Radiation Cytogenetics. Methods and Protocols 978-1-4939-9430-4, 978-1-4939-9432-8

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Methods in Molecular Biology 1984

Takamitsu A. Kato Paul F. Wilson Editors

Radiation Cytogenetics Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Radiation Cytogenetics Methods and Protocols

Edited by

Takamitsu A. Kato Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA

Paul F. Wilson Department of Radiation Oncology, University of California Davis, Sacramento, CA, USA

Editors Takamitsu A. Kato Department of Environmental and Radiological Health Sciences Colorado State University Fort Collins, CO, USA

Paul F. Wilson Department of Radiation Oncology University of California Davis Sacramento, CA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-9430-4 ISBN 978-1-4939-9432-8 (eBook) https://doi.org/10.1007/978-1-4939-9432-8 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface The assessment of chromosomal aberration (CA) levels in cytogenetic spreads prepared of first-division mitotic cells (typically peripheral blood lymphocytes) following exposure to ionizing radiation (IR) remains the “gold-standard” approach for human biodosimetry applications. Here we present concise protocols for preparing metaphase chromosome spreads from both attached cell cultures (e.g., fibroblasts) and suspension-based cell cultures (e.g., mitogen-stimulated lymphocytes), staining them by a variety of classical and molecular methods, and analyzing IR-induced CAs by microscopy and high-throughput approaches. The authors of the different chapters have provided standardized protocols from their laboratories along with extensive notes for readers to conduct these assays in their own laboratories. This book is also beneficial for readers already experienced with cytogenetic analysis to learn additional techniques relevant to radiation biodosimetry and to modify/ develop their own cytogenetic approaches as needed. We would like to thank our doctoral advisor, Dr. Joel S. Bedford of Colorado State University, for patient mentorship and training of not only ourselves but several authors of this volume in the essential techniques of radiation cytogenetics. Fort Collins, CO, USA Sacramento, CA, USA

Takamitsu A. Kato Paul F. Wilson

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v ix

1 Human Lymphocyte Metaphase Chromosome Preparation for Radiation-Induced Chromosome Aberration Analysis . . . . . . . . . . . . . . . . . . . . 1 Takamitsu A. Kato 2 Animal Lymphocyte Metaphase Chromosome Preparation . . . . . . . . . . . . . . . . . . . 7 Kazuki Heishima, Kendon Kuo, Masashi Kimura, and Takashi Mori 3 Micronuclei Formation Analysis After Ionizing Radiation . . . . . . . . . . . . . . . . . . . . 23 Cathy Su, Alexis H. Haskins, and Takamitsu A. Kato 4 G1 Premature Chromosome Condensation (PCC) Assay . . . . . . . . . . . . . . . . . . . . 31 Ryuichi Okayasu and Cuihua Liu 5 G2 Chromosomal Radiosensitivity Assay for Testing Individual Radiation Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Jeremy S. Haskins and Takamitsu A. Kato 6 G2 Premature Chromosome Condensation/Chromosome Aberration Assay: Drug-Induced Premature Chromosome Condensation (PCC) Protocols and Cytogenetic Approaches in Mitotic Chromosome and Interphase Chromatin for Radiation Biology . . . . . 47 Eisuke Gotoh 7 Sister Chromatid Exchange as a Genotoxic Stress Marker . . . . . . . . . . . . . . . . . . . . 61 Shigeaki Sunada, Jeremy S. Haskins, and Takamitsu A. Kato 8 DNA Damage Focus Formation Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Yoshihiro Fujii 9 Nuclear Foci Assays in Live Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Eiichiro Mori and Aroumougame Asaithamby 10 In Situ DNA Damaging Foci Analysis on Metaphase Chromosomes . . . . . . . . . . 87 Chisato Omata and Takamitsu A. Kato 11 PNA Telomere and Centromere FISH Staining for Accurate Analysis of Radiation-Induced Chromosomal Aberrations . . . . . . . . . . . . . . . . . . . 95 Ian M. Cartwright, Jeremy S. Haskins, and Takamitsu A. Kato 12 Modified PNA Telomere and Centromere FISH Protocols. . . . . . . . . . . . . . . . . . . 101 Ian M. Cartwright 13 Directional Genomic Hybridization (dGH) for Detection of Intrachromosomal Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Erin Robinson, Miles J. McKenna, Joel S. Bedford, Edwin H. Goodwin, Michael N. Cornforth, Susan M. Bailey, and F. Andrew Ray

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Contents

Reciprocal Translocation Analysis with Whole Chromosome Painting for FISH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Jeremy S. Haskins and Takamitsu A. Kato Analysis of Radiation-Induced Chromosome Exchanges Using Combinatorial Chromosome Painting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Bradford D. Loucas

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors AROUMOUGAME ASAITHAMBY  Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA SUSAN M. BAILEY  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA JOEL S. BEDFORD  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA IAN M. CARTWRIGHT  Department of Medicine, University of Colorado Denver, Aurora, CO, USA MICHAEL N. CORNFORTH  Department of Radiation Oncology, University of Texas Medical Branch, Galveston, TX, USA YOSHIHIRO FUJII  Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan EDWIN H. GOODWIN  KromaTiD Inc., Fort Collins, CO, USA EISUKE GOTOH  Department of Radiology, Jikei University School of Medicine, Minato-ku, Tokyo, Japan ALEXIS H. HASKINS  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA JEREMY S. HASKINS  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA KAZUKI HEISHIMA  Department of Veterinary Clinical Oncology, Faculty of Applied Biological Sciences, Gifu University, GifuGifu, Japan TAKAMITSU A. KATO  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA MASASHI KIMURA  Kenpoku Livestock Hygiene Service Center of Ibaraki Prefecture, Mito, Ibaraki, Japan KENDON KUO  Department of Clinical Sciences, Bailey Small Animal Teaching Hospital, Auburn University College of Veterinary Medicine, Auburn, AL, USA CUIHUA LIU  National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan BRADFORD D. LOUCAS  Department of Radiation Oncology, The University of Texas Medical Branch, Galveston, TX, USA MILES J. MCKENNA  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA EIICHIRO MORI  Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA TAKASHI MORI  Department of Veterinary Clinical Oncology, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan RYUICHI OKAYASU  National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan CHISATO OMATA  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA F. ANDREW RAY  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA

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Contributors

ERIN ROBINSON  KromaTiD Inc., Fort Collins, CO, USA CATHY SU  Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA SHIGEAKI SUNADA  Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Chapter 1 Human Lymphocyte Metaphase Chromosome Preparation for Radiation-Induced Chromosome Aberration Analysis Takamitsu A. Kato Abstract Radiation-induced chromosomal aberration analysis for metaphase chromosomes is well established and the golden standard for human biodosimetry. This method can estimate doses of human radiation exposure after nuclear accident and unwanted radiation exposure from their lymphocytes. The natural background frequency of dicentric chromosome for human lymphocytes is less than 1% and any increase in dicentric and centric ring chromosomes may be highly associated with radiation exposure. With the appropriate number of metaphase cells, one can detect the exposure of more than 0.1 Gy by observing dicentric and centric ring chromosomes. Dicentric chromosome analysis is relying on morphological changes and may be difficult for researchers without appropriate training. This method is time consuming and labor intensive, but still currently the most reliable technique and analysis needs only light microscopes with high magnification objectives and trained personnel. Recent research enables us to visualize dicentric chromosomes clearly with fluorescent markers for easy detection of dicentric and centric ring chromosomes. This chapter will introduce classical dicentric analysis of human lymphocyte cells with Giemsa staining. Key words Lymphocyte, Mitogenic stimulation, Metaphase, Chromosome aberrations, Dicentrics, Centric ring, Giemsa stain

1

Introduction Radiation induces multiple types of DNA damages including base damages, single strand breaks, crosslinking, and double strand breaks. Notably, the induction of DNA double strand breaks is the most toxic and directly associated with chromosomal aberrations observed at the first postirradiated metaphases. Chromosome aberrations are highly associated with cell killing, mutation, and carcinogenesis. Among radiation-induced chromosomal aberration, dicentric and centric ring chromosomes are an extremely rare event in background level whereas relatively low dose of ionizing radiation induces a substantial amount. For the cells irradiated at G0/G1 phase first-postirradiated mitosis, only DNA double strand break can produce dicentric and centric chromosomes.

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Therefore, dicentric and centric ring chromosomes have been used for biodosimetry to estimate individual radiation exposure. Large cohort studies of chromosome aberrations have been carried out for Japanese atomic bomb survivors in Hiroshima, Nagasaki, and also for residents near Chernobyl to determine the radiationinduced health effects [1–6]. In those studies chromosome aberrations yields were often used to estimate individuals’ radiation exposure [7, 8]. Using peripheral lymphocytes for biodosimetry has several advantages. For example, peripheral blood is easy to draw and less invasive compared to skin biopsy. Ficoll density centrifugation and its alternative commercial kits can easily separate lymphocytes from whole blood as buffy coat [9]. The accessibility of drawing blood provides an excellent abundance of samples while utilization of teeth and hair roots is limited. Lymphocytes are terminally differentiated and can be found abundantly resting at G0 phase. Due to the nature of lymphocytes at the G0 phase, the use of cell-cycle arresting agents is not necessary; this provides an easier and accurate analysis of the first postirradiation metaphase. In addition, the half-life of lymphocytes in the body is also known to be up to years [10]. Therefore, chromosome aberration analysis can be carried out days and months after irradiation event. Giemsa stain-based dicentric and centric ring chromosomes analysis is strong but the limitations exist. Only well-trained individuals can identify morphological abnormalities of chromosomes depicted as dicentric and centric ring chromosomes. Microscopic analysis is labor intense and it requires ample time to analyze thousands of metaphases to quantitatively and qualitatively determine low dose radiation exposure with statistical significance. On the other hand, micronuclei analysis explained in the latter chapter is an alternative for nonlabor intense chromosome aberration analysis. Micronuclei analysis can also be automated for acquisition of images and analysis. Moreover, fluorescent-based probe paint techniques such as whole chromosome painting FISH analysis were designed to overcome difficulty of analysis. However, Giemsa stained chromosome aberration analysis is still the standard of human biodosimetry because standard light microscopes can detect dicentric and centric ring chromosomes without the requirement of fluorescence microscope system, expensive florescence probes, and software.

2 2.1

Critical Parameters Materials

1. RPMI 1640 medium (Sigma). 2. Fetal Bovine Serum (FBS), prior lot testing is required. Some lots increase background level chromosomal aberrations. Heat inactivation at 56  C for 20 min is required (see Note 1).

Human Lymphocyte Metaphase Chromosome Preparation for Radiation-Induced. . .

3

3. Phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 in distilled water and adjust pH 7.4, then autoclave or filter by 0.22 μm pore for sterilization) or purchase PBS ( ) from ThermoFisher, minus means no Calcium, no Magnesium, no Phenol Red. 4. Antibiotics (Antibiotic-Antimycotic (100) Catalog number 15240096, penicillin streptomycin solution or kanamycin 60 mg/ml). 5. 10 μg/ml KaryoMAX Colcemid solution in PBS (Invitrogen, Catalog number 15212012). l

Potassium chloride (KCl) (Sigma): dissolve in distilled water to make 75 mM KCl solution. 2.8 g of potassium chloride in 500 ml of distilled water.

6. Carnoy fixative solution (3:1 mixture of Methanol and Acetic Acid), freshly mixed. 7. 5% Giemsa solution. Mix 2.5 ml of KaryoMAX Giemsa stain solution (GIBCO Catalog number 10092013) with 47.5 ml Gurr solution. Freshly mixed. Gurr solution is made from Gurr buffer tablet (GIBCO, Catalog number 10582013) dissolved in distilled water. 8. BD Vacutainer CPT Mononuclear Cell Preparation tubeSodium Heparin (BD Biosciences, Catalog number 362753), if isolation of lymphocytes is necessary for the experiment. 9. PHA-M phytohemagglutinin (Remel, Lenexa, KS, USA) (see Note 2). 10. GIBCO PB-MAX Karyotyping Medium (GIBCO, Catalog number 125570210 or 12557021) is RPMI medium supplemented with FBS and PHA (see Note 2). 2.2

Equipment

1. Cell culture facility including biosafety cabinet or clean bench, 37  C CO2 controlled incubator, and water bath to keep 37  C. 2. Centrifuge, which can rotate 15 ml tubes. No need of temperature control or brake. 3. Aspirator to remove supernatant and trap waste. 4. Microscope equipped with CCD camera and software. 5. Slide glass, Coverslip, and Cytoseal 60 (Richard-Allan) Mounting Medium.

3

Methods 1. Take 5 ml blood into a heparinized tube. Avoid unnecessary conversation during this process. Chatting during blood draw may increase a risk of contaminations to samples (see Note 3).

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2. Place blood into a 15 ml tube. Add 4 ml RPMI 1640 medium, 1 ml of FBS, 0.2 ml PHA-M, 0.1 ml Colcemid (final 0.05 μg/ml), 0.01 ml Kanamycin (final 60 μg/ml). (a) Alternatively, lymphocytes can be separated from peripheral blood. Centrifuge blood in BD Vacutainer CPT Mononuclear Cell Preparation tube-Sodium Heparin with 3300 rpm for 15 min. Final culture solution should contain 8 ml of RPMI, 2 ml of FBS, 180 μg/ml of phytohemagglutinin (PHA-M) and 0.1 μg/ml Colcemid (GIBCO), and antibiotics. PB-MAX Karyotyping Medium can be also used. 3. Loosen cap of tube and place into CO2 incubator and maintain 37  C. 4. 48 h later, centrifuge (1000 rpm, 5 min) and discard the supernatant. (a) Alternatively, Colcemid can be added last 6 h of incubation period. This way may improve visual chromosome size longer compared to the continuous Colcemid treatment. It is easy to identify chromosome aberrations in the longer chromosomes compared to shrunk chromosomes by the continuous Colcemid treatment, 5. Add 4 ml of 75 mM KCl solution and place tubes into 37  C water bath for 20 min. 6. Add 2 ml Carnoy solution and spin down (1000 rpm/ about 100–200 g, 5 min) by centrifugation, and discard the supernatant by aspiration. 7. Gently tap and break pellet. 8. Then, add 2 ml of Carnoy fixative solution and spin down (1000 rpm, 5 min) by centrifugation, and discard the supernatant, gently tap tubes, and break pellet. Repeat this process three times. 9. Drop 10–100 μl of solution onto clean slides. Let them air dry (see Notes 4–6). (a) One can drop one or two spots. Or one can tilt slides at a 45 angle and drop from the top so the metaphase chromosomes evenly slide down. (b) Too many cells may prevent good metaphase chromosome spread. In that case, add a few drops of Fixative solution to adjust cell concentrations. (c) Do not disturb slide glass till air dry completes. Moving wet slides may cause uneven spread of metaphase cells. 10. Place slides into Coplin Jar with 50 ml of Giemsa solution for 5 min. Rinse slides with running tap water. Let them air dry. 11. Put appropriate size of coverslip with Cytoseal.

Human Lymphocyte Metaphase Chromosome Preparation for Radiation-Induced. . .

5

Fig. 1 Human metaphase chromosome spread with dicentrics and fragment chromsoomes indicated with arrows

12. Observe under a microscope. Find nicely spread metaphase cells. Good metaphase spread has a minimum overlap of chromosomes and not too spread (Fig. 1). 13. Analyze dicentric chromosome yields for 100 cells (for a few Gy analyses). 1000 cells for a few 0.1 Gy analyses. A dicentric chromosome should have a centric fragment in a same cell. (a) Other types of chromosomal aberrations such as chromatid gap, chromatid break, chromatid exchanges, chromosomal interstitial deletion, terminal deletion are not scored for biodosimetry purpose. But for research purpose, these chromosomal aberrations are worth to count.

4

Notes 1. FBS lot number should be also tested to yield maximum mitotic index. Multiple FBS should be purchased with same lot number to maintain experimental quality. 2. If a mitotic yield is low (less than 1%), change PHA-M to different badge. PHA-M quality is important for this experiment. 3. Need appropriate protection for handling human blood. 4. If metaphase chromosomes are too spread, reduce KCl treatment time to 15 min. Alternatively, after fixation, keep tubes at 4  C overnight and drop samples to slides. 5. If metaphase chromosomes are not spread enough, place slide glass into ice cold water before dropping samples to slides. Moisture may help metaphase chromosome spread quality.

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6. If metaphase chromosomes are spread too far, you will not be able to discern where the chromosomes came from, i.e., nearby cell.

5

Anticipated Results Approximately 1–2 dicentric will be found in 1000 metaphase chromosome spread from healthy lymphocyte cells. 1 Gy of radiation exposure can increase a yield of dicentric approximately 110–180 dicentric and centric ring chromosomes per 1000 metaphase cells [11, 12]. However, cells from radiosensitive population may have elevated number of dicentric and centric ring chromosomes. Years after initial irradiation, chromosome aberrations decreased over time [13].

