Techniques in Cell Cycle Analysis Biological Methods Techniques in Cell Cycle Analysis, edited by Joe W. Gray and Zbigniew Darzynkiewicz, 1986 Methods in Molecular Biology, edited by John M. Walker, 1984 Volume I: Proteins Volume II: Nucleic Acids Liquid Chromatography in Clinical Analysis, edited by Pokar M. Kabra and Laurence J. Marton, 1981 Metal Carcinogenesis Testing: Principles and In Vitro Methods, by Max Costa, 7980 Techniques in Cell Cycle Analysis

Edited by Joe W. Gray and Zbigniew Darzynkiewicz

Humana Press • Clifton, New Jersey Library of Congress Cataloging-in-Publication Data Techniques in Cell Cycle Analysis. (Biological methods) Includes index. 1. Flow cytometry. 2. Cell cycle. 3. Cancer cells—Growth. I. Gray, Joe W. II. Darzynkiewicz, Zbigniew. III. Series. QH585.5.F56T43 1986 616.99'4071 86-16111 ISBN 0-89603-097-0 E) 1987 xhe Humana Press Inc. Crescent Manor PO Box 2148 Clifton, NJ 07015

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher.

Printed in the United States of America Preface

Quantification of the proliferative characteristics of normal and malignant cells has been of interest to oncolo­ gists and cancer biologists for almost three decades. This interest stems from (a) the fact that cancer is a disease of uncontrolled proliferation, (b) the finding that many of the commonly used anticancer agents are preferentially toxic to cells that are actively proliferating, and (c) the observa­ tion that significant differences in proliferation characteristics exist between normal and malignant cells. Initially, cell cycle analysis was pursued enthusiastically in the hope of gener­ ating information useful for the development of rational cancer therapy strategies; for example, by allowing identi­ fication of rapidly proliferating tumors against which cell cycle-specific agents could be used with maximum effec­ tiveness and by allowing rational scheduling of cell cycle- specific therapeutic agents to maximize the therapeutic ratio. Unfortunately, several difficulties have prevented realiza­ tion of the early promise of cell cycle analysis: Proliferative patterns of the normal and malignant tissues have been found to be substantially more complex than originally an­ ticipated, and synchronization of human tumors has proved remarkably difficult. Human tumors of the same type have proved highly variable, and the cytokinetic tools available for cell cycle analysis have been labor intensive, as well as somewhat subjective and in many cases inapplicable to humans. However, the potential for substantially improved cancer therapy remains if more accurate cytokinetic infor­ mation about human malignancies and normal tissues can be obtained in a timely fashion. This monograph contains a series of articles describing cytokinetic techniques that have been important to the de­ velopment of our current cytokinetic data base, as well as ui Preface

those that appear to have substantial promise for future cytokinetic studies in model systems and in the clinic. Traditional techniques based on the autoradiographic detection of incorporated tritiated thymidine have provided information about the fraction of cells capable of DNA syn­ thesis (a rough indicator in the proliferative activity of the cell population), about the G1-, S-, and G2M-phase dura­ tions and dispersions therein, and about the growth frac­ tion (fraction of actively proliferating cells in a population). These techniques, their attributes, and their limitations are described in chapters 1 and 2. Chapter 3 focuses on con­ ventional and developing techniques for estimation of the growth fraction. Much of our information on the response of tumors to cytotoxic agents has been derived from measurements of the colony-forming ability of cells from model and human tumors grown in vitro. These studies are reviewed in chapter 4. The majority of this monograph is devoted to flow cy­ tometry and its application to cytokinetics because of the increasing importance of flow cytometry in the field of cytokinetics. Flow cytometry has become the method of choice in many cytokinetic studies within the past decade because of the speed and accuracy with which cellular prop­ erties of cytokinetic importance (e.g., DNA content, RNA content, amount of incorporated bromodeoxyuridine, pro­ liferative status, and so on) can be measured. In addition, many cells can be analyzed in each experiment so that rare subpopulations can be studied. Chapter 5 introduces flow cytometry and reviews several common cytokinetic applica­ tions, including univariate DNA distribution analysis and bivariate analysis of cellular DNA content and amount of incorporated bromodeoxyuridine. Chapters 6 and 7 discuss the techniques necessary to prepare cells for flow cytometric analysis; chapter 6 reviews techniques for dissociation of solid tissues into suspensions of single cells, and chapter 7 reviews a variety of cell staining techniques that are espe­ cially useful for cytokinetic studies. Chapter 8 deals with computer analysis technique for display and cytokinetic analysis of flow cytometric data. Chapter 9 discusses cyto- Preface vii

chemical techniques to allow flow cytometric discrimination of quiescent and proliferating cells. These techniques com­ plement those described in chapter 3. Chapter 10 deals with the combination of flow cytometry and stathmokinesis for cell cycle analysis. Most cytokinetically based cancer therapy optimization attempts have presumed the existence of a battery of effec­ tive cell cycle-specific agents. Chapter 11 introduces the idea that drug-resistant cells existing prior to therapy and/or developed during the course of therapy must also be con­ sidered during the development of cytokinetic therapy strategies. This chapter also suggests several flow cytometric approaches to intracellular drug level quantification. Much of our information about the cell cycle-specific nature of anticancer agents has come from the application of these agents to synchronized cell populations. Several techniques for synchronization of cells grown in vitro are critically compared in chapter 12. These 12 chapters encompass many of the techniques that have been especially useful in cell cycle studies or that hold great promise for the future. Emphasis has been placed on the techniques themselves and on critical review of their attributes and limitations. We offer them here in the hope that they will facilitate selection of appropriate techniques for future studies and will stimulate development of new approaches to remove existing limitations so that the true potential of cell cycle analysis in cancer therapy can be realized. Joe W. Gray Zbigniew Darzynkiewicz Contents

Preface . . . u List of Contributors xvii

CHAPTER 1. Autoradiographic Techniques for Measurement of the Labeling Index .... 1 Linda Simpson-Herren 1. Introduction and History 1 2. Principles of Autoradiography 2 3. Labeling Index 3 3.1. Definition 3 3.2. Discrete or Continuous Model of Cell Cycle 4 3.3. Precursors for Measurement of a Labeling Index ... 5 3.4. Specificity of Precursors 7 3.5. Sources of Artifacts 10 4. Ambiguity of the Labeling Index 10 4.1. Experimental Conditions 10 4.2. Labeling Characteristics of the Tissue 13 4.3. Background Threshold 16 5. Autoradiographic Techniques 19 5.1. Pretest of Emulsion 19 5.2. Background and Grain Development During Exposure 20 5.3. Negative and Positive Chemography 21 6. Conclusions 22 References 24 CHAPTER 2. Percent Labeled Mitosis Curve Analysis 31 Stanley E. Shackney and Paul S. Ritch 1. Experimental Technique 31 ix X Contents

2. PLM Analysis in Kinetically Homogeneous Populations 32 3. PLM Analysis in Kinetically Heterogeneous Populations 34 3.1. General Considerations 34 3.2. PLM Analysis in Relation to the Growth Fraction and the GO Cell Pool 34 3.3. Problems of Characterizing Broad Cell Cycle Time Distributions by PLM Analysis 37 4. Comprehensive PLM and Grain Count Halving Methods 40 5. Conclusions 42 References 44 CHAPTER 3. Tumor Growth Fraction Estimation, Perturbation, and Prognostication 47 Paul G. Braunschweiger 1. Introduction 47 2. Methods for Estimation of Tumor Growth Fraction .... 48 2.1. Pulse-Labeling Methods 48 2.2. Continuous or Repeated [3H]-TdR Labeling 49 2.3. Morphological Methods 50 2.4. Flow Cytometry 51 2.5. PDP Assay 51 2.6. Estimation of GF in Perturbed Tumors 57 3. GF as an Indicator of Chemosensitivity 61 4. Conclusions 64 References 67 CHAPTER 4. In Vitro Assays for Tumors Grown In Vivo: A Review of Kinetic Techniques 73 Janet S. Rasey 1. Introduction 73 2. Review of Techniques 75 2.1. Clonogenic Fraction vs Time 75 2.2. Labeled Microcolony Technique 77 Contents xi

2.3. S-Phase Suicide Technique 79 2.4. Viable Cell Sorting Based on DNA Content 82 2.5. Purification by Centrifugation 83 3. Conclusions 86 References 87 CHAPTER 5. Flow Cytokinetics 93 Joe W. Gray, Frank Dolbeare, Maria G. Pallaoicini, and Martin Vanderlaan 1. Inroduction 93 2. Flow Cytometry and Sorting 94 3. DNA Distribution Analysis 96 4. Radioactivity per Cell (RC) Analysis 99 4.1. Asynchronous Populations 99 4.2. Rate of DNA Synthesis 103 4.3. Perturbed Populations 103 5. Stathmokinetic Analyses 105 6. Bromodeoxyuridine as a Cytokinetic Label 107 6.1. Bromodeoxyuridine (BrdUrd) Quenching of Hoechst Fluorescence 107 6.2. Monoclonal Antibodies Against BrdUrd Ill 7. Cytokinetic Analysis of Cytometrically Distinct Subpopulations 126 7.1. Diploid Cells in the KHT Sarcoma 126 7.2. BrdUrd/DNA Analysis of Murine Myeloblasts and Erythroblasts 127 8. Conclusions 131 References 133 CHAPTER 6. Solid Tissue Dispersal for Cytokinetic Analyses 139 Maria G. Pallaoicini 1. Introduction 139 2. Biochemical Basis of Tissue Disaggregation 140 3. Suggested Guidelines to Obtain Cell Suspensions for Cytokinetic Analyses 144 4. Applications of Guidelines for Tissue Dispersal 146 4.1. Preferential Phase-Specific Cell Selection 146 4.2. Preferential Subpopulation-Specific Selection 147 xii Contents

4.3. Cell Function 150 4.4. Cell Yield 151 4.5. DNA Distributions 152 4.6. Retention of Cell Morphology 152 5. Evaluation of Selected Tumor Dispersal Procedures According to Suggested Guidelines 153 References 156 CHAPTER 7. Multivariate Cell Analysis: Techniques for Correlated Measurements of DNA and Other Cellular Constituents 163 Harry A. Crissman and John A. Steinkamp 1. Introduction 163 2. Fluorescence Principles (Generalized) 165 3. Characteristics of Fluorescent Dyes 165 4. Specificity and Stoichiometry of Fluorochrome Reactions 167 5. Energy Transfer 167 6. Intrinsic Fluorescence of Unstained Cells (Autofluorescence) 169 7. Cell Preparation and Fixation 170 8. Current Flow-System Modifications for Multivariate Analysis 171 9. DNA Content Analysis by Flow Cytometry 173 10. DNA-Specific Cell Staining 175 10.1. Feulgen-DNA Reaction 175 10.2. DNA Reactive Hoechst Dyes, DAPI (4-6-diamidino-2-phenylindole), DIPI [4-6-bis-(2-imidozolynyl-4H, 5H)-2-phenylindole], and LL 585 175 10.3. Viable Cell Staining for DNA With Hoechst 33342 176 10.4. Cell Staining With Mithramycin, Chromomycin, or Olivomycin 179 10.5. Staining With Propidium Iodide (PI) or Ethidium Bromide (EB) 180 10.6. Multiple Fluorochrome Labeling of DNA 181 11. Two-Color DNA and Protein Staining 184 12. Correlated Analysis of DNA, RNA, and Protein 186 Contents xiii 13. Correlation of DNA, RNA, or Protein and Mitochrondria 193 14. Cell Cycle Analyses Using Bromodeoxyuridine (BrdUrd) 196 15. Conclusions 198 References 199

CHAPTER 8. Data Analysis in Cell Kinetics Research 207 Phillip ti. Dean 1. Introduction 207 2. Methods 210 2.1. DNA Distribution Analysis 210 2.2. Kinetic Models 231 3. Conclusions 247 References 250

CHAPTER 9. Cytochemical Probes of Cycling and Quiescent Cells Applicable to Flow Cytometry 255 Zbigniew Darzynkiewicz 1. Introduction 255 1.1. Definition of Quiescence/Classification of Quiescent States 255 1.2. Metabolic Properties of Quiescent Cells 257 2. Cell Size 264 2.1. Dry Weight; Image Analysis 264 2.2. Coulter Volume Measurements 265 2.3. Light Scatter 266 2.4. Cell Morphology by Flow Cytometry 267 3. Protein Content 267 4. RNA Content 268 5. Mitochondrial Probes; Electronegativity of the Mitochondrial Membrane 269 6. Sensitivity of DNA In Situ to Denaturation 270 7. Accessibility of DNA In Situ to the Intercalating Probes 272 xiv Contents

8. Incorporation of BrdUrd 273 9. In Search of Specific Markers 274 References 279 CHAPTER 10. Assay of Cell Cycle Kinetics by Multivariate Flow Cytometry Using the Principle of Stathmokinesis 291 Zbigniew Darzynkiewicz, Frank Traganos, and Marek Kimmel 1. Introduction 291 2. Stathmokinetic Approach for Analysis of the Cell Cycle 293 2.1. Rate of Cell Entry Into Mitosis 293 2.2. Restrictions of the Stathmokinetic Technique .... 293 2.3. DNA Measurements by Flow Cytometry in Stathmokinetic Experiments 295 3. Cell Cultures 296 4. Scheme of the Stathmokinetic Experiment 297 5. