of case reports in renal cell carcinoma (Wersall et al. 2006), melanoma (Kingsley 1975), hepatocellular carcinoma (Ohba et al. 1998), and others (Kaminski et al. 2005). A handful of animal studies modeling the abscopal effect have also been reported, but as discussed below, these experiments differ from the clinically reported cases in several important ways. Nevertheless, a brief review of this literature provides a window through which to begin to examine the immunological effects of radiation.

12.1.1 Immunological Mechanisms Underlying the Abscopal Effect Although the notion that the abscopal effect is a systemic one long suggested the involvement of immunological or hormonal mechanisms, direct evidence for the involvement of the immune system first came through fairly recent work by the Formenti group (Demaria et al. 2004). These studies utilized mice implanted with the breast cancer cell line 67NR, and showed that an abscopal effect could be achieved when local irradiation was applied in concert with Fms-like tyrosine kinase receptor 3 (FLT-3) ligand; an immunomodulator that enhances the production of dendritic cells. These studies provided convincing evidence for an abscopal effect – in that a growth delay of a secondary, out of field mass was reliably observed. This effect was tumor specific, i.e., a secondary mass of A20 lymphoma cells showed no evidence of growth inhibition when a primary mass of 67NR cells was irradiated. Evidence that this effect was immune mediated was twofold: (1) The effect did not occur in animals lacking T cells (athymic nude mice). (2) FLT-3 ligand was essential for out-of-field activity of the treatment regimen. This requirement for an additional immunostimulatory agent might begin to explain why abscopal effects are less commonly observed in a clinical setting, where multiple mechanisms serve to inhibit an antitumor immune response (Drake et al. 2006). In a second series of experiments (Demaria et al. 2005), this group utilized a more common breast cancer line (4T1), which is known to be poorly immunogenic, in that it does not respond to vaccination or single agent immunotherapy. Similar to the previous studies, 4T1 breast cancer cells were injected into the flanks of syngeneic mice, in this case inducing metastatic lesions in the lungs of inoculated mice. Treatment of the primary tumor with radiotherapy resulted in a growth delay of the target lesion, but this was not accompanied by a concomitant increase in overall survival. To enhance the immunological effects of radiotherapy, the group employed a monoclonal antibody that blocks CTLA-4 (an immune checkpoint molecule on the cell surface of CD4 and CD8 T cells that renders such cells relatively nonfunctional) (Korman et al. 2006). Like radiotherapy, anti-CTLA-4 alone had no significant effect on survival, consistent with the nonimmunogenic nature of 4T1 tumors.

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However, when the two treatments were combined, a significant survival increase was observed. This therapeutic effect could be attributed to a decreased incidence of metastatic deposits in the lungs of mice treated with the combination therapy, suggesting an action at a distance, i.e., an in vivo abscopal effect. Further mechanistic studies showed that CD8, but not CD4 T cells were required. Similar to the previous studies (Demaria et al. 2004), the notion that an additional immunomodulation was required to support an abscopal effect is consistent with the notion that evolving tumors appear to induce an immunologically suppressive microenvironment as discussed above.

12.1.2 Nonimmunological Mechanisms Underlying the Abscopal Effect In contrast to the above models, in which radiotherapy alone was insufficient to induce an abscopal effect, the Lewis lung carcinoma (LLC) model shows a significant off-target effect when treated with radiation therapy alone. In an interesting series of studies involving this model, Camphausen et al. (2003) provided an alternative explanation for an observed abscopal effect. LLC were implanted in the flanks of mice, and the mice (but not the tumors themselves) were subjected to a significant (5 × 10 Gy) radiation dose. A delay in the growth of LLC cells implanted at a distant site (the dorsum) could be observed. Interestingly, in this case, the abscopal effect was not tumor specific, when an unrelated tumor (T241 fibrosarcoma) was implanted in the dorsum; a delay in the growth of those masses could also be induced by irradiating the mice. In host animals that lacked p53, an abscopal effect was not observed, consistent with additional studies in which p53 function was pharmacologically inhibited. Although the mechanism involved in these studies was not further elucidated, the authors were not able to completely rule out an immune effect, as the studies were not extended to include animals with either a general or specific immune deficiency. In addition, high-dose irradiation (as in the doses utilized here), has been suggested to release so-called danger signals (McBride et al. 2004), which potentiate immune activation, and which could conceivably be deficient or otherwise attenuated in p53 genetic knockout animals (Murakami et al. 2004).

12.1.3 Counterpoint: Acceleration of Metastatic Disease by Local Irradiation Despite the above data supporting a distant, growth-inhibiting effect of local irradiation, significant data suggest that the opposite effect may occur in some systems, i.e., local irradiation may result in the outgrowth of distant metastases. In one relevant study (Camphausen et al. 2001), mice bearing LLC cells were treated with clinically effective doses of irradiation, effectively eradicating the primary mass.

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The growth of metastatic lesions in the lungs of these mice were not mitigated by an immune-mediated abscopal effect – in fact, lung lesions grew more efficaciously in animals with a treated primary tumor. Earlier studies suggested a mechanism for these results, in that these effects could involve production of antiangiogenic factors by the primary tumor (O’Reilly et al. 1994), whose production was mitigated by efficacious radiotherapy of the primary mass. Some clinical studies corroborate these data; a recent study (Scheer et al. 2008) showed that indolent liver metastases appeared to increase their metabolic activity after surgical removal of the primary mass. Taken together, these data highlight the notion that an immune-mediated abscopal effect can occur, but is only rarely documented in patients treated with radiotherapy. Animal studies suggest two possible explanations for this result: (1) the immunosuppressive local and systemic environment in a cancer-bearing patient may inhibit the induction of a radiation-induced antitumor response, and (2) in some cases, decreased production of antiangiogenic or other tumor inhibiting substances by a treated primary tumor may paradoxically contribute to outgrowth of metastatic lesions.

12.2 Local Immunological Effects of Radiation Despite the complex and potentially counterbalanced clinical effects of local radiotherapy on distant metastatic sites, it has become increasingly clear that radiation of primary lesion induces a number of local changes in both tumor and surrounding stromal cells, and that, such effects may have profound immunological significance (Table 12.1).

12.2.1 Effects of Radiation on Tumor Cells Several studies show that in vitro (Chakraborty et al. 2003) and in vivo (Chakraborty et al. 2004) radiation of tumor cells results in phenotypic changes rendering them more susceptible to immune-mediate killing. The initial studies in this regard Table 12.1 Summary of selected immunological effects of radiation Description Upregulate FAS (death receptor) on tumor cell surface Upregulate MHC Class I on tumor cell surface Alter repertoire of peptides presented in Class I MHC Translocate calreticulin to tumor cell surface, resulting in uptake by antigen presenting cells Release high mobility group box 1 (HMGB1) from dying tumor cells, resulting in dendritic cell maturation

References Chakraborty et al. (2003) Reits et al. (2006) Reits et al. (2006) Obeid et al. (2007) Apetoh et al. (2007)

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involved flow cytometric analysis of murine MC38 adenocarcinoma cells irradiated in vitro, and revealed that radiation results in upregulation of the cell surface molecule Fas, which mediates apoptosis when properly engaged by its ligand (FasL) on cytotoxic T cells. This upregulation of Fas was accompanied by a modest upregulation of the cell surface molecule ICAM-1, a protein involved in lymphocyte adhesion. Interestingly, Class-I MHC, which is required for direct and specific lysis of target cells by cytotoxic CD8 lymphocytes, was not found to be upregulated by increasing radiation doses in this study. To interrogate CTL killing, the authors employed a variant of MC38 that stably expresses carcinoembryonic antigen (CEA), as well as cytotoxic CD8 T cells specific for CEA. Radiation-induced upregulation of Fas appeared to have a functional role, rendering MC38-CEA tumor cells more sensitive to in vitro and in vivo lysis by specific CD8 T cells (Chakraborty et al. 2003). Further work extended these results into more challenging in vivo models (Chakraborty et al. 2004), showing that vaccinia-based tumor vaccines directed against CEA were able to mediate an antitumor effect in mice with implanted MC38-CEA tumors when the mice were treated with radiation therapy, but not in the absence of radiation therapy. Additional evidence for the Fas-dependence of increased sensitivity of irradiated tumors to CTL-mediated lysis came from studies involving tumor cells with a defect in Fas signaling; these tumors did not appear to be preferentially lysed by cytotoxic T cells postradiation. Relevant in vitro studies involving a human melanoma cell line (MelJuSo) reached a slightly different conclusion; here, radiation did appear to increase levels of Class I MHC molecules, providing an alternative mechanism for a potential increase in CD8 T cell recognition and killing post-radiotherapy (Reits et al. 2006). To further elucidate a potential radiation-induced CD8 T cell response, Class I MHC molecules were purified from both control and irradiated melanoma cells, peptides were eluted, fractionated by phase HPLC and analyzed using mass spectrometry. The MHCassociated peptide repertoire of irradiated cells was different from nonirradiated controls, but perhaps most interestingly, a CD8 T cell response to several of these novel peptides could be detected in the peripheral blood of healthy control patients, confirming the immunogenic nature of the radiation-induced peptide repertoire changes. Taken together, these three studies highlight the notion that radiation may induce proimmunogenic changes on tumor cells, but emphasize the observation that the changes involved might be specific to a given tumor type and patient.

12.2.2 Effects on the Tumor-Associated Vasculature Another interesting in vivo study provides an alternative explanation for the potential immunomodulatory effects of radiotherapy (Ganss et al. 2002). In this work, the authors used a transgenic mouse line that spontaneously develops aggressive insulinomas, via tissue-specific expression of the SV40 large T antigen. Adoptive transfer of tumor-specific CD4 T cells is ineffective at retarding tumor growth in this model, presumably because these cells are rendered tolerant by the immunosuppressive

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tumor microenvironment. Surprisingly, a combination of low-dose radiotherapy and adoptive T cell therapy resulted in a significant antitumor effect, clearly reflected in an improved survival. In this model, the immunomodulatory effects of radiotherapy appeared to be mediated by changes in the tumor micro-vasculature, with a reduction in ongoing angiogenesis, and a resultant migration of specific CD4 T cells into the tumor bed (Ganss et al. 2002). These data are important, because they show that irradiation can have enabling effects on the stromal cells of a tumor mass along with direct proinflammatory effects on the tumor cells themselves.

12.2.3 Effects on Local Immune Cells The tumor stroma is not immunologically bland; indeed many types of tumors are relatively infiltrated with a combination of immune cells, including macrophages, mast cells, and various lymphocyte populations (de Visser et al. 2006; Balkwill et al. 2005). Recent data show that these cells may play a role in the genesis of tumors which arise within context of chronic inflammation. In particular, macrophages appear to be critical in the smoldering inflammation more commonly associated with tumorigenesis, although several recent studies have shown a convincing and direct role for B cells (Ammirante et al. 2010; de Visser et al. 2005). In this light, it is perhaps somewhat surprising that few studies have set out to comprehensively characterize the lymphocyte populations that exist in a tumor bed prior to versus post-radiotherapy. Clinical studies in this regard would be challenging, based on the difficulties inherent in obtaining posttreatment biopsy material from patients, but no such limitations would be expected in animal studies, particularly those involving physiologically relevant, transgenic tumors.

12.3 Systemic Immunological Effects of Radiation As discussed above, some experimental and clinical data confirm the existence of systemic immunological effects mediated by local tumor irradiation. The majority of the data in this regard involve circulating cytokine levels, which are relatively straightforward to quantify using either standard enzyme linked immunosorbent assay (ELISA), or more modern microbead based methods, which permit simultaneous quantification of up to 29 cytokines using a small serum sample. The obvious limitation to such studies stems from the fact that many cytokines act in a primarily localized manner, thus systemic levels might not accurately reflect the physiological environment in the treated tumor bed. In addition to these cytokine effects, the immunological effects of radiation may be mediated by changes in the tumor burden, as well as by several more recently recognized mechanisms involving the manner in which dying tumor cells interact with immune cells.

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12.3.1 Effects of Radiation on Cytokine Levels Cytokine levels in patients treated with therapeutic ionizing radiation have primarily been investigated in the context of the hypothesis that levels of these factors can be used as biomarkers of radiation related toxicity or less commonly, of response (Madani et al. 2007; Okunieff et al. 2008). This hypothesis neglects to some degree the profound systemic immunological effects of these cytokines. Interleukin-1 (IL-1) for example, has been shown to augment metastasis in several murine systems (Anasagasti et al. 1997); this may occur through IL-1 mediated upregulation of adhesion molecules on vascular endothelial cells (Lauri et al. 1991). In patients undergoing thoracic radiation, pretreatment IL-1a levels appeared to predict radiation pneumonitis, with patients having higher levels showing a greater tendency toward pneumonitis (Chen et al. 2002). In this small study (24 patients), IL-1a levels did not appear to decrease significantly during treatment, suggesting that changes in circulating levels of IL-1a are perhaps unlikely to mediate systemic immunological effects of radiation (Chen et al. 2002). Still, IL-1 tends to be produced locally, both by tumor cells and by associated macrophages, and it is therefore curious as to why therapeutic radiotherapy failed to affect systemic levels significantly. In terms of radiation pneumonitis, it seems reasonable to hypothesize that IL-1 levels could correlate with a higher baseline level of tumor inflammation, which might predispose to a greater likelihood of postirradiation pneumonitis. Immunohistochemical (IHC) analysis of tumor biopsies would represent one possible methodology by which to test for such an association. Interleukin 6 (IL-6) serum levels have been associated both with tumor outcome and tumor grade in multiple types of cancer (Trikha et al. 2003). IL-6 can be produced by the tumor cells themselves, as well as by tumor-associated macrophages and stromal cells. From an immunological perspective, IL-6 skews CD4 T cells towards a type of cell that produces interleukin 17 (TH17) (Weaver et al. 2006), and which has been associated with both chronic inflammation and tumorigenesis (Kortylewski et al. 2009). In early clinical trials, monoclonal antibodies blocking IL-6 decreased rates of cancer-associated anorexia and cachexia, providing evidence for the pleiotropic effects of this cytokine. If the systemic effects of radiation therapy were mediated by IL-6, then one might expect to be able to correlate outcome in treated patients with a subsequent decrease in circulating levels of IL-6 (Chen et al. 2002). In fact, within the limited context of the few studies performed, ionizing radiation appeared to lead to an increase, rather than a decrease in circulating IL-6 levels, suggesting that the distant effects of radiation therapy may not be mediated by decreased IL-6 levels. However, long-term follow-up of these patients was relatively limited, and it is possible that it could take significant time to “normalize” serum cytokine levels. The cytokine perhaps best associated with radiation-induced pneumonitis is transforming growth factor beta (TGF-b); indeed multiple independent studies showed a correlation between systemic TGF-b levels and radiation induced toxicity (Madani et al. 2007). This association most likely occurs via a role for TGF-b as a “master switch” in the development, and in the persistence of fibrosis (Martin et al. 2000). From an immunological perspective, TGF-b is generally considered to be profoundly immunosuppressive, as it negatively regulates the function of both

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adaptive (T-cell mediated) as well as innate responses (Letterio and Roberts 1998). In this regard it is relevant that, unlike IL-1 and/or IL-6, several studies showed a decrease in TGF-b as a result of therapeutic radiotherapy. To summarize, serum cytokine levels in patients undergoing radiotherapy have been analyzed in a small number of studies. These studies, initiated to test the hypothesis that cytokine levels can predict radiation induced toxicity, were not designed to gather cytokine data in a manner useful for predicting whether distant effects of radiation therapy could be cytokine mediated. Of the several relevant cytokines analyzed, only TGF-b seemed to decrease during radiotherapy of a primary tumor, and thus might represent a systemic factor that mediates immunological effects of radiotherapy. However, more comprehensive time-course analysis of TGF-b in patients with relatively immunologically sensitive tumors (melanoma and renal cell carcinoma) undergoing radiotherapy would be required to more accurately explore its potential role in that context.

12.3.2 Effects on Tumor Burden It has long been appreciated that persistent levels of a particular antigen are associated with T cell tolerance (Ramsdell and Fowlkes 1992), providing one of many possible mechanisms by which tumors escape immunological recognition in intact hosts. Indeed carefully performed animal studies confirmed this notion, showing that tumor-specific CD8 T cells were nonfunctional in tumor-bearing animals, but regained lytic function when transferred into naïve, nontumor bearing hosts (den Boer et al. 2004). Complete removal of the primary tumor may not be necessary for these effects, as demonstrated by studies involving mice with metastatic breast cancer (Danna et al. 2004). In these studies, mice with both localized and metastatic 4T1 breast cancers were treated via surgical removal of the primary tumor. This treatment resulted in a restoration of adaptive immune response (antibody and T cell-mediated), despite the persistence of metastatic disease. These results reflect clinical findings in patients with metastatic renal cell carcinoma (Flanigan et al. 2004), who have a survival advantage when treated with cytoreductive surgery in combination with immunotherapy as opposed to immunotherapy alone. Since clinically efficacious radiotherapy results in a reduction of tumor burden, some of the systemic immunological effects related to treatment might be expected to occur as a result; however, this has yet to be examined in a systematic manner.

12.3.3 Mechanisms Underlying Immune Stimulatory Effects of Radiation Radiation-induced tumor cell death is presumed to occur through apoptotic mechanisms, which, in the past were considered to be immunologically silent (Sauter et al. 2000). However, more recent data cast doubts on this broad generalization, and show

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that additional factors need to be considered in determining the immunological outcome of cancer cell death (Zitvogel et al. 2008). Ionizing radiation, in addition to certain chemotherapy agents, induces the translocation of the chaperone protein calreticulin from the endoplasmic reticulum to the cell membrane of the cancer cell, where its presence facilitates the uptake of the dying cancer cell by antigen presenting cells known as dendritic cells. As these cells are critical in the initiation of an immune response, this process can facilitate the immune recognition of dying tumor cells (Obeid et al. 2007). However, engulfment of dying tumor cells by dendritic cells is not sufficient to stimulate an antitumor immune response. An additional step, dendritic cell maturation, is generally required for full antigen presentation. Exogenous stimuli involved in dendritic cell activation are well known, and include bacterial products such as lipopolysaccharide (LPS) and toll like receptor (TLR) agonists. Important recent studies showed that tumor cell death can release a chromatin binding protein known as high-mobility group box 1 protein (HMGB1), which binds to a TLR (TLR4) on antigen presenting cells, facilitating their maturation, as well as the trafficking of engulfed antigens into pathways which result in efficient cell surface presentation to CD4 and CD8 T cells (Apetoh et al. 2007). Taken together, these data highlight several major mechanisms by which ionizing radiation can prime an antigen-specific antitumor immune response, and suggest that future studies examining the relationship of dose and scheduling of radiotherapy to release of calreticulin and HMGB1 could result in a more complete understanding of the relationship between radiotherapy and immunological recognition of tumors.

12.4 Evidence for Immunological Effects of Radiotherapy in Humans As only a handful of specific tumor antigens have been identified and characterized (Cheever et al. 2009), it is not surprising that very few studies have explicitly asked whether therapeutic radiotherapy can induce a detectable immune response to a tumor-associated antigen. Two recent studies are notable in this regard. In the first, the authors used an open-ended approach to identify new antibody specificities in men with prostate cancer undergoing treatment with standard therapy (including radiation therapy) (Nesslinger et al. 2007). The methodology employed SEREX (serological identification of antigens by recombinant cDNA expression cloning), which involves screening patient sera against plasmids expressing a variety of proteins. Interestingly, new treatment-related antibodies were detected in 14% of patients undergoing radiotherapy, as compared with only 6% of controls. These results were supported by studies in a murine tumor model, where androgen withdrawal also induced new antibody specificities. Open-ended analysis of T cell responses is far more challenging, since T cells recognize antigens as short peptides presented in the context of a specific MHC molecule. Nevertheless, in patients with a known MHC genotype, antigen-specific CD8 T cells can be detected through tetramer technology (Altman and Davis 2003), which fluorescently marks a specific CD8 T cell receptor

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via a construct combining the proper MHC and peptide in a covalent linkage. Thus, tetramers for a particular tumor-associated antigen can be used to quantify tumorspecific CD8 T cells in patients with a known MHC haplotype. To determine whether radiotherapy induced new tumor-specific CD8 T cells, Shaue et al. (2008) used tetramers specific for the tumor antigen survivin. Survivin-specific CD8 T cells could be detected in the periphery of some prostate cancer and colorectal cancer patients. In a few of these patients, an increase in the frequency of survivin-specific CD8 T cells could be detected postradiotherapy, consistent with the notion that radiotherapy augments a tumor-specific CD8 T cell response. As additional tumor antigens are characterized both at the protein and the T cell epitope level, it seems likely that additional, larger studies will be capable of more precisely quantifying a radiation induced increase in the T cell immune response to tumor antigens.

12.5 Clinical Studies Combining Immunotherapy with Radiotherapy With several exceptions, the aggregate data presented above support the notion that therapeutic ionizing radiation may serve to augment an antitumor immune response. This notion has been tested in a small number of clinical trials, involving cervical cancer, hepatoma and prostate cancer (Table 12.2). In an early study, Okawa et al. combined radiotherapy for carcinoma of the uterine cervix with the immune adjuvant LC9018 (a bacterial extract) (Okawa et al. 1989). This was a randomized, Phase II trial, with women assigned to either 15 or 30 Gy radiation, with or without the immune adjuvant. Although the number of patients enrolled was relatively small (49 total), an increase in the objective response rate was reported for patients receiving combination immunoradiotherapy. Another small trial enrolled patients with hepatoma, treating them with 8 Gy of radiation followed by an intratumoral injection of autologous immature dendritic cells 2 days later (Chi et al. 2005). Although only 14 patients were treated in this Phase I trial, the authors noted four minor responses and two partial responses. Specific antitumor immune responses were noted, in the form of a T cell response to a-fetoprotein detected by ELISPOT. In addition, the combined treatment appeared to be well-tolerated and without significant adverse effects. Another related trial focused on men with localized prostate cancer, randomizing 30 patients to receive either a vaccinia-based PSA-directed vaccine plus standard Table 12.2 Clinical trials combining radiotherapy with immunotherapy Description Disease Patients Uterine cervical 24 Randomized Phase II trial combining cancer bacterial immune response modifier with radiotherapy Phase I trial combining intratumoral dendritic Hepatoma 12 cell vaccination with radiotherapy Prostate cancer 30 Randomized Phase II trial combining vaccinia-based prostate cancer vaccine with radiotherapy

References Okawa et al. (1989) Chi et al. (2005) Gulley et al. (2005)

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radiotherapy or radiotherapy alone (Gulley et al. 2005). Randomization was performed on a 2:1 basis, such that 19 men received combination treatment, and 11 received radiotherapy alone. In the radiation alone arm, no significant increases in PSA reactive T cell levels were noted, whereas an increase in T cell reactivity to PSA was detected in 13 out of 17 evaluable patients in the combination treatment arm. Perhaps more interesting, responses to additional prostate-related antigens were noted in the combination treatment arm, supporting the notion that combination treatment induced the phenomena known as antigen-spreading, i.e., a broadening of the immune response to include additional tumor and/or tissue-related antigens. A Phase III study testing the combination of low-dose, “priming” radiotherapy in combination with CTLA-4 blockade using the monoclonal antibody Ipilimumab (BMS) is ongoing; in this study men with castrate resistant, metastatic prostate cancer who have progressed on docetaxel are randomized to radiotherapy +/− immunotherapy. Taken together, this group of trials supports the general concept that radiotherapy may have immune-augmenting systemic properties in cancer patients. However, it is remarkable that the number of combination trials initiated has been relatively small, especially given the abundant preclinical data showing radiotherapy has systemic immunological effects in multiple tumor types.

12.6 Conclusion Ionizing radiotherapy used in cancer therapy has profound effects on tumor cells as well as on the tumor microenvironment – these effects are only beginning to be understood in depth. These phenomena influence an ongoing and potential immune response to cancer in a number of ways, including changes in the tumor cell surface molecules, both local and systemic cytokine levels, in the magnitude of a tolerogenic tumor burden, and in the way in which tumor antigens are processed and presented to the immune system. These changes provide a unique opportunity for immune therapies to synergize with conventional radiotherapy. However, the challenges in this regard are formidable, as we are only beginning to understand the complex interactions between cancer and the immune system. As the field moves forward, a greater level of basic science understanding, as well as careful clinical studies titrating the dose and fractionation of radiotherapy, will be critical in exploiting this knowledge for translational benefit.

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Index

A AACOCF3. See Arachidonyltrifluoromethyl ketone Abscopal effect description, 251–252 immunological mechanisms, 252–253 metastatic disease, 253–254 non-immunological mechanisms, 253 Adenovirus flu-like symptoms and transaminitis, 175 HSV–1 TK, 176 iNOS gene transfer, 180 p53 gene, 177 replication-competent oncolytic, 178–179 TNFa gene therapy, 177–178 Apoptosis cell cycle arrest, 108 CEP–1/p53-mediated, 110 defective mutants, 108 DNA damage-induced, 115–116 g-H2AX markers, 13 ionizing irradiation, 107 p53-dependent, 112 radiation-induced, 109 Arachidonyltrifluoromethyl ketone (AACOCF3) cPLA2 inhibitors, 192–194 description, 191–192 Ataxia-telangiectasia-mutated (ATM) activation BRCA1 recruitment, 56 H2AX phosphorylation, 55 MRN complex, 54–55 ubiquitylation, 55–56 DNA damage signaling ATR activation, 37–39 BRCT-containing proteins (see BRCA1 C-terminal)

checkpoint kinases (see Checkpoint kinases) DSBs response, 43–44 human PIKKs substrates (see Phosphatidylinositol 3-kinase-related kinases) kinase activity, DSB, 35 p53 activation, 46–47 two-tiered kinase cascade, human cells, 36 loss of, 157 PI3 kinase family, 130 repairing DNA, 161 signal, 131–132 Ataxia telangiectasia mutated-and Rad3-related (ATR) activation, 38 ATM and DSBs, 43 substrate specificity, 44 ATM-Chk2 pathway, 41 ATR-interacting protein (ATRIP), 39 Chk1/Chk2 genes, 37 deficient cells, 43 description, 37 Fanconi anemia pathway, 48 functional diversity, 48 hypomorphic mutations, 46 physical interaction, 40–41 RT and, 157 stalled replication forks, 37 ATM. See Ataxia-telangiectasia-mutated ATM and ATR kinases Ataxia telangiectasia gene codes, 24 MDC1-MRN complex, 17 PIKKs, 16

T.L. DeWeese and M. Laiho (eds.), Molecular Determinants of Radiation Response, Current Cancer Research, DOI 10.1007/978-1-4419-8044-1, © Springer Science+Business Media, LLC 2011

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266 B Base excision repair (BER), 149 Bloom’s syndrome protein, 147–148 53BP1. See p53-binding protein 1 BRCA1. See Breast cancer type 1 BRCA1 and BRCA2 gene AZD2281, PARP inhibitor, 161 germline mutations/deletions, 160–161 HR repair, 160 p53 gene, 161 BRCA1 c-terminal (BRCT) 53BP1, 45 C-termini function, 44 description, 44 DNA damage response, 46 MCPH1/BRIT1, 46 MDC1/NFBD1, 46 Breast cancer IL–6, 257 metastatic, 258 4T1, 252 Breast cancer type 1 (BRCA1), 16–17 C Caenorhabditis elegans, radiation responses divergence, vertebrates and nematodes BRCT domains, 113 histone variant H2AX, 113 homology level, 114 NBS1 gene, 114 DNA damage response signalling ARM repeat proteins, 110 CLK–2, orthologue, 109 CLK2/TEL2, 110 GEN1 human, 111 Holliday junction-resolving enzymes, 110 model system, 102 mutational analysis, 111 positional cloning, 110 S-phase regulation, 110 telomere replication, 109 Werner syndrome, 109 worm homologues, ATM and ATR, 109 DNA regulation coactivator, 117 genotoxic agents, 115 mode of action, 116 mutations, 115 neurogenesis, 115–116 null alleles, 116 RBI complex, 117 RNAi knockdown, 116 vertebrates, 113

Index DSB repair BRCA1, 112 com–1 mutant worms, 112 CtIP nuclease, 111–112 foci, 112 gene encoding, 112 ICL repair, 113 molecular substrate, 111 NHEJ genes, 111 RAD–51, 112 synthetic lethal interactions, 113 “uncoordinated” phenotype, 113 experimental system long term storage, 104–105 RNAi screens, 105 self-fertilization, 105 sex chromosome, 106 genetic screens, 101 ionizing radiation (IR), 101 life cycle adult, 102–103 embryogenesis, 102 germ cell proliferation, 104 larval development, 102 oocytes, 104 somatic cell lineage, 102 transition zone, 103 mortal germline (mrt) mutations catalytic subunit, 114 chromosome fusions, 115 mismatch genes, 115 paralogue, 115 progeny numbers, 114 SMC1 nuclease domain, 114–115 nematodes, 104–105 phenotypes apoptosis, 106, 107 Blooms syndrome gene, 106 CED–3 caspase, molecule, 106–107 cell cycle arrest, 108 EGL–1, 107 filtration, 108–109 germ cell apoptosis, 108 irradiated worms, 106 lethality levels, 108 L4 survival assay, 108 meiotic chromosomes, defective, 106 mutants, 105–106 recombination genes, 107 transcription factor, p53, 107 tyrosine residue, 108 physiology, 102 Cancer anticancer agents, APE1, 154–155 clinical trials, 156

Index DNA damage, 179–180 EGFR anti-EGFR mAbs, 206–208 antitumour agents, 204–206 colorectal and lung, 217–218 isesophageal inhibitors and radiation, 214, 216 radiotherapy, 202 resistance biomarkers, 217 TKIs, lung, 218 IR therapy, 187 LPA participation, 195 malignant cell, 179, 181 NSCLC and pancreatic, 152 p53 tumor suppressor gene, 176–177 radiation and cytotoxic drugs effects, 187 therapy, 173–174 therapy (see DNA repair inhibitors and IR response) TNFa gene, 177 CD. See Cytosine deaminase Cell cycle checkpoint activation, 59–60 arrest Artemis defective cells, 72 Chk1 and Chk2 role, 72 DSBs repair, 71–72 genomic stability maintenance, 71 NHEJ proteins, 72 53BP1, 17 cancer, 20 DNA repair and protein signals, 13 and g-H2AX, 18–19 Checkpoint kinases Chk1 ATR-Chk1 pathways, 39 and ATR mutations, 37 BRCA1 expression, 46 cellular viability, 40 Chk2 mutations, 41–42 Claspin interaction, 40 DNA repair, 41 genetic model systems, 40 localization, 41 MK2 function, 42–43 phosphorylation, 40–41 Chk2 ATM pathways, 39 BRCA1, 45 confer cancer predisposition, 37 1100delC design, 42 MK2 function, 42–43 mutations, 41–42 phosphorylate key cell cycle, 42

267 PIKKs, 39 p38MAPK/MK2, 42–43 Chromatin checkpoint kinases, 39 Chk1 protein, 40–41 compaction level, 8–9 DNA damage responses (see DNA damage response (DDR)) g-H2AX homologue in vitro, 18 removal, 10 size, 5–8 relaxation, 46 remodeling complexes, 17–18 factors, 16–18 Core histones synthesis, 5 cPLA2. See Cytosolic phospholipase A2 Cytokine IL–6, 257 TGF-b, 257 Cytosine deaminase (CD) bacteria (bCD) and yeast (yCD), 175 radiosensitizer, 175 Cytosolic phospholipase A2 (cPLA2) lipids and proteins, 191 regulatory mechanisms, 192 D DDR. See DNA damage response Deoxyribonucleic acid (DNA) factors, 134 genes, 134–135 DNA damage response (DDR) activation, 196 BRCT-containing proteins 53BP1 and BRCA1, 45 MCPH1/BRIT1 and MDC1/NFBD1, 46 Caenorhabditis elegans, signalling, 102, 109–111 cellular existence, risk, 4 chromatin histones, 80–81 INO80 remodeling complex, 81–83 nature and remodeling complexes, 81 remodeling, 79–80 DSB repair cell cycle checkpoint, 18–19 chromatin remodeling factors, 16–18 description, 13 foci complexity, 19 PIKKs kinases (see Phosphatidylinositol 3-kinaserelated kinases (PIKKs))

268 DNA damage response (DDR) (cont.) eukaryotics, 101 functions, 71 hypoxic induction angiogenesis role, 132 ATM signal, 131 genomic instability, 133 genotoxic stress, 130 gH2AX role, 133 low oxygen level, 132 p53-dependent apoptosis, 130–131 PI3 kinase family, 130 radiosensitivity syndromes, 130 replication blocked, 131 S phase cell population, 131 INO80 chromatin remodeling, DNA replication, 92 DNA repair factors, DSBs, 87–88 double strand breaks, 83–84 DSBs repair, 85 histone H2AX phosphorylation, 84–85 histones, DSBs, 86–87 homologous recombination, 85–86 ionizing radiation, 83 phosphorylation, Ies4 subunit, 88–89 replication, 91–96 telomere maintenance, 90–91 markers biological process, 20–23 clinical applications, 23–26 DSB induction, 20 phosphorylation and DSB formation apoptosis, 13 characteristics, 5–10 core histones synthesis, 5 genetic information, eukaryotes, 4 immune system development, 11 male fertility, 11 replication/transcription, 12 telomere dysfunction, 12 virus infection, 12–13 RNAi screens, 105 ubiquitylation, 55 DNA double-strand breaks (DSBs) apoptotic cell death, 115 Ataxia telangiectasia, 24 ATM and ATR activation, 43 53BP1, 17 BRCA1, 45, 56 cancer therapeutic treatments cause, 24 cohesion, 18 conversion, 43, 44

Index DNA damages, 228 foci formation, 4 formation and H2AX phosphorylation, 4–5 g-H2AX stoichiometry, 8 Gy X-rays, 66 H2AX phosphorylation, 55 INO80 binding, H2AX phosphorylation, 84–85 DNA repair factors, 87–88 homologous recombination-mediated, 85 remodeling complex, 83–84 INO80-C, 18 ionizing radiation, 229 irradiation-induced, 16 KAP–1 phosphorylation, 67 markers, 112 MDC1 binding, 16 mediator parts, 243 NHEJ and ATM signaling, 65–66 phosphorylation, 7 physiological levels, 26 radiation lethality, 178 radiosensitivity tests, 24 ratio, SSBs, 57 repair Artemis defective cells, 72 deficiency, 10 and DNA damage-signalling defects, 105–109 factors, 9 g-H2AX, 10, 64 inducing, 8 mechanism, 71–72 pathway, 61 and response pathways, 231 routes, 71–72 RFC subunits, 109 sensor use, 25 SSBs generates, 12 stalled replication forks, 44 ubiquitylation, 55 DNA regulation cell death, 117–118 CEP–1-dependent apoptosis, 116–117 coactivator, 117 egl–1 and ced–13, 117 genotoxic agents, 115 mammalian p63 and p73, 115–116 mutations, 117 RBI complex, 117 vertebrates, 113 DNA repair activation, 41 cell cycle checkpoints, 180

Index checkpoint signalling protein, 116 chromosome fragmentations, 112 DSBs factors, INO80 nucleosomes, 88 Rad51 and Rad52 and Mre11, 87–88 EGFR AKT and MAPK, 204 gefitinib, 212 impact, 205 Fanconi anemia pathway, 41 function, 5 g-H2AX, chromatin remodeling factors, 16–18 hyperthermia, 230 mechanisms, 18 mortal germline mutations, 114 pathway function, 45 perturbations, 232 process, 54 protein, 8, 230 radiation-induced ERK activation, 191 tumor cell radiosensitivity, 179 tyrosine 142 dephosphorylation, 13 Werner syndrome patients, 10 DNA repair inhibitors and IR response anti-cancer drugs and IR efficacy, 144 cancer therapy ATM, 161–162 BRCA1 and BRCA2 gene, 160–161 NER and TLS repair, 159 PARP inhibition and p53 gene, 161 reliable biomarkers, 162 Saccharomyces cerevisiae use, 160 sensitivity, 159 “synthetic lethality” principle, 159–160 chemotherapeutic agent use, 144 molecular/genetic and biochemical/ proteomic method, 162 pathways, IR-induced DNA lesions BER, 149–150 DSBs and SSBs, 145 HR, 147–149 MMR, 150–151 NHEJ, 145–147 redundancy level and IR treatment, 144, 145 signal transduction pathway, 144 therapeutic strategy APE1, BER enzyme, 154–155 ATM and ATR, 157–158 caffeine, 157 cell cycle arrest and death, 151 cisplatin, 152 DNA-PK, 154

269 DSBs, 152 flavopiridol, 157 gemcitabine, 152 g-H2AX foci, 158 MGMT, 158 PARP, 155–156 p53 mutations, 157 radiosensitization, chemical inhibitors, 153, 154 RT and cancer drugs, 151 TMZ, 5-FU and capecitabine, 152 DNA replication ATM/ATR target proteins, 48 ATR phosphorylation, 39 base mismatches, 150 BRCA1 phosphorylation, 45 Chk2 activation, 44 chromatin remodeling, 92 cisplatin, 152 defects, 40 DNA breaks, fragile sites, 37 DSB presence, 43 forks, 37, 38 INO80 role chromatin remodeling activity, PCNA ubiquitylation, 95 DNA damage tolerance pathways, 94–95 MMS treatment, Rad51, 95–96 S phase checkpoint, 93–94 suicide genes, 174 toxic lesions, 151–152 UV radiation, 48 DNA single-stranded breaks (SSBs), 12 DSBs. See DNA double-strand breaks E Epidermal growth factor receptor (EGFR) signaling biology approaches, 202 HER family, members, 202 human cancers, 202 inhibitors and radiation, clinical trials anti-EGFR mAbs and TKIs toxicities, 216–217 esophageal cancer, 214, 216 GBM, 216 HNSCC, 213–215 NSCLC, 214 RTOG 0522, 213 radiation induced damage responses (see Radiation induced damage responses, EGFR)

270 Epidermal growth factor receptor (EGFR) signaling (cont.) resistance (see Resistance) signal transduction activity, 179 targeting agents, radiation action mechanisms, 211 angiogenesis, inhibitors, 212 cell cycle, 210–211 cetuximab, 210, 212–213 DNA damage repair, 211 DNA-PK, 211–212 erlotinib-treated tumour xenografts, 213 therapeutic outcomes, 212 tumour vasculature impairment, 212 Extracellular matrix (ECM), 129 F Fanconi anemia pathway, 41, 48 Fms-like tyrosine kinase receptor 3 (FLT–3), 252 G Gene therapy and radiation cancer treatment, 173 DNA repair pathways cell cycle checkpoints, 180 tumor cell radiosensitivity, 179–180 iNOS and PTEN, 180 malignant cells, 174 p53, 176–177 replication-competent oncolytic adenoviruses E1A expression, 178 suicide genes, 179 signal transduction and apoptotic pathways, 179 suicide (see Suicide gene therapy) TNFa, 177–178 Glioblastoma multiforme (GBM), 216 G2/M checkpoint arrest Cdc25A and C, mitotic regulators, 61 initiation ATM-Chk2 and ATM-ATR-Chk1, 61–62 Chk1 vs. Chk2, 62 NHEJ, 61 structure, 62 maintenance aphidicolin and ATR-Chk1 activation, 63 G2 phase cells, 63 structure, 62 mediator proteins role “amplification”, 67 ATM signaling, 66–67

Index “checkpoint” protein, 66 KAP–1 phosphorylation, 67 mitotic progression and Cdk1 activation, 60–61 sensitivity chromosome breakage estimation, 64–65 DSBs requirement, 66 g-H2AX foci, 64 NHEJ and ATM signaling, 65–66 PFGE and PCC, 64 G1/S checkpoint arrest cancer avoidance, 67 maintenance DNA DSB levels, 70 and G2/M checkpoint arrest, limitations, 70 IR doses, 69–70 p53 dependent, 67–68 S phase entry ATM phosphorylation, 69 Cdc25A-dependent process, 68 IR exposure, 68–69 H H2AX function, mediator proteins, 66 gamma (g) DNA DSB levels, 70 DSB repair, 64 INO80 binding genes expression profile, 84–85 gH2AX, 84 phosphorylation, 55 g-H2AX ATR-dependent signaling, 132 biodosimetry and radiosensitivity DSB detection, 23 lymphocytes, 24 radiation, 24 biological processes apoptosis, 13 cellular senescence, 20–21 human cancer model systems, 20 immune system development, 11 male fertility, 11 replication/transcription, 12 RIBE, 21–23 telomere dysfunction, 12 virus infection, 12–13 chemotherapy DSB, 24 phosphorylation, 25

Index chromatin remodeling factors, 16–18 chromosome abnormalities, 132 DNA DSB, 232 DSB stoichiometry, 8 environmental toxins, 25–26 foci, 235 heat shocks, 235–236 MDC1, 237 phosphorylation, 132–133 role, 133 Head and neck cancer (HNC), 213 Head and neck squamous cell carcinoma (HNSCC), 203 Heat shock protein (Hsp70) changes, 235 immunoprecipitation experiments, 234 Herpes simplex virus thymidine kinase (HSV–1 TK) replication-defective adenovirus, 176 wild-type enzyme, 174 Homologous recombination (HR) BLM, 148–149 BRCA1-deficient cells, 112 checkpoint and cyclin dependent kinases CDK activity, 149 CHK1 kinase, 149 double-strand break repair, 149, 151 Chinese hamster cell lines, 135 damage recognition proteins, 147 dissolution, 149 DNA sequence, 148 double-strand DNA breaks, 179–180 DSBs, 62 Fanconi anemia (FANC) family, 148 g-H2AX formation, 18 histone H2AX, 82 Holliday junction (HJ), 148, 149 initiation, 11 INO80, 85–86 oxic/anoxic conditions, 135 PARP inhibitors, 136 primary roles, 19 SSA and BIR, 147 HR. See Homologous recombination Hsp70. See Heat shock protein HSV–1 TK. See Herpes simplex virus thymidine kinase Hyperthermia MRN complex data, 235 heat induced changes, 234 heat shock, 233 hsp70, 234–235 Mre11-Rad50-NBS, 232

271 NPM, 241 radiation and adjuvant therapy, 244 TERs, 229 Hypoxia and cellular radiation response DNA damage response, 130–133 DNA repair pathways cell cycle arrest, 134–135 genomic instability and carcinogenesis, 133–134 oxic/anoxic conditions, 135 environmental exploitation, 135–136 tumor microenvironment characteristics acute and chronic hypoxia, 128 blood vessels, 127–128 cell cycle arrest, 128 chemotherapeutic drugs, 129 ECM, 129 IFP, 128–129 mutator phenotype, 130 OER, 129 Hypoxic environmental exploitation anti-angiogenesis therapies, 135–136 PARP inhibitors oxygen dependent mechanisms, 136 vasodilating effects, 136 I IL–6. See Interleukin 6 Inducible nitric oxide synthase (iNOS), 180 INO80 binding, H2AX phosphorylation expression profile changes, 84–85 gH2AX, 84 DNA damage tolerance pathways, replication checkpoint, S phase, 93–94 chromatin remodeling activity, 95 H2AX phosphorylation, 94 MMS treatment, Rad51, 95–96 PFGE, 94–95 response, 91–92 S phase, 93 DNA repair factor recruitment, DSBs arp8 mutant, 87–88 nucleosomes, 88 histones, DSBs ATP-driven remodeling activity, 86 ChIP analysis, 87 MATa switching, 87 homologous recombination Arabidopsis, 85–86 HO MAT system, 86 mediated, DSBs repair, 85

272 INO80 (cont.) phosphorylation, Ies4 subunit arp8 and nhp10 mutants, 89 ies4 mutants, 88 Mec1/Tel1, DSBs, 89 MMS treatment, 88 remodeling complex ATPase and 3’–5’ helicase activity, 81 bind, DSB, 83–84 molecular functions, 82–83 nuclear functions, 82 subunits, 82 telomere maintenance est1 and ies3 protein, 90–91 ino80, ies3 and arp8 mutant, 91 MRN complex, 90 iNOS. See Inducible nitric oxide synthase Interleukin 6 (IL–6), 257 Interstitial fluid pressure (IFP), 128 Ionizing radiation (IR) chemotherapy, 187 DNA damage, 188 signal transduction pathways, 189–190 vascular targeting agents, 188 IR. See Ionizing radiation K Kinase CHK1, HR, 149, 150 Cyclin B1/Cdk1 and Chk1/Chk2, 60–61 DNA-PKcs, 146 PI3K/Akt and MAPK/ERK, 189–190 PNK, 146 RTK (see Receptor tyrosine kinases) transducer kinases, 58–59 Ku70 DNA repair pathways, 180 NHEJ pathway, 145 L Lewis lung carcinoma (LLC) model cell growth, 253 heterotopic tumor, 194 Lysophosphatidic acid (LPA) cPLA2 activity, 195 receptors, 195 Lysophosphatidylcholine (LPC) conversion, 192 cPLA2a, 193 endothelial cells, 193–194 receptor activation, 193–194

Index M MAFP. See Methyl arachidonyl fluorophosphonate MAPK/ERK. See Mitogen-activated protein kinase/extracellular signal regulated kinase MDC1. See Mediator of DNA damage checkpoint protein 1 Mediator of DNA damage checkpoint protein 1 (MDC1), 15–16 Methyl arachidonyl fluorophosphonate (MAFP) cPLA2 inhibitors, 192 description, 193 O6-Methylguanine DNA-methyltransferase (MGMT), 152, 158 MGMT. See O6-Methylguanine DNA-methyltransferase Mismatch repair (MMR), 150–151 Mitogen-activated protein kinase-activated protein kinase–2 (MK2), 42–43 Mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ ERK) ionizing radiation, 189–190 radiation-induced activation, 191 MK2. See Mitogen-activated protein kinase-activated protein kinase–2 MMR. See Mismatch repair Molecular targeting limitations, 187 signal transduction pathways, radiation induced IR, 188–190 LPC and LPA, 193–195 MAPK/ERK, 191 phospholipids and cPLA2, 191–193 PI3K/Akt, 190–191 spatial cooperation, 187 vasculature, solid tumor development and treatment, 188 MRE11 cytoplasmic localization, 233–234 immunoprecipitation experiments, 234–235 Mre11-Rad50-NBS1 (MRN), 16 N Non-homologous end joining (NHEJ) DNA-PKcs Artemis and PNK, 146 autophosphorylation, 146 description and enzymatic properties, 146

Index polymerases, 147 XRCC4 and DNA ligase IV, 147 Ku70 and Ku80, 145 Non-small cell lung cancer (NSCLC) mutated-p53, 157 RT and gemcitabine, 152 Nucleophosmin (NPM) hyperthermia, 240 isoforms, 240 masking effects, 239–240 proteins impact, 242, 243 O Oncolytic adenoviruses, replication-competent, 178–179 Oxygen enhancement ratio (OER) cell type and inherent radiosensitivity, 129 oxic/anoxic conditions, 135 P p53 activation, upstream kinases DNA damage response, 46 knock-in mouse models, 47 unperturbed cells, 47 canonical pathway, 111 Chk2 mutations, 42 dependent apoptosis, 112, 130–131 dependent G1/S arrest Rb protein phosphorylation, 68 sensitivity and limitation, 68 ubiquitin ligase, MDM2, 67–68 gene mutation, 161 genetic knockout animals, 253 MAML function, 117 phosphorylation, 131 physical interaction, 45 radiosensitization, 157 therapy adenovirus-mediated, 177 apoptosis, 176 transcription factor, 107 upstream signaling pathways, 46 PARP. See Polyadenosine diphosphate-ribose polymerase p53-binding protein1 (53BP1) ATM signaling, 66–67 and BRCA1, g-H2AX, 19 DSB sites, 17 foci, 236–237 functions

273 ATM-dependent pathways, 45 DNA damage signaling, 44 H2A ubiquitylation, 55–56 heat effects, 237 Hsc70, 238 MDC1, 237–238 MRN complexes, 19 physical interaction, 45 radiosensitization, heat, 238–239 TT cells, 238 PCC. See Premature chromosome condensation PFGE. See Pulsed-field gel electrophoresis Phosphatase and tensin homolog (PTEN) delivery, microcapsules, 180 gene therapy and radiation, 180 Phosphatidylinositol 3-kinase/Akt (PI3K/Akt) ionizing radiation, 189–190 radiosensitization, 190–191 RTK inhibitors, 190 vascular endothelial cells, 190 Phosphatidylinositol 3-kinase-related kinases (PIKKs) activation and signaling ATM activation (see ATM) vs. ATR, 57–58 ATR activation, 56–57 signal, 58–59 ATM/ATR phosphorylation, 48 BRCT interaction, 44–46 checkpoint kinases, 39–40 human, diverse substrates, 47–48 PI3K/Akt. See Phosphatidylinositol 3-kinase/Akt PLD. See Potentially lethal damage PNK. See Poly-nucleotide kinase Polyadenosine diphosphate-ribose polymerase (PARP) homologous recombination (HR), 136 inhibitor ABT–888, RT and TMZ, 157 AG14361, 154 AG014699, 156 IR cytotoxicity and DSB repair, 155 phase II study, 156 as radiosensitizers, 155 PARP1 DNA sensing and repair, 155 inhibitor AG014699, 161 overexpression, 156 radiosensitizes malignant tumor cells, 136 vasodilating effects, 136

274 Poly-nucleotide kinase (PNK), 146 Potentially lethal damage (PLD), 174 Premature chromosome condensation (PCC), 64 Prostate cancer CD8 T, 259–260 therapy, 259 vaccinia-based PSA-directed vaccine, 260–261 PTEN. See Phosphatase and tensin homolog PTEN/AKT pathway, 48 p53 therapy. See Protein 53 therapy Pulsed-field gel electrophoresis (PFGE), 8, 64, 94 R Radiation. See also Epidermal growth factor receptor (EGFR) signaling Akt phosphorylation, 190 cell death, vascular endothelial cells, 190 cPLA2, 192 dose and time interval, 188 ERK activation, 191 exposure (see Radiation exposure) response regulation, 211 surgery chemotherapy, 202–203 Radiation exposure cell cycle check point activation, 59–60 checkpoint arrest significance, 71–72 description, DDR processes, 54 DNA repair processes, 54 G2/M checkpoint arrest Cdc25A and C, 61 damage mediator proteins, 66–67 initiation, 61–62 maintenance, 62, 63 mitotic progression and Cdk1 activation, 60–61 sensitivity, 64–66 G1/S checkpoint arrest cancer avoidance, 67 maintenance, 69–70 p53 dependent, 67–68 S phase entry, 68–69 intra-S phase checkpoint arrest, 71 PIKKs activation and signaling, ATM activation, 54–56 vs. ATR, 57–59 Radiation-induced bystander effect (RIBE) “bystander” effects, 23 signaling, 21 DNA damage, 21–22

Index Radiation induced damage responses, EGFR anti-EGFR mAbs antibodies, 207 cetuximab, 206, 208 panitumumab, 206, 208 receptor internalization and degradation, 206 anti-EGFR TKIs gefitinib and erlotinib, 208 inhibition, 208, 210 resistance, 210 cancer therapy, 203 and DNA damage repair AKT and MAPK, 204 cell survival, 203–204 DNA-PK-Ku70/Ku80 complex, 204 impact, 205 inhibitors, 204–206 surgery chemotherapy, 202–203 and tumor cell repopulation, 203 Radiation-induced DNA damage response heat effects, proteins DNA DSB, 230–231 proteotoxic stress, 231–232 masking effect, 239 halo assay, 241 ionizing radiation, 241–242 neutral comet, 240–241 nuclear and chromatin structure, 243–244 pathways, TER improvement HT29 colon carcinoma cells, 245 radiation and adjuvant therapy, 244 proteins perturbation, hyperthermia effects 53BP1, 236–239 clonogenic survival assay, 242 DNA damage cell, 240 g-H2AX, 235–263 masking, 239–240 MRN complex, 232–235 radiosensitization resulting, 243 Radiation-induced immune modulation abscopal effect, 251–254 immunological effects local immune cells, 256 tumor-associated vasculature, 255–256 tumor cells, 254–255 immunological radiation effects cytokine levels, 257–258 immune stimulatory radiation effects, 258–259 tumor burden, 258

Index immunotherapy vs. radiotherapy, 260–261 radiotherapy immunological effects, humans, 259–260 Radiation sensitivity hyperthermia, 229 NPM, 243 proteins, 242 Radiation therapy doses, 196 IL–6, 257 LLC, 253 MC38-CEA tumors, 255 Radiosensitizers CD, 175 chemical, 130 5-fluorouracil (5-FU), 175 HSV–1 TK/GCV suicide gene system, 174 p53 gene therapy, 176–177 TNFa gene therapy, 177 vascular endothelial cells, 191 Radiotherapy (RT) Chk1 inhibitors combination, 161 cisplatin, 152 EGFR expression and activity, 203 HNSCC patients, 206, 213 tissue toxicity, 202–203 vascular normalization, 213 gene therapy, 174 g-H2AX DSBs, 24–25 in vivo lymphocytes irradiation, 24 side effects, 24 human cancer, 181 immunomodulatory effects, 255–256 immunotherapy, 260 NSCLC and pancreatic cancer, 152 PARP inhibitor, 156–157 p53 gene therapy, 177 suicide gene therapy, 175 TGF-b, 258 TMZ, 152, 156 tumor treatment, 252 Receptor tyrosine kinases (RTK) inhibitors, 190 PI3K/Akt, 190 pro-survival signaling pathway, 188 Reoxygenation, 131–133, 135 Repair DNA damage, 176, 179–180 DSB, 178 PLD, 174 Resistance BRCA2-deficient cell lines, 161

275 EGFR inhibitors and genetic mutations, 217–218 and nuclear EGFR, 219 and tyrosine kinase receptors, 218 IR doses, 187 radio cPLA2 contribution, 193 vascular endothelial cell, 195 RIBE. See Radiation-induced bystander effect RT. See Radiotherapy RTK. See Receptor tyrosine kinases S SCD. See SQ/TQ cluster domain Signal transduction pathways LPC and LPA biological processes, 195 cellular responses, 193 cPLA2 activity, 195 endothelial cells, 193–194 pharmacologic inhibition, 195 receptor activation, 193 MAPK/ERK, 191 phospholipids and cPLA2 lipids and proteins, 191 MAFP and AACOCF3 inhibitors, 192–193 regulatory mechanisms, 192 PI3K/Akt endothelial cells, 190 radiosensitization, 190–191 RTK inhibitors, 190 SQ/TQ cluster domain (SCD), 42 SSBs. See DNA single-stranded breaks Suicide gene therapy CD and colorectal adenocarcinoma cells, 175 flu-like symptoms and transaminitis, 175 HSV–1 TK and PLD, 174 metabolic enzymes, 174 primary glioblastoma, 176 prognostic and intermediate-risk, 175–176 T Telomere dysfunction, 12 Temozolomide (TMZ), 152 TER. See Thermal enhancement ratio TGF-b. See Transforming growth factor beta Therapy chemotherapy, 259 immunotherapy, 252 post radiotherapy, 255

276 Therapy (cont.) radiation therapy, 253 radio (see Radiotherapy) radiotherapy, 251–261 Thermal enhancement ratio (TER) 53BP1/MEF, 239 DNA damage stress response pathways HT29 colon carcinoma cells, 245 radiation and adjuvant therapy, 244 hyperthermia, 229 molecular chaperon, 243 radiation dose, 242 radiosensitization, 228 TT cells, 238 Thermotolerant (TT) cells, 238 TMZ. See Temozolomide TNFa. See Tumor necrosis factor alpha

Index Toxicities, anti-EGFR mAbs and TKIs HNSCC and NSCLC treatment, 216 side effects, 217 skin, cetuximab therapy, 217 Transforming growth factor beta (TGF-b), 257 TT cells. See Thermotolerant cells Tumor necrosis factor alpha (TNFa) investigational therapy, 178 radiosensitizer, 177 TNFerade, 178 Turcot’s syndrome, 159 W Werner’s syndrome protein (WRN), 148 WRN. See Werner’s syndrome protein