References 1. Cardis E, Vrijheid M, Blettner M, Gilbert E, Hakama M, Hill C, Howe G, Kaldor J, Muirhead CR, Schubauer-Berigan M et al (2007) The 15-country collaborative study of cancer risk among radiation workers in the nuclear industry: estimates of radiation-related cancer risks. Radiat Res 167:396–416 2. Douple EB, Mabuchi K, Cullings HM, Preston DL, Kodama K, Shimizu Y, Fujiwara S, Shore RE (2011) Long-term radiation-related health effects in a unique human population: lessons learned from the atomic bomb survivors of Hiroshima and Nagasaki. Disaster Med Public Health Prep 5(Suppl 1):S122–S133 3. Hsu WL, Preston DL, Soda M, Sugiyama H, Funamoto S, Kodama K, Kimura A, Kamada N, Dohy H, Tomonaga M et al (2013) The incidence of leukemia, lymphoma and multiple myeloma among atomic bomb survivors: 1950–2001. Radiat Res 179(3):361–382 4. Preston DL, Pierce DA, Shimizu Y, Cullings HM, Fujita S, Funamoto S, Kodama K (2004) Effect of recent changes in atomic bomb survivor dosimetry on cancer mortality risk estimates. Radiat Res 162:377–389 5. Preston DL, Shimizu Y, Pierce DA, Suyama A, Mabuchi K (2012) Studies of mortality of atomic bomb survivors. Report 13: solid cancer and noncancer disease mortality: 1950–1997. 2003. Radiat Res 178: AV146–AV172 6. Walsh L, Jacob P, Kaiser JC (2009) Radiation risk modeling of thyroid cancer with special

emphasis on the Chernobyl epidemiological data. Radiat Res 172:509–518 7. Lindholm C, Salomaa S, Tekkel M, Paile W, Koivistoinen A, Ilus T, Veidebaum T (1996) Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH. Int J Radiat Biol 70:647–656 8. Snigiryova G, Braselmann H, Salassidis K, Shevchenko V, Bauchinger M (1997) Retrospective biodosimetry of Chernobyl clean-up workers using chromosome painting and conventional chromosome analysis. Int J Radiat Biol 71:119–127 9. Noble PB, Cutts JH (1967) Separation of blood leukocytes by Ficoll gradient. Can Vet J 8:110–111 10. McLean AR, Michie CA (1995) In vivo estimates of division and death rates of human T lymphocytes. Proc Natl Acad Sci U S A 92:3707–3711 11. Kanda R, Jiang T, Hayata I, Kobayashi S (1994) Effects of colcemid concentration on chromosome aberration analysis in human lymphocytes. J Radiat Res 35:41–47 12. Norman A, Sasaki MS (1966) Chromosomeexchange aberrations in human lymphocytes. Int J Radiat Biol Relat Stud Phys Chem Med 11:321–328 13. Norman A, Sasaki MS, Ottoman RE, Fingerhut AG (1966) Elimination of chromosome aberrations from human lymphocytes. Blood 27:706–714

Chapter 2 Animal Lymphocyte Metaphase Chromosome Preparation Kazuki Heishima, Kendon Kuo, Masashi Kimura, and Takashi Mori Abstract Metaphase chromosome analysis of lymphocytes is the gold standard for biodosimetry to estimate the levels of radiation exposure in various animals as well as humans. Animals, including experimental, companion, and wild animals, are powerful and indispensable models for researching radiation injury, safety, and therapy. Moreover, biodosimetry of animal models can be used to support human biodosimetry data and may be useful for estimating environmental contamination by radioactive materials. The basic restraint procedure and venipuncture technique are different depending on each animal type. The general procedure evaluating metaphase chromosomes is similar to the human blood technique except for a minor modification in the initial culture. This chapter will introduce basic mouse, rat, rabbit, dog, cat, cow, horse, goat, pig, and wild boar venipuncture and blood sampling techniques for metaphase chromosome preparation and analysis. Key words Metaphase chromosome analysis, Lymphocytes, Radiation exposure, Biodosimetry, Animal models

1

Introduction Animals, including experimental, companion, and wild animals, are powerful and indispensable models for researching radiation injury, protection, and therapy. Moreover, biodosimetry of animal models can be used to support human biodosimetry data and may be useful for estimating environmental contamination by radioactive materials. Each animal model has different advantages. Rodents, such as mice and rats, are well-established experimental animals suitable for a broad range of in vivo experiments. Rodents are advantageous because they are small and easy to handle, and have high reproductive capacity coupled with a short maturation time. Moreover, their genetic backgrounds are well studied with minimal individual genetic variation in most of the established research breeds. Companion animals, such as dogs and cats, are suitable models both for evaluating the effects of radiation therapy and for estimating the environmental contamination by radioactive materials. They

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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spontaneously develop a wide range of diseases including cancers, many of which are treated with radiation therapy similar to the equivalent human cancer. Furthermore, they share similar environmental factors with humans; thus, the information gained from biodosimetry of companion animals is useful for evaluating the contamination of radioactive materials in human environments. Large animals, such as cows, horses, goats, pigs, and wild boars, are suitable models for estimating the effect of environmental contamination due to radioactive materials on food production, such as meat and milk, which directly impact human health. Each researcher should select an appropriate animal model that is most suitable for their individual experimental objectives. Although the animal models share many similarities, there are several significant differences between each model. For example, studies have shown that lymphocytes from different species have different radiosensitivities. These differences, which were initially speculated to be due to the number of chromosomes in the species [1], may be due to the differences in DNA repair capabilities among various species [2]. This chapter will focus on the methodology of collecting and analyzing blood samples from mice, rats, rabbits, dogs, cats, cows, horses, goats, pigs, and wild boars.

2

Materials 1. Anesthetizing or sedative agents. (a) Isoflurane (Forane). (b) Pentobarbital (Nembutal). (c) Xylazine (Sedazine). (d) Medetomidine (Domitor). (e) Butorphanol (Stadol). (f) Ketamine (Ketalar). (g) Mafoprazine mesylate. (h) Azaperone (Stresnil). (i) Droperidol (Inapsine). (j) Midazolam (Dormicum). 2. Lymphocyte Separation Medium (Organon Teknika, Durham, NC). 3. Culture media. (a) RPMI 1640 Dutch Modification (Gibco, Thermo Fisher Scientific, MA, USA). (b) PB-MAX™ Karyotyping Medium (Gibco).

Animal Lymphocyte Metaphase Chromosome Preparation

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(c) Ham’s F10 medium (Gibco). (d) Parker Medium (Gibco). 4. Fetal bovine serum (FBS, Gibco). 5. L-glutamine (Gibco). 6. Mitogen. (a) Phytohemagglutinin (PHA-M, Wellcome Diagnostics, Beckenham, Kent, UK). (b) CON-A (Type IV, Sigma, MO, USA). (c) Lipopolysaccharides (LPS, Sigma). (d) Phorbol myristate acetate (PMA, Sigma). (e) Ionomycin (Sigma). (f) Lectin from Phytolacca americana (pokeweed, Sigma). 7. Antibiotics. (a) Penicillin-streptomycin solution (Invitrogen, Thermo Fisher Scientific, MA, USA). (b) Kanamycin (Gibco). 8. PBS (Gibco). 9. Colcemid (Gibco). 10. 75 mM KCl solution (2.8 g of KCl in 500 ml of distilled water). 11. Carnoy fixative solution (3:1 mixture of Methanol and Acetic Acid). This solution should be freshly mixed before the procedure. 12. 5% Giemsa solution. Mix 2.5 ml of KaryoMAX Giemsa stain solution (Gibco) with 47.5 ml Gurr solution. Gurr solution is made from Gurr buffer tablet (Gibco) dissolved in distilled water. This solution should be prepared just before staining. 13. Cytoseal™ (Richard-Allan Scientific, Thermo Fisher Scientific). 2.1

Equipment

1. Blood sampling. (a) 18–26 G needles (or butterfly needles) (b) Syringe pump. (c) Restrainers. l

Hog catcher.

l

Rodent handling gloves.

(d) Animal warming chamber. (e) Sterile cotton balls. (f) Capillary tubes. (g) 70% ethanol. (h) Surgical scalpel blades.

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(i) Heparinized tubes. l

l

BD Vacutainer® Plus Green BD Hemogard™ plastic plasma tube—Lithium Heparin (BD Biosciences, NJ, USA). BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube—Sodium Heparin (BD Biosciences).

2. Metaphase chromosome examination. (a) Microscope. (b) Centrifuge. (c) 37  C water bath. (d) Micropipette. (e) CO2 incubator. (f) Culture tubes. (g) Glass slides. (h) Coplin staining jar. (i) Coverslips.

3

Methods Methods for collecting the blood are summarized in Table 1. The method should be selected that is suitable for the individual objective, experimental design, and animal type. It is important to minimize

Table 1 Summary of blood sampling methods for various animals Non-terminal

Terminal

Mouse

Tail vein, Orbital sinus

Caudal vena cava, Cardiac puncture

Rat

Tail vein, Orbital sinus

Caudal vena cava, Cardiac puncture

Rabbit

Marginal ear vein

–

Dog

Cephalic vein, Jugular vein, Lateral and medial saphenous vein

–

Cat

Cephalic vein, Jugular vein, Medial saphenous vein

–

Cow

Jugular vein, Tail vein

–

Horse

Jugular vein

–

Goat

Jugular vein

–

Pig

Jugular vein, Caudal auricular vein

–

Wild boar

Medial saphenous vein

Cardiac puncture

Animal Lymphocyte Metaphase Chromosome Preparation

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stress when collecting blood samples as stress may affect the outcome of the study. Therefore, blood samples may require collection under anesthesia or with appropriate restraint (see Note 1). 3.1 Blood Sampling from Mice and Rats

Blood samples are taken from the tail vein, orbital sinus, caudal vena cava, or heart (cardiac puncture) in mice. The tail vein and orbital sinus are used for multiple blood sampling, while blood sampling from the caudal vena cava or heart are terminal procedures for taking a single, large volume, and high quality sample. In rats, most procedures are identical, although a larger volume of blood can be obtained compared to mice. If necessary, anesthetize the animal by inhalation of 1–3% isoflurane or intraperitoneal injection of pentobarbital at the dose of 40–50 mg/kg.

3.1.1 Tail Vein

The tail vein is useful for multiple samplings to obtain 50–100 μl of blood sample in mice (up to 2 ml in rats). 1. Restrain the animal manually or with a suitable restraint device. 2. If the tail vein is not visible, warm the tail by dipping the tail into 39  C warm water or using a heat lamp up to 10 min. 3. Sterilize the surface of the tail with 70% ethanol. 4. Insert a 26 G needle with 1 ml syringe into the tail vein in mice (21 G needle with 3 ml syringe in rats). 5. Stop bleeding by gently applying pressure to the collection site.

3.1.2 Orbital Sinus

This procedure is suitable for multiple, but infrequent, sampling to obtain up to 200 μl of samples at once in mice (up to 4 ml in rats). General anesthesia is necessary to perform this procedure. This method may cause severe or slight ocular damage such as hematoma, corneal ulceration, keratitis, and pannus formation. Sampling should be performed at a minimum frequency of 2 week intervals. This technique is also called retro-orbital, peri-orbital, and orbital venous plexus bleeding. 1. Gently scruff the neck and pull the skin taut around the eye until the eyeball protrudes out. 2. Insert a capillary tube into the medial canthus of the eye at a 25–30-degree angle to the nose. Subsequently, add slight pressure to puncture into the plexus/sinus tissue. 3. Slightly pull back the capillary tube so that blood flows out into the tube. 4. After an adequate volume of blood is obtained, restraint can be loosened and the capillary tube can be gently removed. 5. Wipe excess blood with a sterile cotton ball and stop bleeding with gentle pressure. 6. Carefully monitor for adverse effects after the procedure.

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3.1.3 Caudal Vena Cava and Heart

Caudal Vena Cava

These methods are terminal procedures suitable for obtaining a large volume (up to 1 ml in mice or up to 15 ml in rats) and a high quality sample, although the sample can be taken only once. The animal should be deeply anesthetized or euthanized prior to starting the procedure. 1. Anesthetize the animal and confirm the anesthetic depth by checking the toe pinch and corneal reflex. 2. Place animal in dorsal recumbency (on back) and immobilize the four limbs using cellophane tape. 3. Wet the skin on the abdomen with 70% ethanol. 4. Make a V-cut to open the abdominal cavity. 5. Move aside the internal organs to visualize the caudal vena cava. 6. Carefully remove the connective tissues around the caudal vena cava by gently swiping the surface with sterilized cotton. 7. Insert 23 G needle with 1 ml syringe (21 G for rats) into the vena cava and advance the needle to the part draining the renal vein. Insert the needle bevel side down when entering the vein. 8. Gently apply negative pressure to the syringe plunger to collect the blood sample. Avoid collapsing the vena cava by applying too much negative pressure. If the blood does not flow, carefully rotate the needle slowly. 9. After an adequate sample is obtained, remove the needle and if necessary perform euthanasia, such as cervical dislocation or exsanguination, and ensure the animal is deceased.

Cardiac Puncture

1. Anesthetize the animal and confirm the anesthetic depth by checking the toe pinch and corneal reflex. 2. Place animal in dorsal recumbency (on back) and immobilize the four limbs with cellophane tape. 3. Wet the skin on the abdomen with 70% ethanol. 4. Make a V-cut through the abdomen to the upper ribs to open the thoracic cavity along the costal cartilage. 5. Insert 23 G needle with a 1 ml syringe (21 G in the rat) into the right ventricle. 6. Gently apply negative pressure to the syringe plunger, but not enough to collapse the heart chamber. If no blood appears, slowly rotate the needle or move it slightly in or out. 7. After an adequate sample is obtained, remove the needle and if necessary perform euthanasia, such as cervical dislocation or exsanguination, and ensure the animal is deceased.

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3.2 Blood Sampling from Rabbits

Blood samples are commonly taken from the marginal ear vein, and this method is the least invasive method in rabbits. Up to 10 ml of blood can be collected at once by using this method. Seven or eight samples can be taken in a 24-h interval.

3.2.1 Marginal Ear Vein

1. Local anesthetic cream may be applied to the skin of the ear 20–30 min prior to sampling. 2. Use a restrainer for immobilizing the rabbit. 3. To dilate the marginal ear vein, warm the ear using a heat lamp or by gently stroking the ear. 4. Sterilize the surface of the ear with 70% ethanol. 5. Insert a 26 G butterfly needle with syringe into the marginal ear vein to collect the blood sample. Blood is usually taken from the tip of the ear. Otherwise, cut the marginal ear vein with a clean scalpel blade and collect the sample under local anesthesia. 6. Stop bleeding by gently applying pressure to the collection site.

3.3 Blood Sampling from Dogs and Cats

In both dogs and cats, blood samples are taken from the cephalic, jugular, or medial saphenous vein. Blood can also be taken from the lateral saphenous vein in dogs. Anesthesia is usually not necessary to obtain a sufficient blood sample if the manual restraint is adequate.

3.3.1 Cephalic Vein

The cephalic vein is an easily accessible superficial vein in the forelimb from which 1–5 ml of blood can be taken. This method is suitable for multiple sampling; up to 8 samples may be taken in a 24-h period. 1. Have an assistant manually restrain the animal. 2. Occlude the cephalic vein by adding pressure on the vein or using a tourniquet. 3. If necessary, shave the area where the cephalic vein is most prominent. 4. Wet the area about to be punctured with 70% ethanol. 5. Insert a 23 G needle with syringe into the cephalic vein and pull back the syringe plunger after a small amount of blood enters the tip of the syringe. 6. After obtaining the sample, remove the needle and release the pressure over the vein. 7. Apply pressure over the venipuncture site with a sterile cotton ball to stop the bleeding.

3.3.2 Jugular Vein

The jugular vein is used for obtaining larger volumes (2–20 ml) of high quality of blood samples. Multiple samples can be taken (up to 8 samples) in a 24-h period.

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1. Have an assistant manually restrain the animal. 2. If necessary, shave the collection site. 3. Wet the skin with 70% ethanol. 4. Visualize the vein by digitally applying pressure to the thoracic inlet. 5. Insert a 21 G needle with syringe to collect a blood sample. 6. After obtaining the sample, remove the needle and release pressure over the vein. 7. Apply pressure over the venipuncture site with a sterile cotton ball to stop the bleeding. 3.3.3 Medial Saphenous Vein

The medial saphenous vein is used for obtaining small volume blood samples in dogs and cats when the cephalic and jugular veins are not available or should be avoided for blood sampling. Multiple samples can be taken from this vein. 1. Restrain the cat in lateral recumbency. 2. Wet the medial aspect of the leg with 70% ethanol. 3. Occlude the vein with pressure in the inguinal region to visualize the medial saphenous vein. 4. Insert a 23 G needle with syringe into the vein and collect the necessary volume of blood. 5. After obtaining the sample, apply pressure to the venipuncture site to stop the bleeding.

3.3.4 Lateral Saphenous Vein

The lateral saphenous vein is used for obtaining small volume blood samples in dogs when the cephalic and jugular veins are not available or should be avoided for blood sampling. Multiple samples can be taken from this vein. 1. Have an assistant restraint the animal on lateral recumbency. 2. To visualize the lateral saphenous vein, the assistant holds and encircles the pelvic limb just below the knee by hand or using a tourniquet. 3. If necessary, shave the collection site on the pelvic limb. 4. Puncture the vein using a 23 G needle with syringe and collect the necessary volume of blood. 5. Apply pressure to the venipuncture site to stop the bleeding.

3.4 Blood Sampling from Cows

In cows, blood samples are obtained from the jugular vein or tail vein. For safe sampling, it is important to properly immobilize the animal by roping or using a suitable restrainer. If necessary, use sedation by intramuscularly administering 0.05 mg/kg of xylazine.

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Ten to twenty milliliter of blood sample can be taken at once. Multiple samples (up to 5–10 samples) may be taken in any 24-h period. 1. Restrain the cow and hold the head up so that the jugular groove is accessible. 2. Visualize the jugular vein by adding firm pressure to the base of the jugular groove. 3. Insert an 18 G needle with syringe into the prominent vein just above the digit applying pressure at a 20-degree angle to the skin. 4. Gently apply negative pressure to the syringe plunger to draw blood after a small amount of blood enters the tip of the syringe. 5. After obtaining the sample, remove the needle and apply pressure to the venipuncture site to stop the bleeding.

3.4.2 Tail Vein

Two to three milliliter of blood sample can be taken at once. Multiple samples (up to 5–10 samples) may be taken in any 24-h period. 1. Immobilize the head of the animal. 2. Raise the tail vertically, so it is parallel to the ground. 3. Insert a 20 G needle with syringe to the vein at approximately 10 cm from the tail base (3rd–4th coccygeal vertebra). The needle should be inserted slightly to the right of midline at a 30- to 45-degree angle. 4. Gently apply negative pressure to syringe plunger to draw blood after a small amount of blood enters the tip of the syringe. 5. Apply pressure to the venipuncture site to stop the bleeding.

3.5 Blood Sampling from Horses

In horses, blood samples are commonly obtained from the jugular vein. It is important to relax the horse properly before obtaining a blood sample. A properly trained assistant should gently restrain the head of horse by holding the halter and rope. Next, the operator should approach while calmly talking to the horse. Avoid using stressful devices such as a twitch. If necessary, administer 4–6 μg/kg of medetomidine intravenously with 0.05 mg/kg of butorphanol intramuscularly, or alternatively administer 2.5 mg/kg of ketamine intravenously for sedation.

3.5.1 Jugular Vein

Please refer to Subheading 3.4.1

3.6 Blood Sampling from Goats

In goats, blood samples are commonly obtained from the jugular vein. Goats can be easily immobilized by manual restraint, and blood samples are easy to obtain due to their thin skin. For

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restraint, an assistant holds the head of the animal with their hands. The jugular vein is easily accessed if the assistant pushes the neck of the goat to the side with their body. Sedation is usually not necessary for goats. 3.6.1 Jugular Vein

Please refer to Subheading 3.4.1

3.7 Blood Sampling from Pigs (see Note 2)

In pigs, blood samples are taken from the jugular vein or caudal auricular vein. The jugular vein is commonly used for blood sampling from pigs 8 weeks or older. The caudal auricular vein is used for blood sampling from adult pigs, especially from breeds having large ears such as Landrace and Duroc. A hog catcher is useful for immobilizing pigs. For sedation, the single administration of mafoprazine mesylate, azaperone, droperidol, or a combination of xylazine and butorphanol, or medetomidine and midazolam, is commonly used. Mafoprazine mesylate, which has weaker actions on the extrapyramidal system compared to azaperone, is intramuscularly administered into the root of the ear at the dose of 0.3 mg/kg. Azaperone is intramuscularly administered at the dose of 1–4 mg/ kg. It should be noted that azaperone does not sufficiently suppress sensory stimulation such as auditory, tactile sensation, and pain; thus, slight stimulation can awaken pigs during the procedure. Droperidol is intramuscularly administered at the dose of 0.1–0.4 mg/kg. Xylazine and butorphanol are concurrently administered at the dose of 2 mg/kg and 0.2 mg/kg by intramuscular injection, respectively. Medetomidine and midazolam is concurrently administered at the dose of 40 μg/kg and 0.2 mg/kg by intramuscular injection, respectively. The combination of medetomidine and midazolam accomplishes strong hypnogenesis, chemical immobilization, and reduced reactivity from environmental stimulus such as auditory, tactile sensation, and pain. Intramuscularly administering atipamezole at the dose of 160 μg/kg quickly reverses the strong sedative effects.

3.7.1 Jugular Vein

The external jugular vein is commonly used for obtaining blood samples from pigs 8 weeks or older. This vein is suitable for multiple samplings and a sample up to 20 ml of blood can be collected at once. 1. Restrain the pig by using a hog catcher. 2. To visualize the vein, turn the head upward and retract the thoracic limb caudally to sufficiently stretch the neck. 3. After confirming the correct puncture site, which is in the jugular groove between the medial sternocephalic and lateral brachiocephalic muscles, perpendicularly insert a 20 G needle with syringe into the vein through the skin. The needle should

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be inserted in its full length due to the thick adipose tissue above the vein. 4. Gently apply negative pressure to the syringe plunger to draw blood after a small amount of blood enters the tip of the syringe. 5. Apply pressure to the venipuncture site to stop the bleeding. 3.7.2 Caudal Auricular Vein

The caudal auricular vein is used for blood sampling from adult pigs with large ears. One to two milliliter of blood may be obtained from this vein at once. It should be noted that the auricular vein might continue to bleed for up to several minutes after venipuncture. 1. Restrain the pig with a hog catcher. 2. Swipe the surface of the auricular vein with 70% ethanol. 3. Occlude the vein by adding pressure at the base of the ear or using a tourniquet. 4. Insert a 20 G butterfly needle with syringe into the vein from the tip of the ear. 5. Gently apply negative pressure to the syringe plunger to draw blood after a small amount of blood enters the tip of the syringe. 6. Apply pressure to the venipuncture site to stop the bleeding.

3.8 Blood Sampling from Wild Boars (see Note 2)

3.8.1 Cardiac Puncture

In the wild boar, blood samples are commonly taken from heart (cardiac puncture) following euthanasia. If the blood sample has to be taken from live wild boars, the medial saphenous vein is recommended for blood sampling. It is difficult to obtain samples from other veins such as jugular, cephalic, and lateral saphenous veins due to their hard skin. The wild boar should be under adequate anesthesia prior to sampling to ensure operator safety. A combination of medetomidine and ketamine can be administered intramuscularly for sedation and anesthesia at the dose of 80 μg/kg and 5 mg/kg, respectively. Additional dosage may be required depending on the body weight. 1. Euthanize the wild boar with an appropriate method. 2. Open the thoracic cavity and insert an 18 G needle with syringe into the heart as soon as possible before the blood coagulates. Blood samples from wild boars may coagulate rapidly compared to blood samples from other animals. 3. Transfer the blood sample to appropriately sized heparinized tubes.

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3.8.2 Medial Saphenous Vein

1. Anesthetize the wild boar prior to the procedure. 2. Restrain the wild boar in lateral recumbency. 3. Shave the hair with hair clippers and wet the medial aspect of the leg with 70% ethanol. 4. Occlude the vein with pressure in the inguinal region to visualize the medial saphenous vein. 5. Insert a 22 G needle with syringe into the vein and collect the necessary volume of blood. 6. After obtaining the sample, apply pressure to the venipuncture site to stop the bleeding.

3.9 Method for Evaluating Lymphocyte Metaphase Chromosomes (for Mice, Rats, Dogs, Cats, Cows, Horses, Goats, Pigs, and Wild Boars)

This method is suitable to evaluate lymphocyte metaphase chromosome for mice [3–6], rats [5], dogs [7, 8], cats [9], cows [10], horses [11], goats [10], pigs [10, 12–14], and wild boars [15]. 1. Blood sampling: Transfer 5 ml of the blood sample into a normal heparinized tube. Alternatively, you may use a heparinized tube with a gel barrier for separation of mononuclear cells (BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube—Sodium Heparin, BD Biosciences, NJ, USA) to minimize variability from sample processing. 2. Lymphocyte separation: Separate the lymphocytes from other blood components with Lymphocyte Separation Medium (Organon Teknika, Durham, NC) [5]. If you use blood collection tube with a separating agent, centrifuge the tube according to the manufacturer’s protocol. 3. Resuspension: Resuspend the lymphocytes at a concentration of 7  105 cells/ml in culture medium (RPMI 1640 Dutch Modification, Gibco, Thermo Fisher Scientific, MA, USA) containing 20% fetal bovine serum (FBS), 2 mM of L-glutamine, antibiotics (75 IU—75 μg/ml of penicillinstreptomycin), and mitogen (180 μg/ml of phytohemagglutinin, PHA-M, Wellcome Diagnostics, Beckenham, Kent, UK). PB-MAX™ Karyotyping Medium (Gibco) or Ham’s F10 medium may be used instead of RPMI 1640 culture medium. Kanamycin may be used at the dose of 60 μg/ml instead of penicillin-streptomycin. Six μg/ml of CON-A (Type IV, Sigma, MO, USA) and/or 50 μg/ml of lipopolysaccharides (LPS, Sigma) may be used instead of or with PHA as mitogen. For pigs and wild boars, 50 ng/ml of phorbol myristate acetate (PMA, Sigma) and 1 μg/ml of ionomycin (Sigma) must be used instead of PHA as mitogen. For horses, lectin from Phytolacca americana (pokeweed, Sigma) must be used instead of PHA as mitogen [11]. 4. Incubation: Put 1 ml of the medium containing lymphocytes into a culture tube. After loosening the cap of the tube, place

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the tube into a CO2 incubator and maintain at 37  C with 5% CO2 for 47 h. Add the 0.1 μg/ml of colcemid (Gibco) during the last 6 h of the incubation period; this technique helps to yield a bigger size of chromosome than with the continuous treatment of colcemid. After 48 h, centrifuge the tubes and discard the supernatant. 5. Hypotonic treatment: Add 4 ml of 75 mM KCl solution and place the tubes into a 37  C water bath for 20 min. 6. Fixation: Add the 2 ml of Carnoy solution to the lymphocytes. Spin down the cells at 200 x g for 5 min, and discard the supernatant. Subsequently, gently tap to loosen the pellet. Repeat this process three times. 7. Giemsa stain: Add and air dry the 10–100 μl of solution on a clean slide. Place the slides into a Coplin staining jar with Giemsa solution for 5 min. After rinsing the slides with running tap water, let them air dry. 8. Mounting: Place an appropriately size coverslip with mounting solution such as Cytoseal™ (Richard-Allan Scientific, Thermo Fisher Scientific). 9. Microscopic examination and analysis: Observe the slides under a microscope. Find the area with a nice spread of metaphase cells (see Note 3). Ideal metaphase spread has minimal overlapping chromosomes that are not too extensively spread out (Fig. 1) (see Notes 4 and 5). Analyze dicentric chromosome yields for 100 cells (experiments with over 1 Gy of radiation exposure) or 1000 cells (experiments with 0.1–0.9 Gy of radiation exposure). A dicentric chromosome should have a centric

Fig. 1 Properly spread metaphase chromosomes of a peripheral lymphocyte. The metaphase chromosomes from a peripheral lymphocyte properly spread with minimum overlaps of chromosomes. The black arrows indicate dicentric chromosomes or acentric fragments

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fragment within the same cell. Mouse and dog chromosomes are acrocentric. The C-band technique can be used to stain centromeres darker, which may improve the analysis of dicentric chromosomes. 3.10 Method for Evaluating Lymphocyte Metaphase Chromosomes (for Rabbits)

The original method by Evans et al. [16] may be ineffective for rabbit lymphocytes. This modified method is thus used for yielding a better outcome when using rabbit lymphocytes [17]. 1. Blood sampling*. 2. Preparation of culture medium: Prepare the 4 ml of Parker Medium containing 75 μg/ml of penicillin-streptomycin, 1 ml of FBS, 0.3 mg/ml of L-glutamine, and 180 μg/ml of PHA (Wellcome). 3. Incubation: Add 0.4 ml of blood into the culture medium. Subsequently, incubate the sample at 38.5  C for 15 h. Add 0.3 μg/ml of colcemid the last 3 h. After the incubation, refresh the medium to a medium without PHA. 4. Separation of the lymphocyte: Separate the lymphocytes by using a micropipette or other equipment. Centrifugation is usually not necessary because the cells naturally sediment at the bottom of the culture tube. 5. Hypotonic treatment*. 6. Fixation*. 7. Giemsa stain*. 8. Mounting samples*. 9. Microscopic examination and analysis*. *Please see the description above.

3.11 Anticipated Results

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Approximately 1–2 dicentric chromosomes will be found per 1000metaphase chromosome spread from healthy cells. One Gy of radiation exposure can increase the yield of dicentric chromosomes to about 100 dicentric chromosomes per 1000 metaphase cells for cats, and 150 dicentrics chromosomes per 1000 metaphase cells for dogs [2].

Notes 1. Obtaining blood from animals requires adequate animal handling skills. 2. Blood from pigs and wild boars can clot easily; appropriate and timely handling is required [18]. 3. If the mitosis yield is low, change PHA-M to a different lot or product. PHA-M quality is important for this experiment.

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4. If metaphase chromosomes are too spread out, reduce the KCl treatment time to 15 min. 5. If metaphase chromosomes are not spread out enough, place glass slide into ice cold water. Moisture may help the metaphase chromosome spread quality.

Acknowledgments We thank Dr. Furuhama for kindly giving us his protocols for blood sampling. References 1. Sankaranarayanan K (1976) Evaluation and re-evaluation of genetic radiation hazards in man. II. The arm number hypothesis and the induction of reciprocal translocations in man. Mutat Res 35(3):371–386 2. Fujii Y, Yurkon CR, Maeda J, Genet SC, Kubota N, Fujimori A, Mori T, Maruo K, Kato TA (2013) Comparative study of radioresistance between feline cells and human cells. Radiat Res 180(1):70–77. https://doi.org/10. 1667/RR3194.1 3. Takeshita T, Conner MK (1984) Accumulation and persistence of cyclophosphamide-induced sister chromatid exchange in murine peripheral blood lymphocytes. Cancer Res 44 (9):3820–3824 4. Nishi Y, Hasegawa MM, Ohkawa Y, Inui N (1986) Mouse peritoneal lymphocytes, a new target for analyzing induction of sister chromatid exchanges on in vivo exposure to a genotoxic agent. Cancer Res 46(7):3341–3347 5. Erexson GL, Kligerman AD, Bryant MF, Sontag MR, Halperin EC (1991) Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes. Mutat Res 253(2):193–198 6. Kanda R, Shang Y, Tsuji S, Eguchi-Kasai K, Hayata I (2004) An improved culture system of mouse peripheral blood lymphocytes for analysis of radiation-induced chromosome aberrations. Biosci Rep 24(6):641–650. https://doi.org/10.1007/s10540-005-27984 7. Catena C, Conti D, Villani P, Nastasi R, Archilei R, Righi E (1994) Micronuclei and 3AB index in human and canine lymphocytes after in vitro X-irradiation. Mutat Res 312 (1):1–8 8. Leonard A, Decat G, Fritz TE (1982) Radiosensitivity and life span of dog peripheral blood lymphocytes. Mutat Res 92(1–2):257–263

9. Stephan G, Adler ID, Schwartz-Porsche D, Hollihn KU, Obe G (1979) Characterization of feline whole-blood cultures and determination of the frequency of radiation-induced dicentrics in human and feline lymphocytes. Int J Radiat Biol Relat Stud Phys Chem Med 35(4):351–359 10. Leonard A, Gerber GB, Papworth DG, Decat G, Leonard ED, Deknudt G (1976) The radiosensitivities of lymphocytes from pig, sheep, goat and cow. Mutat Res 36 (3):319–332. https://doi.org/10.1016/ 0027-5107(76)90242-6 11. Raudsepp T, Santani A, Wallner B, Kata SR, Ren C, Zhang HB, Womack JE, Skow LC, Chowdhary BP (2004) A detailed physical map of the horse Y chromosome. Proc Natl Acad Sci 101(25):9321–9326. https://doi. org/10.1073/pnas.0403011101 12. McFee AF (1977) Chromosome aberrations in the leukocytes of pigs after half-body or wholebody irradiation. Mutat Res 42(3):395–400. https://doi.org/10.1016/S0027-5107(77) 80044-4 13. Remy J, Martin M, Haag J (1984) Radiosensitivity of swine lymphocytes: in vitro modification of the cell cycle and kinetics of the appearance of chromosomal aberrations. Mutat Res 126(2):169–175. https://doi.org/ 10.1016/0027-5107(84)90059-9 14. Bianchi MS, Bianchi NO, Larramendy M, Garcia-Heras J (1981) Chromosomal radiosensitivity of pig leucocytes in relation to sampling time. Mutat Res 80(2):313–320. https://doi.org/10.1016/0027-5107(81) 90104-4 15. Bosma AA (1976) Chromosomal polymorphism and G-banding patterns in the wild boar (Sus scrofa L.) from the Netherlands. Genetica 46(4):391–399. https://doi.org/10. 1007/bf00128086

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16. Evans HJ (1965) A simple microtechnique for obtaining human chromosome preparations with some comments on dna replication in sex chromosomes of the goat, cow and pig. Exp Cell Res 38:511–516 17. Bajerska A, Liniecki J (1975) The yield of chromosomal aberrations in rabbit lymphocytes after irradiation in vitro and in vivo. Mutat

Res 27(2):271–284. https://doi.org/10. 1016/0027-5107(75)90088-3 18. Kostering H, Mast WP, Kaethner T, Nebendahl K, Holtz WH (1983) Blood coagulation studies in domestic pigs (Hanover breed) and minipigs (Goettingen breed). Lab Anim 17(4):346–349. https://doi.org/10. 1258/002367783781062262

Chapter 3 Micronuclei Formation Analysis After Ionizing Radiation Cathy Su, Alexis H. Haskins, and Takamitsu A. Kato Abstract Micronuclei are formed by broken chromosome fragments or chromosomes, which were not appropriately separated into the daughter cells’ nuclei after division. The appearance of micronuclei is typically a sign of genotoxic events. Majority of micronuclei are formed by broken acentric fragments, but some micronuclei are formed by centric chromosome fragments which were not appropriately separated to daughter cells’ nuclei. Because researchers only need to measure visible micronuclei in binucleated cells, micronuclei analysis is much easier than metaphase chromosome aberration analysis discussed in the previous chapter. This method does not require professional training compared to metaphase chromosome aberration analysis. In addition, one can analyze many samples in a relatively short time. Not only ionizing radiation, but other genotoxic stress also induces micronuclei formation. The background frequency of micronuclei is noticeably higher than chromosome aberrations. But researchers can easily analyze 300–1000 binucleated cells per data point to obtain statistically significant differences of irradiated samples. In this chapter, we will discuss the advantages and preparation of micronuclei samples. Key words Micronuclei, Cytochalasin B, Binucleated cells, Cytokinesis, Acentric fragments, Genotoxicity

1

Introduction Genotoxic damage is not limited to ionizing radiation exposure. Some industrial activities and occupations associated with chemicals also affect human genetic integrity by various chemical and physical genotoxic stresses, which eventually cause damage in DNA of the cell [1, 2]. The cytogenetic damages observed in such an environmental and industrial pollution and occupational hazards are often associated with elevated chromosome aberrations with a high risk of cancers [3]. Therefore, it is important to develop a rapid and sensitive method for screening the potential genotoxic agents. Examination of metaphase chromosomes treated with potential genotoxic agent with chromosomal aberrations is a classical method for investigating cytogenetic damages [2]. However, the metaphase chromosome aberrations analysis requires trained personnel and time. Moreover, automation analysis of chromosome

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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aberrations is not available yet. In contrast to this classical method, observing the frequency of micronuclei in testing cells provides a sensitive and rapid way for screening genotoxic agents [2]. Moreover, micronuclei analysis needs less training compared to chromosome aberration analysis. The automation detection of binucleated cells and micronuclei has been developed and shortened time to result [4, 5]. In general, micronuclei originate from the acentric fragments of chromosomes or lagging acentric chromosomes, which are not properly incorporated into daughter nuclei during mitosis due to non-attachment of microtubules [2, 6]. Acentric fragments at mitosis can be formed by double strand breaks at G0/G1 phase (interstitial deletions and terminal deletions), a replication collapse during S-phase by single strand breaks or DNA repair deficiency and base damages formed at G0/G1 phase (chromatid type breaks and exchanges), and double strand breaks during G2 phase (chromatid type breaks and exchanges). Loss of DNA damage repair increases background and induced micronuclei formation. On the other hand, the formation of micronuclei with centromere signals is originated from the missegregation of a whole chromosome or a part of a chromosome with a centromere [6]. This can be detected by centromere specific FISH probes. Cytochalasin B is an agent that has been most widely used to block cytokinesis and analyze micronuclei formation; however, other actin inhibitor agents can be used if necessary. Cytochalasin B inhibits actin polymerization and cytokinesis, Therefore, it prevents separation of daughter cells at the end of mitosis, incompletion of cytokinesis and leads to a binucleated cell formation. It is important to examine micronuclei frequency in binucleated cells. Binucleation is evidence that the cells have reached cytokinesis. Researchers should not analyze tetranucleated cells, because these cells had two cytokinesis events and may have altered micronuclei frequency compared to binucleated cells. Centromere signals can be detected with FISH (fluorescence in situ hybridization) probes or immunochemical staining against the centromere region by CREST serum [7]. A centromere positive signal can be used as a modification of the micronuclei analysis method. Radiation-induced micronuclei are often present without centromere signals. On the other hand, Vinblastine, a tubulin inhibiting anti-tumor drug, presents many micronuclei with centromere signals [8]. Therefore, dysfunction of chromosome segregation during mitosis increases micronuclei with centromere signals [6, 9, 10]. Radiation-induced micronuclei are unstable with or without centromeres. Micronuclei frequency declines to the basal level 7 days after 3 Gy irradiation to lymphoblstoid cells [11]. Therefore as a biodosimetry, a time limitation may exist for micronuclei formation analysis. However, micronuclei formation is

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a great indicator of chromosomal aberrations and chromosome mis-segregation, which leads to genetic abnormality and instability [2, 6].

2

Materials 1. Appropriate cell culture medium such as MEM, alphaMEM, DMEM, and RPMI1640. 2. Fetal Bovine Serum (FBS), prior lot testing is required. Some lots increase background level chromosomal aberrations. Heat inactivation at 56  C for 20 min is required. 3. Phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 in distilled water and adjust pH 7.4, then autoclaved for sterilization) or purchase PBS ( ) from ThermoFisher (minus means no Calcium, no Magnesium, no Phenol Red). 4. Antibiotics (Antibiotic-Antimycotic (100) Catalog number 15240096, penicillin streptomycin solution or kanamycin 60 mg/ml). 5. Trypsin-EDTA (0.05% Trypsin/0.002% EDTA-4Na in Hanks Balanced Salt Solution). 6. 1 mg/ml stock solution of Cytochalasin B (Sigma) dissolved in PBS. Final concentration should be in a range of 2–8 μg/ml (see Note 1). 7. Potassium chloride (KCl) (Sigma): dissolve in distilled water to make 75 mM KCl solution. 2.8 g of potassium chloride in 500 ml of distilled water. 8. Carnoy solution (3:1 mixture of Methanol and Acetic Acid), freshly mixed. 9. 5% Giemsa solution. Mix 2.5 ml of KaryoMAX Giemsa stain solution (GIBCO Catalog number 10092013) with 47.5 ml Gurr solution. Freshly mixed. Gurr solution is made from Gurr buffer tablet (GIBCO, Catalog number 10582013) dissolved in distilled water.

2.1

Equipment

1. Cell culture facility including biosafety cabinet or clean bench, 37  C CO2 controlled incubator, and water bath to keep 37  C. 2. Centrifuge, which can rotate 15 ml tubes. No need of temperature control or brake. 3. Aspirator to remove supernatant and trap waste. 4. Microscope equipped with CCD camera and software. 5. Slide glass, coverslip, and Cytoseal 60 (Richard-Allan) Mounting Medium.

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Methods 1. Cell cycle population is important for micronuclei assay. Cells should be used in exponentially growing log phase or synchronized population. (a) e.g., Mitotic shake off or contact inhibition can synchronize cell cycle population into G1 or G0/G1 phase. If lymphocytes are used, all lymphocytes are G0 phase without PHA stimulus. 2. After ionizing radiation, add cytochalasin B for a final concentration of 2–8 μg/ml. Then cells should be kept in CO2 incubator and maintain 37  C. (a) For lymphocytes, PHA stimulus should be done after irradiation with cytochalasin B treatment. 3. Incubate cells, at least one cell cycle time. (a) Lymphocyte, 48–60 h. (b) Human cancer cells, typically 24–40 h. (c) Hamster cells, 12–20 h. 4. For adhesive cells, aspirate medium, add PBS, aspirate PBS, and add trypsin-EDTA. After a few minutes of trypsin treatment, cells should be detached from dish/flask and single cell suspension should be transferred to a tube. Obtained single cell suspension or lymphocyte cells should be centrifuged and discard the supernatant (see Note 2). 5. Gently break pellet and add 4 ml of 75 mM KCl solution. Then add 2 ml of Carnoy solution (see Notes 3 and 4). 6. Spin down and discard the supernatant. Gently disturb pellet by tapping. 7. Add 2 ml of Carnoy solution. Spin down and discard the supernatant. (a) Compared to the metaphase chromosome spread preparation, only one or two fixations are enough for micronuclei samples because complete loss of cellular membrane will cause difficulty in identifying binucleated cells. 8. Drop 10–100 μl of solution onto clean slides. (a) Adjust cellular concentration by adding fixative solution if necessary. 9. Let them air dry. 10. Slides will be placed into Coplin Jar with Giemsa solution for 5 min. Rinse with tap water (see Note 5). 11. Put on coverslip with Cytoseal.

Micronuclei Formation Analysis After Ionizing Radiation

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Fig. 1 Two binucleated CHO cells. One has no micronuclei, another has one micronucleus

12. Observe under a microscope with objective lens, at least 40 magnification. 13. Find binucleated cells and count micronuclei formation. (a) Micronuclei cannot be formed if cells do not reach cytokinesis. So, do not count single nucleus cells. (b) Binucleated cells should have two nuclei in a cell (Fig. 1). (c) Do not count tetra nucleated cells because these cells tried to divide twice and may carry more micronuclei than binucleated cells. 14. Obtain micronuclei (MN) per binucleated (BN) cell. (a) Count number of micronuclei per binucleated cell. If two micronuclei are observed in a single binucleated cell, count as 2. If three micronuclei are observed in a single binucleated cell, count at 3, etc.

4

Notes 1. If binucleated cell frequency is too low, change concentration of Cytochalasin B. Optimal concentrations should be in the range of 2–6 μg/ml. If concentrations are too high, cytotoxic effects may arise. 2. Adhesion cells can be fixed without trypsinization by methanol, Carnoy’s solution, or paraformaldehyde.

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3. Longer KCl solution treatment may improve the quality of cells by swelling them, but it may reduce cytoplasmic staining to distinguish binucleated cells. 4. If cellular membrane is completely destroyed and morphology of binucleated cell is lost, add 20 μl of 37% formaldehyde solution after final centrifugation. It may keep cellular membrane intact when cells are dropped onto slides. 5. Alternatively, Acridine Orange staining can be used with fluorescence microscope. Also, anti-actin antibody fluorescent immunostaining with DAPI can be applied with fluorescence microscope with proper filter sets.

5

Anticipated Results Approximately 1–3% of binucleated cells contain micronuclei in the natural background level of healthy human lymphocyte cells [4, 12, 13]. 0.5 Gy of radiation exposure can increase the yield of micronuclei to approximately 85 micronuclei per 1000 binucleated cells [4]. 1 Gy of radiation exposure can increase the yield of micronuclei to approximately 90 micronuclei per 1000 binucleated cells [12]. 2 Gy of radiation exposure can increase the yield of micronuclei to approximately 200 micronuclei per 1000 binucleated cells [13]. Micronuclei can be stained by centromere or telomere signals to investigate micronuclei in depth [14, 15].

References 1. Fenech M (1993) The cytokinesis-block micronucleus technique and its application to genotoxicity studies in human populations. Environ Health Perspect 101(Suppl 3):101–107 2. Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW, Salamone MF (1983) The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency GeneTox Program. Mutat Res 123:61–118 3. Picciano D (1979) Cytogenetic study of workers exposed to benzene. Environ Res 19:33–38 4. Bertucci A, Smilenov LB, Turner HC, Amundson SA, Brenner DJ (2016) In vitro RABiT measurement of dose rate effects on radiation induction of micronuclei in human peripheral blood lymphocytes. Radiat Environ Biophys 55:53–59 5. Repin M, Pampou S, Karan C, Brenner DJ, Garty G (2017) RABiT-II: implementation of a high-throughput micronucleus biodosimetry

assay on commercial biotech robotic systems. Radiat Res 187:492–498 6. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond DA, Tucker JD, Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125–132 7. Caria H, Chaveca T, Laires A, Rueff J (1995) Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes. Mutat Res 343:85–94 8. Salassidis K, Huber R, Zitzelsberger H, Bauchinger M (1992) Centromere detection in vinblastine- and radiation-induced micronuclei of cytokinesis-blocked mouse cells by using in situ hybridization with a mouse gamma (major) satellite DNA probe. Environ Mol Mutagen 19:1–6

Micronuclei Formation Analysis After Ionizing Radiation 9. Bakhoum SF, Genovese G, Compton DA (2009) Deviant kinetochore microtubule dynamics underlie chromosomal instability. Curr Biol 19:1937–1942 10. Gisselsson D (2008) Classification of chromosome segregation errors in cancer. Chromosoma 117:511–519 11. Ramirez MJ, Surralles J, Puerto S, Creus A, Marcos R (1999) Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells. Mutat Res 440:163–169 12. Fenech M, Morley AA (1985) Measurement of micronuclei in lymphocytes. Mutat Res 147:29–36

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13. Fujii Y, Yurkon CR, Maeda J, Genet SC, Kubota N, Fujimori A, Mori T, Maruo K, Kato TA (2013) Comparative study of radioresistance between feline cells and human cells. Radiat Res 180:70–77 14. Lindberg HK, Falck GC, Jarventaus H, Norppa H (2008) Characterization of chromosomes and chromosomal fragments in human lymphocyte micronuclei by telomeric and centromeric FISH. Mutagenesis 23:371–376 15. Miller BM, Werner T, Weier HU, Nusse M (1992) Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes. Radiat Res 131:177–185

Chapter 4 G1 Premature Chromosome Condensation (PCC) Assay Ryuichi Okayasu and Cuihua Liu Abstract Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage. Key words Premature chromosome condensation (PCC), G1-phase, Cell fusion, Sendai virus, Polyethylene glycol (PEG), DNA double strand breaks

1

Introduction The phenomenon of premature chromosome condensation (PCC) was first described in early 1970s by Johnson and Rao [1]. Since then the PCC technique has been applied to detect interphase (mainly G1 and G2 phases) chromosome damage and repair in mammalian cells exposed to relatively low doses of ionizing radiation [2–10]. As this chapter focuses on G1 type PCC, we discuss the method to induce PCC by fusing G1 type cells with mitotic cells [11]. The basic principle for G1 PCC induction would be that mitosis-promoting factor (MPF) can transfer from mitotic cells to G1 cells after cell fusion and help condense the chromosomes of G1 cells. Thus G1 type chromosomes can be visible as in metaphase cells since they are fully condensed. There are basically two methods to fuse cells, either using polyethylene glycol (PEG) or inactivated Sendai virus; instead of using Sendai virus itself, the envelope of the virus called HVJ-E (hemaglutinating virus of Japan) could be used for the same purpose [6, 9]. We have shown that the results are identical whichever fusion method one uses [11]. Another

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_4, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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necessary factor for G1-type PCC is mitotic cell for PCC induction. Any kind of mammalian cells could be used for this purpose, but depending on the MPF activity of a particular cell line, the number of G1 PCC breaks in irradiated cells may be different [12]. In general two cell lines, CHO (Chinese hamster ovary) and HeLa (human cervical cancer) cells, are most commonly used to obtain mitotic cells. Although some preparations such as mitotic cells and a cell fusing agent are needed to detect G1 type chromosomes by PCC, this technique can provide immediate chromosome damage (mainly breaks) at G1 phase and repair kinetics of chromosome damage can also be assessed. By observing ring type chromosomes in G1 PCC samples, one can also study a degree of mis-rejoining in irradiated G1 cells [13]. Evaluation of these end points is not possible with the conventional chromosome aberration method at metaphase as one has to wait until interphase (G1 or G2) cells going through the cell cycle to reach mitotic stage. The PCC method is especially useful for cells with complex DNA damage which might be induced by high linear energy transfer (LET) radiation as some of the damaged cells by high LET may not advance to metaphase stage. As DNA double strand break (DSB) is thought to be the key damage for various biological consequences following radiation exposure and chromosome aberrations mainly observed at metaphase are closely correlated with biological end points including cell survival, PCC assay could reveal biological processes in between DNA DSBs and chromosome aberrations generally observed at metaphase. Furthermore, by combining G1 PCC and fluorescence in situ hybridization (FISH), observation of mis-rejoined chromosomes at G1 stage is readily obtained (Fig. 1) [14–16].

2 2.1

Materials Cell Culture

1. Cells: Any mammalian cells at G1 or G0 stage can be used for G1 PCC assay. 2. Mitotic inducer cells for PCC: CHO, HeLa, XP2OS (repairdeficient xeroderma pigmentosum cells) and other cell lines could be used. We describe the case for harvesting mitotic HeLa cells below. CHO mitotic cells are produced easily with shake-off procedure in colcemid or nocodazole treated cells. 3. Culture medium: McCoy’s 5A (Modified) (Gibco BRL, Bethesda, MD, USA) with fetal bovine serum (FBS) (10–15%) and without FBS or any other kinds of medium could be used.

G1 Premature Chromosome Condensation (PCC) Assay

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Fig. 1 An example of G1 PCC combined with FISH technique. G1/G0 phase AG-1522 cells were fused with HeLa mitotic cells and G1 PCC was induced. Human chromosomes 1 and 3 were painted with chromosome 1 (green) and 3 (red) probes. G1-type chromosomes can be observed with two colors as shown by arrows. Corresponding chromosomes in mitotic HeLa cells were also stained, but they are not considered for the analysis 2.2 Chemicals, Buffers, and Fusion Agent

1. Colcemid (Gibco BRL, Bethesda, MD, USA) Use this chemical to stop cells at mitosis. 2. Giemsa stain solution (Wako, Osaka, Japan) in Gurr’s Buffer (Gurr buffer tablet Gibco BRL, Bethesda, MD, USA) to stain fixed chromosomes. 3. 75 mM KCl solution. 4. Hank’s Balanced Salt Solution (Life Technologies, Tokyo, Japan). 5. Fixative solution: methanol: glacial acetic acid ¼ 3:1 (v/v) (see Note 1). 6. Sendai virus HVJ-E (Hemagglutinating virus of Japan (HVJ) Envelope) (Ishihara Sangyo, Osaka, Japan) for cell fusion (see Note 2). Store at 80  C. 7. Polyethylene glycol (PEG) (M.W.1540; Boehringer Mannheim GmbH, Germany) in PBS solution.

2.3 Microscope and Microscope Supplies

1. Microscope (Carl Zeiss, Olympus, Nikon or any other makers). 2. Slide glass (Matsunami, Osaka, Kishiwada, Japan), cover glass (Matsunami, Osaka, Kishiwada, Japan), and Multi Mount (Matsunami, Osaka, Kishiwada Japan). 3. Immersion oil (Sigma, St. Louis, MO, USA) for microscope observation.

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Tubes Etc

1. Round bottom tube (BD Falcon, Franklin Lakes, NJ, USA). 2. Refrigerated Centrifuge (Kubota, Tokyo, Japan).

2.5 Collection of HeLa Meta-Phase Cells by Synchronization

1. Plate 2.5  106 cells into T150 flasks in alpha MEM + FBS (15%). 2. After 12–25 h, add thymidine (TdR) (2.5 mM final concentration) (see Note 3). 3. After 16–18 h, rinse flasks with TdR-free medium twice, then add alpha MEM + 15% FBS. 4. After 5–12 h, add thymidine as before (see Note 4). 5. After 16–17 h, rinse flask with TdR-free medium as before, and add alpha MEM + 15% FBS + colcemid (0.2 μg/ml final concentration) (see Note 5). 6. After 12–15 h from the last rinse, shake off mitotic cells (should be nearly all of the population). 7. Take small aliquot from shaken off cells. Count cells and check mitotic index (MI). MI should be >95% (see Note 6). 8. Centrifuge at 500  g for 5 min at 4  C and aspirate the supernatant. 9. Resuspend the cells in 10 ml Hank’s Balanced Salt Solution + 0.2 μg/ml colcemid. 10. Repeat centrifugation and freeze cells at 80  C (see Note 7).

3

Methods

3.1 Protocol for G1 PCC with Sendai Virus

1. 106 G1 or G0 cells are mixed with an equal number of mitotic HeLa or CHO cells (mitotic index >90%, frozen and thawed) in a polypropylene tube under cold temperature (4  C) (see Note 8). 2. The cells are centrifuged at 500  g (Use cold temperature such as 4  C) for 5 min and aspirate the supernatant (see Note 9). 3. Cell pellets are washed in 10 ml ice-cold serum-free medium, and then centrifuged again as in step 2 (see Note 10). 4. Add 2–4 μl of hemagglutinating virus of Japan envelope (HVJ-E; also known as Sendai virus) to the cells immediately and mix well. Then add 0.7 ml cold serum-free medium with colcemid and mix very well (see Note 11). 5. The HVJ-E-treated cells are kept on ice for 15 min to allow the virus envelope to attach and then placed in a water bath at 37  C for 3 min. 6. The samples in tubes are incubated in a CO2 incubator (37  C) in warm (37  C) water bath for 1 h to allow cell fusion and induction of PCC to occur (see Note 12).

G1 Premature Chromosome Condensation (PCC) Assay

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Fig. 2 Examples of Giemsa stained G1 PCC (left: nonirradiated control, right: X-ray irradiated with 2 Gy). Human lymphocytes were fused with mitotic CHO cells and G1 PCC was induced (spaghetti-like appearances). In control sample on the left, 46 chromosomes are observed, while fragmented G1-type chromosomes are observed in the irradiated sample on the right ref. 12

7. After 1 h of incubation, mix the sample slightly, then the samples are carefully resuspended in 8 ml of 75 mM KCl solution for 15–20 min at 37 Cwater bath. 8. 2 ml of freshly prepared fixative solution (methanol:glacial acetic acid ¼ 3:1 v/v) is slowly added to the solution, and the cells were centrifuged as in step 2. 9. Pour the supernatant and add 8 ml of fixative solution to the samples and centrifuge again at 500  g for 5 min and aspirate the supernatant. 10. After one or two further washes in the fixative solution, chromosomes are dropped onto wet slide glasses at room temperature and air dried. 11. The cells are then stained with 5% Giemsa for 7 min in staining jar and rinsed two times with distilled water (see Note 13). 12. Specimens of prematurely condensed chromosomes were sealed with cover glass and analyzed under a light microscope (Fig. 2) (see Note 14). 3.2 Protocol for G1 PCC with PEG (This Procedure Is Basically the Same as 3.2 for the Sendai Virus Fusion Method After the Sample Incubation at 37  C)

1. 106 mitotic XP2OS cells, which are produced by 14–18 h incubation in the presence of 0.05 mg/ml demecolcine (Wako Pure Chemical Industries Ltd), are mixed with an equal number of target G1 cells in a polypropylene tube (BD Falcon, 352059). 2. The mixed cells are centrifuged at 500  g for 5 min. Aspirate the supernatant.

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3. Cells are washed once in 5 ml of PBS. Then they were again centrifuged at 500  g for 5 min, and the supernatant is aspirated. 4. The pellet in each tube was exposed to 0.15 ml of 50% (w/v) polyethyleneglycol (PEG) in 75 mM Hepes for 1 min to allow cell fusion to occur. 5. Then 4 ml of PBS is gently added to the tube, and the cell suspension is centrifuged at 500  g for 5 min and the supernatant is aspirated. 6. The pellet in each tube is resuspended in 5 ml of MEM containing 0.1 μg/ml demecolcine and incubated in a CO2 incubator at 37  C for 1 h. 7. Subsequently, the cells are treated with 75 mM KCl solution which is preheated at 37  C for 20 min. 8. Then cells are fixed with 3:1 ¼ methanol: acetic acid solution. After being centrifuged at 500  g for 5 min, the pellet is resuspended in the fixing solution again. 9. The cell suspension is dropped onto clean slides and stained with 5% Giemsa solution for 7 min in staining jar and washed two times with distilled water. 10. Specimens of prematurely condensed chromosomes are sealed and analyzed under a light microscope.

4

Notes 1. Fixative solution should be freshly made each time. 2. HVJ-E is available outside of Japan using Cosmo Bio company. Freeze-dry HVJ-E material is available from them. The original publication for Sendai virus propagation is “Production of Sendai virus for cell fusion” by Giles and Ruddle [17] . 3. Thymidine arrests all DNA synthesis. Cells originally in G2, M, or G1 accumulate at the G1/S border, while cells in S phase remain there. 4. This second block accumulates all cells at the G1/S border. 5. This step lets all cells progress through S and G2 and blocks in mitosis at metaphase. 6. If the mitotic index 20%) than mitotic index (usually 1–2%, but will be often much lower) that facilitates the observation and analysis of chromosome much easier [2]. Therefore, drug-induced PCC has been becoming popular and many articles using the technique have been published [9, 30–49] (as ref.39, also see Note 3). In addition, the drug-induced PCC has an outstanding merit that allows the interphase chromatin (i.e., G1-, S-, and G2-phase chromatin) to be visualized as a condensed form of chromosome structure [2, 28]. Hence, the drug-induced PCC method will be a useful way not only as an alternative method for mitotic chromosome analysis but also as a new analytical tool for interphase chromatin cytogenetics [2]. In this chapter, the detailed protocol of druginduced PCC is described. The protocol is very simple and easily recognized by the unskilled person in chromosome preparation and hopefully the drug-induced PCC will be used in wider ranges in cytogenetics and related fields [50–52]. In addition, some special techniques for analysis of chromosome repair dynamic following ionizing irradiation exposure are described.

2

Materials 1. Calyculin A (C50H81N4O15P, MW ¼ 1009.17, Wako Chemicals cat. No. 038-14453 10 μg, cat. No. 032-14451 100 μg) (see Note 4): (a) Reconstitution of calyculin A for 100 μM stock solution: For 100 μg of calyculin A (Wako Chemicals cat. No. 032-14451) lyophilized in a vial provided from the supplier, dissolving with 1 mL of solvent (DMSO or 100% ethanol must be used because calyculin A does not dissolve in water) will make ~100 μM stock solution (M.W. of calyculin A is 1009.17). Aliquot in 100 μL or your preferred volume in a microcentrifuge tube and store at

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20  C or under, and it will be stable for at least 3 months (see Notes 5 and 6). 2. 0.075 M KCl: (a) 7.45 g KCl (M.W. 74.55): Dissolve in 100 mL MilliQ water to make a 1 M KCl stock solution. (b) Take 7.5 mL of the 1 M stock solution, make it up to 100 mL with MilliQ water, and mix well to obtain a 0.075 M KCl working solution. Usually do not autoclaving for using hypotonic cell treatment. 3. Carnoy’s fixative (methanol:glacial acetic acid ¼ 3 parts:1 part): (a) Take 3 parts of methanol and 1 part of glacial acetic acid (for example 150 mL of methanol and 50 mL of acetic acid to make 200 mL fixative) and mix together well. The temperature of mixture will be somehow increased according to exothermic reaction of alcohol and acid. (b) It is preferable to prepare just before use; however the fixative can be stored at 20  C for a month. After storing for longer time even at low temperature, esterification will proceed resulting in production of methyl acetate and water. At that time, the fixative smells some typical fragrance of ester, and dropped on a glass slide fixative does not spread well circularly and some water will be remained after evaporation. In this case, discard the old fixatives and new fixative must be re-prepared. 4. 10 mM Potassium phosphate buffer (pH 6.8 at 25  C): (a) Solution A: Dissolve 136.09 g KH2PO4 with 1 L MilliQ water to make 1 M stock solution. (b) Solution B: Dissolve 174.18 g K2HPO4 with MilliQ water to make it up to 1 L for 1 M stock solution. (c) Mix 50.3 mL solution A with 49.7 mL solution B and make it up to 1 L with MilliQ water for making 10 mM working solution. If desired, adjust the pH before the final mess up to 1 L. If you want to prepare other pH phosphate buffer, appropriate different volume ratios of solution A and solution B should be used as described elsewhere in biochemistry textbook (for example, Molecular Cloning: A Laboratory Manual. Second Edition [53]). (d) Gurr Buffer tablet (one tablet for 1 L solution, pH 6.8, available from Invitrogen, Cat. No. 10582013) or phosphate buffer powder (for 1 L solution, 1/15 M, pH 6.8, available from Wako Chemicals, Cat. No. 163-16471) is

G2 Premature Chromosome Condensation/Chromosome Aberration Assay. . .

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for example also used well for convenient substitution for phosphate buffer. 5. Giemsa dye solution (Merck KGA Cat. No. 1.09204.0100): Dilute with 10 mM phosphate buffer (pH 6.8 at 25  C) to make 5% (4–6%) solution (freshly prepare just before use) (see Note 7). 6. Glass slides and coverslips: Glass slides are soaked in methanol until use. Wipe out methanol with KIMWIPES. In the present time, this procedure may not be necessary. Earlier, glass slides provided by factories were somewhat polluted with oil which might be spilled during cutting the glass; however currently available slides are substantially clean for use in chromosome preparation, and thus degrease with methanol will not be necessary.

3

Methods

3.1 Drug-Induced Premature Chromosome Condensation (PCC) for Adherent Cells 3.1.1 Cell Culture, Premature Chromosome Condensation, and Harvest

3.1.2 Chromosome Preparation

Plate the cells in culture dish 1–2 days before the experiment in order for the cells to reach 70–80% confluence and grow exponentially at the time of induction of PCC to achieve a high PCC index (see Note 8). One 35 mm diameter culture dish of cells is usually sufficient to obtain a substantial number of PCC spreads for analyzing one data point. Add 1/2000 volume of 100 μM calyculin A stock solution (i.e., 50 nM final concentration) to the culture medium, then incubate for another 30 min, and keep at 37  C. After incubation, cells are gently pipetted with medium using a 1000 mL Pipetman with a blue tip, and cells may easily detach and float in the medium. Neither trypsinization nor use of a scraper for detaching cells is required as usually done following colcemid treatment, because the cells become round and attach very loosely on the dishes or float off from the dish after calyculin A exposure. If the cells do not become round and still attach tightly on the dish while growing, it may suggest that calyculin A loses activity and does not work. In such a case, replace new aliquot of calyculin A. Simply transfer the medium with suspended cells into the centrifuge tube, centrifuge at 250  g for 5 min, and discard the supernatant by inverting the tube (see Note 9). Loosen the cell pellet by quickly shaking the tube and add gently prewarmed (37  C) 1.5 mL 0.075 M KCl to suspend the cells for hypotonic treatment. Incubate at 37  C for 20 min. Fixate the cells by slowly adding with the same volume of cold Carnoy’s fixative to the medium and mix well gently (capping and inverting the tube

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couple of times). Centrifuge at 250  g for 5 min, discard the supernatant, loosen the cell pellet, gently add 1.5 mL of fixative, and centrifuge. Repeat three to four times these steps for complete fixation. After fixation, cells were finally suspended in adequate volume of fixative (usually 200–300 μl for 35 mm diameter dish culture scale of cells). Chromosome suspension (~15 μL using a Pipetman using a yellow tip) was dropped on a glass slide and air-dried. To make sure that the cells are certainly condensed prematurely, you can observe the chromosome spreads under a microscope in low magnification without staining. As usual, many chromosome spreads are seen like fireworks in the sky, and 10–20% PCC index will be easily achieved. Stain with 5% Giemsa dye solution, covered by a coverslip and mounted with sealant for observation (see Note 7). Usually, one sample slide may give a substantial number of PCCs for karyotyping or chromosome aberration analysis. Unstained chromosome specimens will be stored in a desiccated container and will be subjected to several special banding procedures (G-, Q-, or R-banding), in situ hybridization including FISH (single- or multicolor FISH), or immunostaining examination. 3.2 Drug-Induced PCC for Suspension Cells 3.2.1 Cell Culture, Premature Chromosome Condensation, and Harvest

The protocol for suspension cells such as peripheral blood lymphocytes is basically the same as that for adherent cells. For suspension cells, okadaic acid will also work as well as calyculin A (see Note 6). Prepare the suspension cell culture in an appropriate cell concentration (i.e., 105–106 cells/mL, depending on cell types used) to maintain exponential growing at the time of PCC induction (see Note 8). Prior to the PCC induction, it may be convenient to transfer the cells in a centrifuge tube. One milliliter of cell culture is sufficient to obtain a substantial number of PCCs for analyzing one data point (i.e., total 10 mL of culture can be used for ten data points). Add 50 nM final concentration of calyculin A to the medium, and process as same as the protocol for attached cells.

3.2.2 Chromosome Preparation

After 30 minutes incubation, harvest the cells by centrifuging, then cells are subjected to hypotonic treatment, cell fixation, and spreading on the glass slides as same as adheret cells (see Subheading 3.1.2).

3.3 Drug-Induced PCC Protocols for Radiation Biology

One of the most success of drug-induced PCC technique is apply for radiation biology field; such as irradiation biodosimetry in accidental exposure and chromosome repair kinetics following irradiation exposure damages. Many of the literatures that applied drug-induced PCC in radiation biology studies have been published during 20 years. Basically, PCC protocol for the radiation biology is the same as that for the usual cytogenetic studies as described above (see Subheadings 3.1.2 and 3.2.2), but several

G2 Premature Chromosome Condensation/Chromosome Aberration Assay. . .

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different tips/cares are required for different studies. In this section, several technical tips for biodosimetry and repair kinetics are briefly described. 3.3.1 Technical Tips for Cytogenetic Biodosimetry for High-Dose Exposure Accident

In the accidental irradiation exposure, the assessment of absorbed dose in victims must be done as quickly as possible for following rescue lives. Over several Gys of γ-irradiation, however, it was very difficult or impossible to estimate the dose because obtaining mitotic chromosomes was difficult as the damaged cells arrested the cell cycle and did not enter in mitosis. Drug-induced PCC method has first overcome this problem and has made it possible to estimate the absorbed dose up to 40 Gy of γ-irradiation. During dose assessment for the accidental exposure case, the following points should be taken care of: (1) The blood sample must be collected as soon as possible after accident, because peripheral lymphocytes soon diminish from the blood after large-dose irradiation exposure [54, 55]. (2) Manipulate the blood samples under clean condition to prevent the samples from loosing by contamination. (3) If there is no capacity for handling the blood to make chromosome samples, send quickly to the appropriate institute for the dose assessment. (4) During shipping of the sample to the institute, do not chill the blood and keep at room temperature; otherwise mitotic index (PCC index) will be decreased as the activity of lymphocytes will be lost during cold temperature [42]. For more details about the cytogenetic biodosimetry for accidental ionizing radiation exposure, see for example the following text [38, 56–58].

3.3.2 Technical Tips for Repair Kinetic Analysis Following Irradiation Exposure

Chromosomes are easily damaged by ionizing exposure. Damaged chromosomes are then repaired by cellular process. Basically repair commences immediately after the damages are introduced and continues for several hours to complete the damages. Chromosome damages are usually analyzed by observation of chromosomes. Using the conventional colcemid block method, it is however clearly impossible to detect the early phase of repair kinetics, because it takes 2 or more hours to arrest mitotic chromosomes using the colcemid protocol. It is also difficult to detect the earlier phase of repair process even with the use of fusion-mediated PCC, because this method also takes hours including fusion manipulation and following colcemid treatment. On the other hand, druginduced PCC is a very simple technique and handling time is very short compared with the mitotic arresting method or fusionmediated PCC method. Therefore, drug-induced PCC is very useful in particular for detecting the very early stage of repair process.

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Usually for the kinetics of repair process, to obtain PCC after irradiation, calyculin A is added in the medium at individual data points after irradiation to obtain PCC samples. Detecting the very early phase (from 0 to 10 min after exposure) of repair process requires some technical tips which may sound a little bit tricky. (1) Following exposure to 50 nM of calyculin A, the cells start chromosome condensation at 5 min after and condensation completes after 20–30 min. (2) After condensation of chromosomes, chromosomes are relatively resistant to ionizing irradiation exposure and the damages will not be introduced in chromosomes. (3) Therefore, at least 5-min duration is required to fix the DNA damages in condensed chromosomes. This 5 min can be neglected for longer sampling time data (i.e., more than 30 min) analysis; however for the shorter sampling time data less than 30 min, in particular from 0 to 10 min after irradiation, early-phase repair proceeds during chromosome condensation. This may influence the number of chromosome damages. Therefore, calyculin A should be added in the medium at least 5 min prior to irradiation, and then cells should be harvested at individual sampling times (i.e., 0, 5, 10, 15, 30 min). For the details of this protocol, please see for example Gotoh et al. [37].

4

Notes As easily recognized, the drug-induced PCC protocol is very simple with substitute use of calyculin A to colcemid in established chromosome preparation protocol. However, there are some keys for succeeding in achieving high PCC index and good-quality PCCs. Following are the keys that I have experienced through my years of study: 1. Premature chromosome condensation (PCC) usually means the phenomenon or method, and the condensed interphase chromatin is called as prematurely condensed chromosomes (PCCs). They give the similar abbreviations that are usually used interchangeably. 2. Calyculin A, okadaic acid, or other phosphatase inhibitors induce PCC predominantly in G2 and S phase but only few in G1 and G0 phase [2]. Presumably drug-induced PCC exploits the activation of intracellular MPF by phosphatase inhibitors, but the amount of intracellular maturation promoting factor (MPF, also known as p34cdc2/cyclinB complex) [62–66] is very low in G1 or G0 phase [2, 67]. In the cases that PCC analysis is requested in G0 or G1 phase of cells, the

G2 Premature Chromosome Condensation/Chromosome Aberration Assay. . .

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conventional fusion-PCC is still worthwhile to do. Another approach of drug-induced PCC in G0-phase peripheral blood lymphocytes was reported using adenosine triphosphate (ATP) and p34cdc2/cyclin B with okadaic acid [68]. The protocol of fusion-PCC or okadaic acid:p34cdc2/cyclin B:ATP-induced PCC has been described previously [3, 13, 15, 68, 69] (also see Note 10). 3. Usually, calyculin A solely works efficiently and gives a high PCC index. Combinational use of colcemid with calyculin A will be sometimes effective in obtaining the higher PCC index [35]. 4. Wako Chemicals is in Japan (Osaka, Japan). Calyculin A is now available from many other chemical suppliers (Subheading 2). 5. It is hard to see and weigh out the lyophilized powder in the vial. So, pour the solvent (ethanol or DMSO) directly into the vial using a Pipetman, gently slant and swirl the vial to dissolve the whole chemicals, and transfer the solution to the microcentrifuge tube (e.g., Eppendorf tube). If dissolved in ethanol, the screwed lid with a rubber seal-type tube is preferable (Subheading 2); otherwise ethanol will escape in vapor and the concentration of calyculin A will change. Accordingly, DMSO is more preferable for dissolving calyculin A. 6. Okadaic acid, its salts or derivatives, or other inhibitors endothall or cantharidine can also be used as PCC inducers [27, 59, 60], but these chemicals work only in suspension-type cells (for example peripheral blood lymphocytes) but not in adherent cells. The reason is still unclear but presumably due to the different cell sensitivities to these chemicals. Therefore, the use of calyculin A is recommended for PCC induction both in suspension and adherent types of cell. If okadaic acid is used in suspension cells, 100 nM final concentration rather than 50 nM will be much better for work (Subheadings 3.1.1 and 3.2.1). 7. The tint of Giemsa staining varies depending on the pH of phosphate buffer shifts; color turns from reddish purple (pH 6.8) to bluish purple (pH 7.2). Which pH of phosphate buffer is depend on the individual favorite, but reddish purple color seems to much good for observation and taking wellcontrasted images under the microscope (see Subheadings 3.1.2 and 3.2.2). 8. The PCC efficiency is highly dependent on cell types, cellgrowing condition, dose, or incubation time of calyculin A. Usually, 50 nM final concentration of calyculin A works very well in many kinds of cells and a sufficient number of PCCs will be obtained following 30 min of incubation. However, some cells are resistant to calyculin A but others are hypersensitive to calyculin A. In such cases, start the experiment first with 50 nM

OH

O O

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(c) Cantharidin CH2 H3C H3C

(e) Endothal

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Fig. 1 Chemical structures of several major protein phosphatase inhibitors. (a) Calyculin A: Source: marine sponge (Discodermia calyx), chemical structure: C50H81N4O15P, molecular weight (M.W.): 1009.18. (b) Okadaic acid: Source: marine sponge (Halichondria okadai (Kadota)), C44H68O13, M.W. 805.2. (c) Cantharidin: Source: blister beetle (Meloe corvinus), C10H12O4, M.W. 196.2. (d) Cantharidic acid: C10H14O5, M.W. 214.2. (e) Endothall: C8H10O5, M.W. 186.2. (f) Lasonolide A: Source marine sponge (Forcepia sp.), C41H60O9, M.W. 696.90

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concentration and for 30-min incubation time as a default condition, and then increase or decrease the dose and/or the incubation time depending on the cell response (appearance of cell, detachment from the dish) and resulting PCC index. Keep in mind that the critical point is that cells should be growing exponentially at the induction of PCC; otherwise PCC index will drop down. In the case of adherent types of cells, cells should be re-plated 1–2 day(s) before the experiment to reach 70–80% confluence at the time of PCC induction. Avoid re-plating the cells on the day of the experiment. The higher the dose and the longer the incubation time, the PCC index is higher but the resulting PCCs become hyper-condensed. Therefore, the appropriate dose and incubation time should be taken on balance depending on the experiment design [61]. PCC starts 5 min after beginning of calyculin exposure, and 10 min of incubation will induce a substantial number of PCCs [9, 29, 37] (Subheadings 3.1.2 and 3.2.2). 9. A round-bottomed centrifuge tube (for example Becton & Dickinson 5 mL polystyrene centrifuge tube with snap cap, Cat. No. 352058) is preferable for chromosome preparation, so the cells are less packed in tube bottom after centrifugation so as to avoid cell aggregation (Subheadings 3.1.1 and 3.2.1). 10. Lasonolide A is a new potent PCC inducer which can induce PCC even in G0 phase [70]. Therefore, lasonolide A will be expected to open a new approach of drug-induced PCC in chromosome science approach. Chemical structure of several PCC agents including calyculin A and lasonolide A is shown in Fig. 1.

Acknowledgments I wish to express my thanks to Prof. Paul Wilson of Brookhaven National Laboratory and Prof. Takamitsu Kato of Colorado State University for their recommendation to give me a chance to contribute to this chapter. References 1. Vagnarelli P (2012) Mitotic chromosome condensation in vertebrates. Exp Cell Res 318 (12):1435–1441 2. Gotoh E, Durante M (2006) Chromosome condensation outside of mitosis: mechanisms and new tools. J Cell Physiol 209:297–304. and Cover page 3. Johnson RT, Rao PN (1970) Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature 226 (247):717–722

4. Sperling K, Rao PN (1974) The phenomenon of premature chromosome condensation: its relevance to basic and applied research. Humangenetik 23(4):235–258 5. Sperling K, Rao PN (1974) Mammalian cell fusion. V. Replication behaviour of heterochromatin as observed by premature chromosome condensation. Chromosoma 45(2):121–131 6. Rao PN, Wilson B, Puck TT (1977) Premature chromosome condensation and cell cycle analysis. J Cell Physiol 91(1):131–141

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7. Mullinger AM, Johnson RT (1983) Units of chromosome replication and packing. J Cell Sci 64:179–193 8. Ito S et al (2002) Epstein-Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular. J Gen Virol 83(Pt 10):2377–2383 9. Gotoh E (2007) Visualizing the dynamics of chromosome structure formation coupled with DNA replication. Chromosoma 116 (5):453–462 10. Cornforth MN, Bedford JS (1983) X-rayinduced breakage and rejoining of human interphase chromosomes. Science 222 (4628):1141–1143 11. Cornforth MN, Bedford JS (1983) High-resolution measurement of breaks in prematurely condensed chromosomes by differential staining. Chromosoma 88(4):315–318 12. Pantelias GE, Maillie HD (1984) The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry. Radiat Res 99(1):140–150 13. Durante M et al (1996) Rejoining and misrejoining of radiation-induced chromatin breaks. I. Experiments with human lymphocytes. Radiat Res 145(3):274–280 14. Hittelman WN (1990) Direct measurement of chromosome repair by premature chromosome condensation. Prog Clin Biol Res 340B:337–346 15. Pantelias GE, Maillie HD (1983) A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol. Somatic Cell Genet 9(5):533–547 16. Lamadrid Boada AI et al (2013) Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes. Mutat Res 757(1):45–51 17. Rao PN, Johnson RT (1970) Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature 225(228):159–164 18. Rao PN, Johnson RT (1971) Mammalian cell fusion. IV. Regulation of chromosome formation from interphase nuclei by various chemical compounds. J Cell Physiol 78(2):217–223 19. Hittelman WN (1986) Visualization of chromatin events during DNA excision repair in XP cells: deficiency in localized but not generalized chromatin events. Carcinogenesis 7 (12):1975–1980 20. Pantelias GE (1986) Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation. Radiat Res 105(3):341–350

21. Iliakis GE, Pantelias GE (1990) Production and repair of chromosome damage in an X-ray sensitive CHO mutant visualized and analysed in interphase using the technique of premature chromosome condensation. Int J Radiat Biol 57(6):1213–1223 22. Pantelias GE et al (1993) Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes. Int J Radiat Biol 63(3):349–354 23. Schlegel R, Pardee AB (1986) Caffeineinduced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science 232(4755):1264–1266 24. Schlegel R, Belinsky GS, Harris MO (1990) Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine. Cell Growth Differ 1(4):171–178 25. Yamashita K et al (1990) Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells. EMBO J 9(13):4331–4338 26. Gotoh E (2009) Drug-induced premature chromosome condensation (PCC) protocols: cytogenetic approaches in mitotic chromosome and interphase chromatin. In: Chellappan SP (ed) Methods in molecular biology, Chromatin protocols, vol 523, 2nd edn. Humana Press, New York, pp 83–92 27. Gotoh E (1995) Agents and a method of chromosome preparation using protein phosphatase inhibitors induced premature chromosome condensation (PCC) technique. Gotoh, E.: USA Patent 28. Gotoh E, Asakawa Y, Kosaka H (1995) Inhibition of protein serine/threonine phosphatases directly induces premature chromosome condensation in mammalian somatic cells. Biomed Res 16(1):63–68 29. Bryant PE, Mozdarani H (2007) A comparison of G2 phase radiation-induced chromatid break kinetics using calyculin-PCC with those obtained using colcemid block. Mutagenesis 22(5):359–362 30. Ravi M, Nivedita K, Pai GM (2013) Chromatin condensation dynamics and implications of induced premature chromosome condensation. Biochimie 95(2):124–133 31. Ono T, Yamashita D, Hirano T (2013) Condensin II initiates sister chromatid resolution during S phase. J Cell Biol 200(4):429–441 32. Roukos V, Burgess RC, Misteli T (2014) Generation of cell-based systems to visualize chromosome damage and translocations in living cells. Nat Protoc 9(10):2476–2492

G2 Premature Chromosome Condensation/Chromosome Aberration Assay. . . 33. Gotoh E, Asakawa Y (1996) Detection and evaluation of chromosomal aberrations induced by high doses of gamma-irradiation using immunogold-silver painting of prematurely condensed chromosomes. Int J Radiat Biol 70(5):517–520 34. Asakawa Y, Gotoh E (1997) A method for detecting sister chromatid exchanges using prematurely condensed chromosomes and immunogold-silver staining. Mutagenesis 12 (3):175–177 35. Durante M, Furusawa Y, Gotoh E (1998) A simple method for simultaneous interphasemetaphase chromosome analysis in biodosimetry. Int J Radiat Biol 74(4):457–462 36. Johnson RT et al (1999) Targeting doublestrand breaks to replicating DNA identifies a subpathway of DSB repair that is defective in ataxia-telangiectasia cells. Biochem Biophys Res Commun 261(2):317–325 37. Gotoh E, Kawata T, Durante M (1999) Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation. Int J Radiat Biol 75(9):1129–1135 38. IAEA (2001) Cytogenetic analysis for radiation dose assessment. A manual. Technical reports series no. 405. International Atomic Energy Agency, Vienna 39. Bezrookove V et al (2003) Premature chromosome condensation revisited: a novel chemical approach permits efficient cytogenetic analysis of cancers. Genes Chromosomes Cancer 38 (2):177–186 40. El Achkar E et al (2005) Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites. Proc Natl Acad Sci U S A 102(50):18069–18074 41. Srebniak MI et al (2005) The usefulness of calyculin a for cytogenetic prenatal diagnosis. J Histochem Cytochem 53(3):391–394 42. Gotoh E, Tanno Y (2005) Simple biodosimetry method for cases of high-dose radiation exposure using the ratio of the longest/shortest length of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC). Int J Radiat Biol 81(5):379–385 43. Gotoh E, Tanno Y, Takakura K (2005) Simple biodosimetry method for use in cases of highdose radiation exposure that scores the chromosome number of Giemsa-stained druginduced prematurely condensed chromosomes (PCC). Int J Radiat Biol 81(1):33–40 44. Mochida A et al (2005) Telomere size and telomerase activity in Epstein-Barr virus

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(EBV)-positive and EBV-negative Burkitt’s lymphoma cell lines. Arch Virol 150 (10):2139–2150 45. Deckbar D et al (2007) Chromosome breakage after G2 checkpoint release. J Cell Biol 176 (6):749–755 46. Kanda T et al (2007) Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids. J Cell Sci 120(Pt 9):1529–1539 47. Pathak R, Prasanna PG (2014) Premature chromosome condensation in human resting peripheral blood lymphocytes without mitogen stimulation for chromosome aberration analysis using specific whole chromosome DNA hybridization probes. Methods Mol Biol 1105:171–181 48. Romero I et al (2014) Shortening the culture time in cytogenetic dosimetry using Pcc-R assay. Radiat Prot Dosim 163(4):424–429. pii: p. ncu258 49. Miura T et al (2014) A novel parameter, cellcycle progression index, for radiation dose absorbed estimation in the premature chromosome condensation assay. Radiat Prot Dosim 159(1–4):52–60 50. Zabka A et al (2015) The biphasic interphasemitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa. J Plant Physiol 174:62–70 51. Samaniego R, de la Torre C, de la Espina SMD (2002) Dynamics of replication foci and nuclear matrix during S phase in Allium cepa L. cells. Planta 215(2):195–204 52. Rybaczek D et al (2002) Induction of premature mitosis in root meristem cells of Vicia faba and Pisum sativum by various agents is correlated with an increased level of protein phosphorylation. Folia Histochem Cytobiol 40 (1):51–59 53. Sambrook J, Fritsch EF, Maniatis T (1989) Phosphate buffers. In: Molecular cloning, vol 3. Cold Spring Harbor Laboratory Press, New York, p Appendix B.21 54. Coleman CN et al (2009) Medical response to a radiologic/nuclear event: integrated plan from the office of the assistant secretary for preparedness and response, department of health and human services. Ann Emerg Med 53(2):213–222 55. Kanda R, Hayata I, Lloyd DC (1999) Easy biodosimetry for high-dose radiation exposures using drug-induced, prematurely condensed chromosomes. Int J Radiat Biol 75 (4):441–446

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56. Gotoh E (2012) Cytogenetic biodosimetry for accidental emergency irradiation exposure preparedness, in particular merit of the use of drug-induced premature chromosome condensation (PCC) with calyculin A. Deep Insight for the Atlas of Genetics and Cytogenetics in Oncology and Haematology. http:// atlasgeneticsoncology.org/Deep/ VisuDynChID20114.html 57. IAEA (2011) Cytogenetic dosimetry: applications in preparedness for and response to radiation emergencies. A manual. Technical reports. Technical reports series. International Atomic Energy Agency, Vienna 58. del Rosario Perez M et al (2009) A new handbook on triage, monitoring and treatment of people following malevolent use of radiation. Health Phys 98(6):898–902 59. Gotoh E (1994) Agents and a method of chromosome preparation using protein phosphatase inhibitors induced premature chromosome condensation (PCC) technique. Gotoh E: UK, Germany, Italy, France, Netherland, Sweden 60. Gotoh E (1995) Agents and a method of chromosome preparation using protein phosphatase inhibitors induced premature chromosome condensation. Gotoh, E.: Canada Patent 61. Miura T, Blakely WF (2013) Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies. Cytometry A 79(12):1016–1022 62. Masui Y (1974) A cytostatic factor in amphibian oocytes: its extraction and partial characterization. J Exp Zool 187(1):141–147 63. Masui Y (2001) From oocyte maturation to the in vitro cell cycle: the history of discoveries of

maturation-promoting factor (MPF) and cytostatic factor (CSF). Differentiation 69(1):1–17 64. Dunphy WG et al (1988) The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54(3):423–431 65. Gautier J et al (1988) Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell 54(3):433–439 66. Maller J et al (1989) Maturation-promoting factor and the regulation of the cell cycle. J Cell Sci Suppl 12:53–63 67. Doree M, Galas S (1994) The cyclin-dependent protein kinases and the control of cell division. FASEB J 8(14):1114–1121 68. Prasanna PG, Escalada ND, Blakely WF (2000) Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry. Mutat Res 466(2):131–141 69. Prasanna PG, Blakely WF (2005) Premature chromosome condensation in human resting peripheral blood lymphocytes for chromosome aberration analysis using specific whole-chromosome DNA hybridization probes. In: Keohavong P, Grant SG (eds) Methods in molecular biology, Molecular toxicology protocols, vol 291. Humana Press, Totowa, NJ, pp 49–57 70. Zhang YW, Ghosh AK, Pommier Y (2012) Lasonolide a, a potent and reversible inducer of chromosome condensation. Cell Cycle 11 (23):4424–4435

Chapter 7 Sister Chromatid Exchange as a Genotoxic Stress Marker Shigeaki Sunada, Jeremy S. Haskins, and Takamitsu A. Kato Abstract Sister chromatid exchange (SCE) is the phenomenon of partial DNA exchange during DNA replication. SCE detection has been developed through eliciting DNA’s semiconservative replicative nature. Thymidine analogues such as 50 -bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU) are incorporated into the newly synthesized DNA for two cell cycles. The addition of Colcemid to the culture blocks and synchronizes cells at mitosis, and conventional cytogenetic preparations are made. Differential staining methods with Hoechst dye and Giemsa (Fluorescence Plus Giemsa staining), antibody detection against BrdU, or highly specific Click reaction to EdU, allow the newly synthesized DNA within a chromatid to be recognized. SCEs represent a point of DNA template exchange during DNA synthesis, visualized by differential chromatid staining or harlequin chromosomes. We will introduce three basic protocols in this chapter including non-fluorescence and fluorescence methods for SCE microscopic analysis. SCE is a very sensitive marker of genotoxic stress during replication. Key words SCE, Genotoxicity, BrdU, EdU, Fluorescence, DNA replication

1

Introduction Sister chromatid exchange (SCE) is the phenomenon of partial DNA exchange during DNA replication. The same amount of DNA is exchanged at the same location in each chromatid. Therefore, SCE requires that cells go through DNA synthesis. SCEs were first observed by Taylor et al., using autoradiography on cells that had undergone one cycle of radioactive tritiated thymidine incorporation followed by a replication cycle in nonradioactive medium [1]. Currently, 50 -bromodeoxyuridine (BrdU), an analog of thymidine, is used in place of radiolabeled thymidine to avoid using radioisotopes. SCEs, typically, have been studied for the detection of DNA damage and genotoxic stress [2]. Exposure of cells to carcinogenic or mutagenic chemicals, ultra violet light, or ionizing radiation has been known to induce SCEs. Nagasawa and Little utilized SCE as a sensitive indicator of bystander effects after extremely low fluence of alpha-particle exposure [3]. Bloom’s syndrome, an inherited disease that induces chromosomal fragility, has

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_7, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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been known to increase SCE frequency dramatically [4]. On the other hand, the frequency decreases in cells lacking homologous recombination repair after DNA damage and/or even in spontaneous levels [5]. Although SCEs are generated in S phase, recent study showed SCEs could be formed during G2 phase, too [6]. The use of BrdU or EdU is a quicker and much more efficient method than the use of radioactive thymidine for labeling newly synthesized DNA. Moreover, BrdU or EdU is incorporated into the newly synthesized DNA in the place of thymidine; however, concentrations of BrdU or EdU should be closely monitored because subtoxic levels may induce unwanted sister chromatid exchanges in the background level [7–9]. Thus, accurately controlling and optimizing concentrations of EdU and BrdU is necessary. These controls will include establishing thresholds of toxicity by adjusting either the amount of BrdU or the time of BrdU exposure for each cell line and experimental conditions. Furthermore, cells treated with BrdU are extremely light sensitive; thus, the cell cultures treated with BrdU should avoid light and be maintained in the absence of light during incubation period [10]. Additionally, it is critical to avoid unnecessary exposure of cell cultures, which have BrdU incorporated, to the light of a microscope for checking cell culture. The BrdU incorporated strand becomes very fragile after Hoechst 33258 dye and UV exposure. The Fluorescent plus Giemsa (FPG) technique is based on the theory that the fluorescent dyes can promote selective degradation of BrdU-substituted DNA strand. After two cell cycle with BrdU incorporation, one chromatid contains more BrdU compared to the other chromatid. SCEs represent a point of DNA template exchange during strand synthesis, visualized as differential chromatid staining or “harlequin” chromosomes. The fluorescence-based monoclonal BrdU antibody staining method does not require Hoechst treatment and following UV exposure and high salt solution treatment with heat. However, because BrdU is incorporated into DNA double helix, heat denature or acid treatment is required for the antibody detection to the BrdU incorporated strand. Due to heat denaturing with formamide, which causes an alteration of chromosome structure, temperature and time length of heat denature have to be optimized for the investigators’ experimental outcome. Ethynyldeoxyuridine (EdU), a synthetic thymidine analog and alternative staining method, has overcome this burden by bypassing the need of denaturing DNA. An alternative method that omits the denaturation step is a relatively new protocol that implements EdU and a Click-azide reaction. The EdU-Click-azide reaction does not require volatile heating or acid treatment used for antibody staining. Therefore, direct chemical reaction of EdU Click-azide reaction may preserve chromosome structure better than FPG staining and fluorescence BrdU detection with heat or acid denaturation.

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Materials

2.1 Reagents and Setup

1. Appropriate cell culture medium such as MEM, alphaMEM, DMEM, or RPMI1640. 2. Fetal Bovine Serum (FBS), prior lot testing is required. Some lots increase background level chromosomal aberrations. Heat inactivation at 56  C for 20 min is required. 3. Phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 in distilled water and adjust pH 7.4, then autoclave for sterilization) or purchase PBS () (Ca2+ and Mg2+ free) from ThermoFisher. 4. Antibiotics (penicillin streptomycin solution from Invitrogen or kanamycin 60 mg/ml) (Invitrogen). 5. 5-Bromodeoxyuridine (BrdU; Sigma): dissolved in PBS to make 3 mg/ml and filter to prepare sterile stock solution. Keep away from direct light in 20  C freezer. 6. Ethynyldeoxyuridine (EdU) (Life sciences): dissolved in DMSO to make 10 mM stock solution. Keep stock solution in 20  C freezer. 7. 10 μg/ml KaryoMAX Colcemid solution in PBS (Invitrogen, Catalog number 15212012). 8. Trypsin-EDTA (0.05% Trypsin/0.002% EDTA-4Na in Hanks Balanced Salt Solution). 9. Potassium chloride (KCl) (Sigma): dissolved in distilled water to make 75 mM KCl solution. 2.8 g of potassium chloride in 500 ml of distilled water. 10. Carnoy fixative solution (3:1 mixture of Methanol and Acetic Acid), freshly mixed. 11. 20 SSC (SSC ¼ sodium chloride and sodium citrate solution): dissolve 175.3 g of sodium chloride and 88.2 g of sodium citrate in 900 ml of distilled water. Adjust the pH to 7.0 using sodium hydroxide or hydrochroric acid, then make up to 1 l with more distilled water. This can be stored at room temperature for up to 6 months. For other concentrations, dilute this stock. 12. 2 SSC: mix 1 part of 20 SSC with 9 parts distilled water to make 2 solution. 13. Denaturing solution: 70% formamide in 2 SSC in Coplin Jar: (a) 5 ml 20 SSC. (b) 35 ml formamide solution. (c) 10 ml dH2O. 14. Gurr solution: dissolve Gurr buffer tablet (GIBCO, Catalog number 10582013) in distilled water.

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15. 5% Giemsa solution. Mix 2.5 ml of KaryoMAX Giemsa stain solution (GIBCO, Catalog number 10092013) with 47.5 ml Gurr solution. Freshly mixed. 16. NP40, Igepal or Tween 20. 17. Hoechst 33258 working solution. (a) Stock solution (75 μg/ml): Dissolve 7.5 mg Hoechst 33258 (e.g., Molecular Probes) in 100 ml water. Store in 4  C. Protect from direct light. (b) Working solution: Combine 50.0 ml Hoechst stock solution with 25.0 ml PBS. 18. Mouse monoclonal anti-BrdU antibody (BU1/75 (ICR1), BU-1, or B44). 19. Alexa 488 conjugated goat anti-mouse IgG antibody (Invitrogen). 20. EdU staining Solution (360 μl Water, 40 μl 10 reaction buffer, 16 μl 100 mM CuSO4, 1 μl Alexa Fluore Azide, 5 μl 10 Reaction buffer additive). Freshly made and do not store once mixed. 21. DAPI in SlowFade (ThermoFisher, S36938). 2.2

Equipment

1. Cell culture facility including biosafety cabinet or clean bench, 37  C CO2 controlled incubator, and 37  C water bath. 2. Centrifuge, which can rotate 15 ml tubes. No need of temperature control or brake. 3. Aspirator to remove supernatant and trap waste. 4. Microscope equipped with CCD camera and software. 5. Slide glass, coverslip, and cytoseal. UV light source, e.g., the transilluminator light box with black light (UVA).

3

Methods

3.1 Cell Synchronization and Irradiation

1. Synchronize cells into G0/G1 or G1 phases by densely contact inhibition for human fibroblasts or by serum starvation or isoleucine depletion medium for multiple cell lines. 2. Irradiate cells. SCE frequency decreases in above lethal doses of low LET radiation [11]. This is due to the fact that cells are receiving such a high dose of radiation that, simply, no cells survive through a second mitotic division, thereby diminishing all cell viability. Therefore, researchers have to optimize testing dose ranges (trial-and-error).

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1. To cell culture, add BrdU to a final concentration of 5 μg/ml or EdU to a final concentration of 10 μM. Since BrdU is a photosensitizer, keep the cultures in the dark by covering flasks in aluminum foil. Return the flask to the incubator to incorporate BrdU or EdU for two cell cycles. Depending on the nature of the experiment, a period of two cell cycles in medium with thymidine analogs may be required. It takes 20–24 h for CHO cells (12 h doubling time), 40 h for human lymphocyte (after PHA stimulation) and 40–48 h for normal human fibroblast and typical cancer cells (24 h doubling time). For typical cancer cells (24 h doubling time) 40–48 h. Irradiation slows down cell doubling time. If cells have DNA repair deficiency mutation, cell doubling time tends to be longer. Therefore, extra incubation times are required for high dose irradiated samples or DNA repair deficient cells. 2. Add sufficient amount of 10 μg/ml Colcemid stock to the culture to give a final concentration of 0.1 μg/ml. Return the flask to the incubator and continue incubating for 3–6 h. Avoid light exposure if BrdU is used as thymidine analogue.

3.3 Staining and Evaluation

1. Harvest the cells and prepare cytogenetic suspensions. Make metaphase spreads on clean glass slides as the previous chapter. (a) Fluorescence plus Giemsa Method l

Put 50 μl Hoechst 33258 solution onto slides and place coverslip (24  60 mm) on.

l

Incubate slides in Hoechst 33258 working solution for 10–15 min at room temperature in the dark, then rinse briefly in PBS.

l

Place slides onto gel imaging UV trans-illuminator and cover it with aluminum foil, then expose to UV light for 15 min, then rinse in fresh PBS and water. Single 15 W black light needs 10–15 min exposure from 1 cm distance. If one uses UVB or UVC lamp, longer exposure may be required.

l

l

Treat slides with 2 SCC in the Couplin jar at 65  C for 15 min. Stain slides by immersing them in a Coplin jar containing 5% Giemsa stain for 5 min at room temperature. Rinse slides in water, then visualize by light microscopy.

The sister chromatid with BrdU incorporation will show decreased staining with Giemsa. l

Score the metaphases for sister chromatid exchange (see Fig. 1).

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(b) Fluorescence anti BrdU antibody Method l

Rinse slides in water, then denature slides with 70% formamide in 2 SSC at 80  C for 3 min.

l

Put slides in 2 SSC at room temperature for 5 min, rinse slides in PBS.

l

Apply mouse monoclonal antibody against BrdU (BD B44) with 1:100 dilution in 10% goat serum in PBS with parafilm. Incubate slides for 1 h at 37  C.

l

Wash slides by immersing them in a Coplin jar containing 5% Giemsa stain for 5 min at room temperature. Repeat this three times.

l

*Apply secondary antibody goat anti-mouse monoclonal antibody conjugated with Alexa 488 (Life Technology) with 1:500 dilution in 10% goat serum in PBS with parafilm. Incubate slides for 1 h at 37  C.

l

Wash slides by immersing them in a Coplin jar containing 5% Giemsa stain for 5 min at room temperature. Repeat this three times.

l

Mount slides with coverslip and Slowfade with DAPI.

l

Score the metaphases for sister chromatid exchange (see Fig. 1).

Fig. 1 Sister chromatid exchange formation after 1 Gy of high LET radiation in CHO cells

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(c) Fluorescence EdU Click reaction Method l

4

Apply freshly prepared 50 μl EdU staining solution to each slide and cover slides with parafilm.

l

Incubate for 30 min at room temperature in the dark.

l

Wash slides with PBS for 5 min in a Coplin Jar.

l

Mount slides with coverslip and Slowfade with DAPI.

l

Score the metaphases for sister chromatid exchange (see Fig. 1).

Notes The detection of exchanges requires that the investigator is proficient in visually detecting subtle differences between lightly or heavily stained chromatids. If the Giemsa staining is too strong, the subtle color differences of each chromatid will be hard to distinguish; in such cases, one should reduce the stain time or Giemsa stain concentration. There should be a discernable “blackand-white,” “cut-and-dry” color difference. Slides can be washed with 100% Ethanol to remove Giemsa stain. Researchers can also increase black light exposure time after Hoechst staining. The investigator should also consider whether the concentration of BrdU was sufficient to label the new DNA. As mentioned above, caution should be taken when establishing BrdU concentrations, since it can induce unwanted sister chromatid exchanges. If UV exposure is too strong, staining may be too weak. In this case, adjust the strength and time of UV exposure. For BrdU-based detection, low concentration of BrdU uptake will cause weak signals. Insufficient denature process may result in no BrdU signals. The management of temperature and time for the denature process should be carefully conducted. Over-denaturing will cause morphological destruction of chromosomes. For EdU-based detection, if EdU stock solution is kept in 20  C for a long time, EdU may not be viable anymore. EdU staining solution should be prepared freshly.

5

Anticipated Results If sister chromatid exchanges are present, they should be detectable at 40 objective lens or higher magnification. The sister chromatids should be exchanged in a reciprocal manner. That means genetic information is equally exchanged between two sister chromatids at spatially homologous chromosome regions. The anticipated results will depend on the nature of the experiment. The frequency of exchanges can be determined and

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used in a correlative fashion (e.g., varying exposure to different drugs or to radiation). Typically, approximately 6–10 SCEs were observed per mammalian diploid metaphase spread [12]. Cancer cells and DNA repair deficient cells typically show elevated SCE frequency in natural background. Homologous recombination repair deficient cells show normal or reduced SCE frequency in the natural background level [13]. Ionizing radiation can induce up to 16 SCEs per cell but it will decline with dose at high dose range. UV-C exposure, alkaline, and crosslinking agents induce SCEs [14]. However, it is very difficult to analyze more than 100 SCE per cell. References 1. Taylor JH, Woods PS, Hughes WL (1957) The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidinee. Proc Natl Acad Sci U S A 43:122–128 2. Gebhart E (1981) Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing. Hum Genet 58:235–254 3. Nagasawa H, Little JB (1992) Induction of sister chromatid exchanges by extremely low doses of alpha-particles. Cancer Res 52:6394–6396 4. Ellis NA, Proytcheva M, Sanz MM, Ye TZ, German J (1999) Transfection of BLM into cultured bloom syndrome cells reduces the sister-chromatid exchange rate toward normal. Am J Hum Genet 65:1368–1374 5. Wilson DM III, Thompson LH (2007) Molecular mechanisms of sister-chromatid exchange. MutatRes 616:11–23 6. Conrad S, Kunzel J, Lobrich M (2011) Sister chromatid exchanges occur in G2-irradiated cells. Cell Cycle 10:222–228 7. Gutierrez C, Gonzalez-Gil G, Hernandez P (1983) Analysis of baseline and BrdUdependent SCEs at different BrdU concentrations. Exp Cell Res 149:461–469 8. Heartlein MW, O’Neill JP, Preston RJ (1983) SCE induction is proportional to substitution

in DNA for thymidine by CldU and BrdU. Mutat Res 107:103–109 9. Suzuki H, Yosida TH (1983) Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA. Mutat Res 111:277–282 10. Hazen MJ, Villanueva A, Juarranz A, Canete M, Stockert JC (1985) Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plusGiemsa (FPG) chromosome technique. Histochemistry 83:241–244 11. Nagasawa H, Little JB (1979) Effect of tumor promoters, protease inhibitors, and repair processes on X-ray-induced sister chromatid exchanges in mouse cells. Proc Natl Acad Sci U S A 76:1943–1947 12. Su C, Allum AJ, Aizawa Y, Kato TA (2016) Novel glyceryl glucoside is a low toxic alternative for cryopreservation agent. Biochem Biophys Res Commun 476:359–364 13. Cartwright IM, Kato TA (2015) Role of various DNA repair pathways in chromosomal inversion formation in CHO mutants. Int J Radiat Biol 91:925–933 14. Nagasawa H, Fornace D, Little JB (1983) Induction of sister-chromatid exchanges by DNA-damaging agents and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in synchronous Chinese hamster ovary (CHO) cells. Mutat Res 107:315–327

Chapter 8 DNA Damage Focus Formation Assay Yoshihiro Fujii Abstract Advanced techniques allow investigating cellular DNA damage measurements. Ionizing radiation produces multiple DNA damages. Among them, DNA double strand breaks are most toxic to cells. DSBs can form mutations, chromosome aberrations, and cell killing. Although DSBs in cells can be detected directly by neutral elution, pulse field gel electrophoresis, and premature chromosome condensation, recent technologies like cellular immunocytochemistry-based fluorescence detection allow us to visualize the DSBs in cells. Here, we describe gamma-H2AX and Rad51 focus formation assay, which play an important role in DNA damage responses. Key words DNA damage focus formation assay, Immunocytochemistry, DNA double strand breaks (DSBs), Gamma-H2AX, Rad51

1

Introduction

1.1 Background Information

DNA is routinely damaged internally and externally by reactive oxygen species (ROS), ultraviolet, natural radiation, and chemical substances [1]. Moreover, we have possibility to be exposed to radiations following DNA damages in diagnosis for some diseases and injuries at a hospital by CT scan, nuclear medicine examination and nuclear accidents. Thus we are damaged by radiations at various kinds of situation in our daily life. Damaged DNA is caused to physical dysfunction following cell death and to cancer following mutation. Generally there are some kinds of DNA daamge such asbase damage, base loss, single strand breaks (SSBs) and double strand breaks (DSBs). While cells have various DNA repair mechanisms for these damages such as base excision repair (BER), nucleotide excision repair (NER), in addition, non-homologous end joining (NHEJ) and homologous recombination repair (HRR) work for double strand breaks (DSBs) [2–4]. As DSBs are the most severe damage, it is very important to investigate the mechanism(s) of these two repair systems closely.

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_8, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Fig. 1 An example of DNA damage focus formation assay by gamma-H2AX (green). Cells were irradiated with X-rays and nucleus was stained with DAPI (blue)

Fig. 2 An example of DNA damage focus formation assay by Rad51 (green). Cells were irradiated with X-rays and nucleus was stained with DAPI (blue)

One of the most useful methods of investigating DSBs repair mechanism is the DNA damage focus formation assay. DNA damaging foci detection for gamma-H2AX (Fig. 1), phosphorylated ATM, 53BP1, and Rad51 (Fig. 2), which is a very important role to DNA damage responses (DDS), can directly visualize location and the number of specific DNA damage responses [5, 6]. Observation of these foci was direct evidence of protein accumulation and activity in situ. Specially, among them, gamma-H2AX is the most

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useful and the direct DSBs marker. One Gamma-H2AX foci is thought to be one to one correspondence to one DSB [6]. Therefore, we can observe gamma-H2AX foci with fluorescence as a shape of circle by a microscope and know the correct number of DSBs quantitatively. Moreover, it has been reported that high linear energy transfer (LET) radiations like alpha-ray, neutron, and heavy particle beam have a strong biological effect compared to low LET radiations, and high LET raditions form bigger foci, therefore the size of focus or complex of foci indicates the severity of the damage [7]. These DNA damaging foci can be persisted to metaphase chromosomes after initial DNA damage at interphase. The persistence of foci is associated with DNA repair capacity and time after initial damage formation. The long persistence of gamma-H2AX foci means that the repair of damages does not go well, meanwhile the short residual of them means that the repair of damages goes well. Therefore, it is possible to know the radio-sensitivity of the damaged cells indirectly by the degree of residual of gamma-H2AX foci [8–10]. While, although foci at the metaphase chromosomes are not always formed at the time of initial irradiation, and it can be produced during DNA replication, therefore we can observe even some normalspontaneous foci. We should be very careful to analyze for the foci [11].

2

Materials

2.1 Chemicals, Buffers, and Antibodies

1. 4% Paraformaldehyde solution in Phosphate buffered saline (PBS, GIBCO). 2. 0.2% Triton X-100 solution (Sigma) in PBS for gamma-H2AX foci. 3. 0.1% SDS, 0.5% Triton X-100 solution in PBS for Rad51 foci. 4. 10% goat serum in PBS. 5. Phospho-Histone H2AX antibody (Millipore Ser129). Mouse monoclonal antibody (Upstate, Charlottesville, VA, USA). 6. Rad51 antibody. Rabbit polyclonal antibody. 7. Alexa 488 conjugated goat anti-mouse IgG antibody (Molecular Probes, Eugene, OR, USA). 8. Alexa 592 conjugated goat anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA). 9. Prolong Gold with DAPI (Molecular Probes).

2.2

Equipment

1. Olympus FV-300 confocal laser fluorescent microscope (Olympus, Tokyo, Japan).

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Methods 1. Cells should be cultured on chamberslides. Alternatively, clean 22  22 coverslip after 70% Ethanol wash, placed on P-35 dishes, can be used (see Note 1). 2. After irradiation and sufficient repair time, cells should be washed with PBS and fixed in 4% Paraformaldehyde solution for 15 min. 3. Wash with PBS and permeabilize in 0.2% Triton X-100 solution for 5 min. (a) For Rad51 foci detection, use 0.1% SDS 0.5% Triton X-100 solution instead. 4. Slides should be stored with 10% Goat serum solution for blocking for 1 h at 37  C (see Notes 2 and 3). 5. Apply mouse monoclonal antibody against Phopho-Histone H2AX antibody (1:500 dilution) or Rad51 antibody. Rabbit polyclonal antibody with 1:100–1:500 dilution in 10% goat serum in PBS with parafilm. Incubate slides for 1 h at 37  C (see Notes 4 and 5). 6. Wash slides by immersing them in a Coplin jar containing PBS for 5 min at room temperature. Repeat this three times. 7. Apply secondary antibody (e.g., Alexa488 anti-mouse goat antibody, 1:500 dilution for gamma-H2AX, Alexa592 antirabbit antibody, 1:500 dilution for Rad51) in 10% Goat serum for 1 h at 37  C (see Notes 4 and 5). 8. Wash slides by immersing them in a Coplin jar containing PBS for 5 min at room temperature. Repeat this four times. 9. Mount slides with coverslip and Prolong Gold with DAPI. 10. Score the foci location and the number of them on the cells (see Figs. 1 and 2, Note 6).

4

Notes 1. As described above, foci are not always formed at the time of initial irradiation, and it can be produced during DNA replication. Therefore, it is difficult to tell the foci that you would like to observe from the foci that are produced intrinsically on the cells during DNA replication. To prevent this confusing situation, cells should be synchronized by such as mitotic shake off method and drugs which can regulate cell cycle phase before culturing them on chamber slides. 2. Background foci may be reduced by overnight blocking at 4  C.

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3. High passage normal human fibroblasts, cancer cells, DNA repair deficient cells may have higher background foci than normal health cells. 4. Background fluorescence can be reduced with lower concentration of antibody. 5. Primary and secondary antibody reactions may be achieved in 4  C overnight treatment if any time limitation happened. 6. Z-stage microscope or confocal microscope can obtain multistack layered images if high resolution image is necessary.

5

Anticipated Results If foci on cells are present, they should be detectable at 40 or higher magnification. Typically, a few number of background foci per cell can be seen, and gamma-H2AX foci can be formed approximately 40 foci per cell by 1 Gy of X-ray irradiation.

References 1. Cadet J, Wagner JR (2013) DNA base damage by reactive oxygen species, oxidizing agents, and uv radiation. Cold Spring Harb Perspect Biol 5(2). https://doi.org/10.1101/ cshperspect.a012559 2. Mu D, Park CH, Matsunaga T, Hsu DS, Reardon JT, Sancar A (1995) Reconstitution of human DNA repair excision nuclease in a highly defined system. J Biol Chem 270 (6):2415–2418 3. Scott TL, Rangaswamy S, Wicker CA, Izumi T (2014) Repair of oxidative DNA damage and cancer: recent progress in DNA base excision repair. Antioxid Redox Signal 20(4):708–726 4. Duda´sˇova´ Z, Duda´sˇ A, Chovanec M (2004) Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol Rev 28:581–601 5. Costes SV, Chiolo I, Pluth JM, Barcellos-Hoff MH, Jakob B (2010) Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization. Mutat Res 704:78–87 6. Kuo LJ, Yang LX (2008) Gamma-H2AX - a novel biomarker for DNA double-strand breaks. In Vivo 22(3):305–309

7. Nakajima NI, Brunton H, Watanabe R, Shrikhande A, Hirayama R, Matsufuji N, Fujimori A, Murakami T, Okayasu R, Jeggo P, Shibata A (2013) Visualisation of γH2AX foci caused by heavy ion particle traversal; distinction between core track versus non-track damage. PLoS One 8(8):e70107 8. Yuan J, Adamski R, Chen J (2010) Focus on histone variant H2AX: to be or not to be. FEBS Lett 584(17):3717–3724 9. Celeste A, Difilippantonio S, Difilippantonio MJ, Fernandez-Capetillo O, Pilch DR, Sedelnikova OA, Eckhaus M, Ried T, Bonner WM, Nussenzweig A (2003) H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell 114(3):371–383 10. Sak A, Stuschke M (2010) Use of γH2AX and other biomarkers of double-strand breaks during radiotherapy. Semin Radiat Oncol 20 (4):223–231 11. Gagou ME, Zuazua-Villar P, Meuth M (2010) Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1. Mol Biol Cell 21(5):739–752

Chapter 9 Nuclear Foci Assays in Live Cells Eiichiro Mori and Aroumougame Asaithamby Abstract DNA double strand breaks (DSBs) are a serious threat to genome stability and cell viability. Accurate detection of DSBs is critical for the basic understanding of cellular response to ionizing radiation. Recruitment and retention of DNA repair and response proteins at DSBs can be conveniently visualized by fluorescence imaging (often called ionizing radiation-induced foci) both in live and fixed cells. In this chapter, we describe a live cell imaging methodology that directly monitors induction and repair of single DSB, recruitment kinetics of DSB repair/sensor factors to DSB sites, and dynamic interaction of DSB repair/sensor proteins with DSBs at single-cell level. Additionally, the methodology described in this chapter can be readily adapted to other DSBs repair/sensor factors and cell types. Key words Nuclear foci, Live cell imaging, DNA double strand breaks, 53BP1, FRAP

1

Introduction Cell lethality, mutations, chromosomal translocations, apoptosis, and cancer induced by ionizing radiation (IR) result principally from an inefficient or inaccurate repair of DNA double strand breaks (DSBs). Recruitment and retention of DNA repair and response proteins at DSBs can be visualized by fluorescence imaging (often called ionizing radiation-induced foci [IRIF]). The H2AX is immediately phosphorylated at the sites of DSBs, and the phosphorylated H2AX (γH2AX) can be visualized in situ by immunostaining with a γH2AX specific antibody [1–4]. In addition to γH2AX, phosphorylated DNA-dependent protein kinase (DNA-PK) also localizes precisely at the DSB sites and serves as an ideal maker for visualizing DSBs in situ [5]. p53 binding protein 1 (53BP1), a DNA damage response protein, responds to DSBs, forms discrete nuclear foci upon exposure to IR, and colocalizes with γH2AX and DNA-PK; it also serves as a surrogate marker for DSBs [6–9]. These indirect immunostaining techniques have been widely used to visualize DSBs generated by low- and high-linear energy transfer (LET) IR in fixed cells [10–12].

Takamitsu A. Kato and Paul F. Wilson (eds.), Radiation Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1984, https://doi.org/10.1007/978-1-4939-9432-8_9, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Though indirect immunostaining has significantly improved our understanding of induction and repair of DSBs, some of the challenging questions in the study of IR-induced DNA damages cannot yet be answered. For instance, how fast is the recognition of various types of damaged DNA? Which proteins arrive first to the sites of DNA damage? What is the affinity of different repair proteins for different DNA lesions? How do distinct proteins recognize different types of DNA lesions? Due to the use of green fluorescent proteins (GFP) and their spectral variants [13–16] and advancements in microscopy and digital imaging technology, it is now possible to study the spatio-temporal aspects of different DNA damages in live cells [9, 17, 18]. To this end, multiple DNA damage response and repair factors fused to fluorescent proteins have been used in understanding the spatio-temporal aspects of different DNA lesions in live cells [9, 19–21]. For example, 53BP1 and NBS1 are used as markers for DSBs for live cell imaging [6, 7, 19] (Fig. 1). Further, X-ray repair complementing defective repair in Chinese hamster cells (XRCC1) [8, 17] and aprataxin (APTX) are used as surrogate markers for single strand breaks [22, 23], and 8-oxoguanine DNA-glycosylase 1 (OGG1) is used as a surrogate marker for base damages [17] (Fig. 1). Multiple DNA damage inducing sources have been utilized in understanding the cellular response to DNA lesions in living cells. For example: (1) Low-LET IR, including X- and γ-rays [9, 19, 20]; (2) High-LET IR, including Tandem Van de Graaff [24] and heavy ion particles [12, 19, 25, 26] (NASA Space Radiation Laboratory, Brookhaven National Laboratory, New York), and Universal Linear Accelerator (Gesellschaft fu¨r Schwerionenforschung mbH, Germany) [22]; (3) Ultra-violet (UV) lasers of 365 and 405 nm [17, 18], to introduce focused DNA damages in the nucleus in the field of interests; (4) Microbeam irradiation is another tool for inducing localized DNA damages [27–29]; and (5) UV microspot irradiator combined with a charged-particle microbeam irradiator (Radiological Research Accelerator Facility, Columbia University) [30], are some of the widely used radiation sources. DNA repair research has been boosted substantially by the development of several methods to inflict different types of DNA lesions in living cells, enabling the direct visualization of fluorescent protein-tagged repair/sensor factors. Further, quantitative live cell imaging techniques combined with methods to induce local DNA damage in a small region of the nucleus are contributing substantially to unraveling the molecular mechanisms underlying the cellular response to DNA damage. However, development of instruments together with irradiation facility and imaging tools has been challenging, and continued development of innovative imaging tools is required to meet the emerging radiation platform. Therefore, to accurately predict the biological effects of IR, an assay must be sensitive enough to measure levels of different DNA

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Fig. 1 Live cell images show recruitment of fluorescent protein-tagged DNA repair proteins to the sites of DNA damage. (a) Representative images show

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lesions at the single-cell level. Here, we describe a live cell imaging methodology that directly monitors induction and repair of single DSB, recruitment of DSB repair/sensor factors to DSBs, and dynamic interaction of DSB repair/sensor proteins with individual DSB site at the single-cell level. Additionally, the methodology described in this chapter can be readily adapted to other DSBs repair/sensor factors and cell types.

2

Materials 1. Growth medium: Supplement Minimum Essential Medium (MEM) alpha (SH3026502, GE Healthcare HyClone) with 5% fetal bovine serum (S11150, Atlanta Biologicals), 5% newborn calf serum (SH3011803, GE Healthcare HyClone), 100 μg/mL streptomycin and 100 U/mL penicillin (SV30010, GE Healthcare HyClone) (see Note 1).

2.1 Cell Culture and Establishment of Stable Cell Lines

2. Incubator: Use humidified CO2 (5%) cell culture incubator maintained at 37
C (see Note 2). 3. Cell line: We routinely use HT1080 cells (CCL-121™, ATCC®) to stably express fluorescent protein-tagged DSB repair/sensor factors (see Note 3). 4. Plasmid DNA: Mammalian expression plasmids encoding fluorescent protein-tagged DNA repair/sensor factors. Here, we will use mammalian expression pcDNA3.F2-EYFP-53BP1 plasmid. 5. Transfection reagents: We use two different transfection methods: a. Nucleofection procedure using a Nucleofector instrument (Amaxa); and b. Lipofectamine® 2000 reagent (Invitrogen). 6. Antibiotics for selecting stable clones: Use 500 μg/mL of G418 (neomycin, Invitrogen) for selecting stable HT1080 clones and 200 μg/mL of G418 for maintaining stable HT1080 clones (see Note 4). ä Fig. 1 (continued) recruitment of EYFP-53BP1 (top) and EGFP-XRCC1 (bottom) to the sites of DNA damage induced by 1 Gy γ-rays. (b) Representative images show recruitment of EYFP-53BP1 (top) and EGFP-XRCC1 (bottom) to the sites of DNA damage induced by 1 Gy Iron (Fe) particles (1GeV/n). (c) Representative images show recruitment of EGFP-XRCC1 (top) and EGFP-OGG1 (bottom) to the sites of DNA damage induced by micro-UV laser. Cells were grown on glassbottomed 35 mm dishes, imaged prior to DNA damage (pre-IR) and the same cells were imaged 30 min after the DNA damage

Dynamics of DNA Damage Foci in Live Cells

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1. Transfection: For Lipofectamine 2000 mediated transfection, seed ~1  106 cells onto a 100 mm cell culture dish and allow cells to attach for 24 h. Then, transfect cells with 5 μg of pcDNA3.F2-EYFP-53BP1, according to the manufacturer’s instructions. For nucleofection, resuspend ~1  106 cells in solution V, mix with 10 μg of pcDNA3.F2-EYFP-53BP1 plasmid, and transfect the cells using X-001 program of the Nucleofector instrument. 2. Selection: 24 h after the transfection, trypsinize cells and seed 1000, 5000, and 10,000 cells per 100 mm dish in triplicate. Next day, add 500 μg/mL G418 containing medium to the transfected cells. Change G418 containing medium every 2 days for 12 days. Seeding different concentrations of transfected cells is very critical for getting enough number of well dispersed individual stable clones. 3. Colony picking: Circle well separated clones that are visible to naked eyes on the bottom of the culture dishes using a black marker pen. Soak precut small circular Whatman filter papers in 0.25% Trypsin-EDTA (25200-072, Invitrogen). Next, wash the cells with 1 PBS, place a pre-soaked filter paper on the top of a marked clone, and wait for 1–2 min. Lift the filter paper using a sterile forceps and place it in a single well of a 24-well culture dish containing 1 mL of 200 μg/mL G418 growth medium. Pick up ~40–50 colonies per transfection. Grow the cells for 3–4 days. 4. Colony screening: Check the cells under a fluorescent microscope for the presence of fluorescent signal in the nuclei of all cells. Select the clones that are positive for fluorescent signal in the nuclei of all cells. 5. Colony expansion: Trypsinize fluorescent signal positive cells and transfer to a 6-well plate. Subsequently, transfer cells to a T25 flask. All clones may not grow at the same rate, therefore, expand the clones accordingly. 6. Selecting appropriate clone(s) for further experiments: Optimal expression of fluorescent-tagged protein is critical for the accurate detection of foci in live cells. Higher fluorescent signal will mask real foci from the background fluorescent signal and low fluorescent signal will lead to under estimation of ionizing radiation induced foci. Therefore, it is critical to select clones those express medium levels of fluorescent protein-tagged DNA damage repair/sensor factors.

2.3 Microscope Setup, Irradiation, and Live Cell Imaging

1. Microscope setup: We use an LSM 510 Meta laser scanning confocal microscope equipped with a 63  1.4 NA PlanApochromat oil immersion objective for live cell imaging. Set

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the tube current at 6.1A and the laser power to 0.3–1% transmission with the pinhole opened to 1–2 Airy units. For the detection of EYFP fluorescence, use a 458/514 nm dichroic beamsplitter and place an additional 530–600-nm bandpass emission filter in front of the photomultiplier tube. Other microscopes equipped with appropriate objectives and filters can also be used for live cell imaging. 2. Irradiation source: We use 137Cs irradiator (Mark 1 irradiator, JL Shepherd & Associates) to induce DNA lesions in cells. For doses up to 100 mGy, we place lead attenuators between the source and the samples to reduce the dose rate by 50–80% (4 mGy/min). For higher doses, we do not use any attenuators. To verify dose accuracy, we routinely perform dosimetry using thermoluminescence dosimetry devices (Landauer Inc., Glenwood, IL) (see Note 5). 3. Cell seeding: Seed ~50,000 cells onto a glass-bottomed 35 mm culture dish (P35G-0-14-C, MatTek Culture ware). Allow the cells to grow for a minimum of 72 h. 4. Medium for live cell imaging: Use either CO2-independent (18045–088, Invitrogen) or regular growth medium for live cell imaging (see Note 6). Change regular medium to CO2independent medium 2–3 h prior to live cell imaging.

3

Methods

3.1 Direct Visualization of Appearance and Disappearance of EYFP-53BP1 Foci in Live Cells

1. Seed ~50,000 cells onto a glass-bottomed 35 mm culture dish and allow cells to grow for 3–5 days. Cells need to be ~70–80% confluent at the time of experiment. 2. Change the medium to CO2-independent medium, if you are using a microscope that is not equipped with a stage-top incubator. 3. Position the culture dish on the microscope stage and mark the location of the culture dish and the stage. This is critical for proper re-positioning of the culture dish on the microscope stage after IR (see Note 7). 4. Identify 5–7 locations of the culture dish and mark the locations of the cells using “mark and find” function of the LSM Meta 510 software (see Note 8). 5. Acquire cell images just before exposure to IR. This will give a measure of background EYFP-53BP1 foci number in each cell (pre-IR). For each field of cells, take 15–20 “Z”-stack images of 0.5 μm thickness each. 6. Remove the culture dish from the microscope stage and expose cells to desired doses of IR.

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7. Accurately re-position the culture dish on the microscope stage. 8. Record the images of the same cell population immediately after IR using the “mark and find” option of the LSM Meta 510 software. Acquire images every 10 min for 8–12 h using identical imaging parameters (see Note 9). 9. For the quantification of EYFP-53BP1 foci dissolution kinetics, first de-convolute all the “Z” stack images using either ImageJ or Imaris software (Bitplane). Then assign a number to all the cells in a given field. Subsequently, enumerate the number of foci in the same cell at different time points. Record all the details in a Microsoft excel spreadsheet (see Note 10). 10. Calculate appearance and disappearance of 53BP1 foci in each cell at different post-IR times. Average the number of 53BP1 foci in ~100 cells. 3.2 Direct Monitoring of Kinetics of Recruitment of EYFP-53BP1 to DSBs in Live Cells

1. To gain insight into protein redistribution at DSBs in response to IR, real-time assay can be used to monitor accumulation kinetics of fluorescent protein-tagged DNA repair/sensor factors on the damaged DNA. 2. Follow steps 1–7, as described in Subheading 3.1. 3. Start time-lapse image acquisition immediately after the induction of DSBs to obtain post-irradiated cell images. Acquire images of the same cells every 5 min for up to 2 h (see Note 11). 4. To measure the average fluorescent intensity of DSBs sensor/ repair factors at the damaged DNA, randomly select 2–5 IR induced foci in a cell using region of interest (ROI) function of the LSM510 Meta software (see Note 12). 5. Subsequently, convert the signal intensity of accumulated EYFP-53BP1 fluorescence at the DSBs at different post-IR times into a numerical value using LSM510 Meta software. 6. To compensate for nonspecific fluorescent bleaching during the repeated image acquisition, in every image, first measure the average fluorescent intensity of the EYFP-53BP1 focus as a function of time, and then divide it by the average fluorescent intensity measured elsewhere in the cell (background) as a function of time. 7. To get normalized EYFP-53BP1 accumulation curve for each focus, calculate the EYFP-53BP1 fluorescent intensity (RF) at a DSB site by the following formula: RF(t) ¼ [(I  Ipre-IR)/ (Imax  Ipre-IR)], where Ipre-IR is the fluorescent intensity of the focus region before irradiation, and Imax represents the maximum fluorescent intensity of the EYFP-53BP1 focus at a DSB site. Average normalized fluorescent intensity curves from 20 individual cells.

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Eiichiro Mori and Aroumougame Asaithamby

3.3 Visualization of Dynamic Interaction of EYFP-53BP1 with DSB by Fluorescence Redistribution After Photo-Bleaching (FRAP) Technique

1. FRAP technique is used to understand the dynamic interaction between DNA repair/sensor factor and the damaged DNA. 2. To monitor dynamics of DSB sensor/repair factors at IR induced DSBs, image cells prior to irradiation (pre-IR), expose cells to desired doses of IR, and acquire images of the same preIR cell population immediately after IR (post-IR) by following steps 1–7. 3. For photo-bleaching, set 514-nm line of an argon laser at 100% power transmission and 6.1A tube current. Set scanning speed to maximum and the number of frames per scanning to minimum. 4. Acquire images of a cell that showed IR induced 53BP1 foci (pre-bleach) every 10 s to 2 min for 1–10 min. Subsequently, select a single IR induced focus using ROI function of LSM 510 Meta software and photo-bleach the fluorescent signal of the selected focus at multiple “Z” planes (see Note 13). 5. Immediately after photo-bleaching, acquire new images (bleach) and continue to image the same cell every 10 s to 2 min for 30 min using similar image acquisition parameters (see Note 14). 6. Subsequently, measure the average fluorescent intensity of the photo-bleached focus before photo-bleaching and immediately after photo-bleaching as a function of time, as described in Subheading 3.2 (steps 4 and 5). 7. To compensate for nonspecific fluorescent bleaching during the repeated image acquisition, in every image, first measure the average fluorescent intensity of the photo-bleached EYFP53BP1 focus as a function of time, and then divide it by the average fluorescent intensity measured elsewhere in the cell (background) as a function of time. 8. To get normalized FRAP curve for each focus, divide the fluorescent intensity after photo-bleaching by the pre-bleach intensity, and set the pre-bleach intensity to one. Calculate normalized FRAP curves from 20 individual cells and average the FRAP curves.

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Notes 1. Type of culture medium is dependent on the nature of the cell lines. Optimal cell culture conditions are needed for each cell line and experimental setting. 2. The percentage of oxygen depends on the nature of cell type, i.e., primary (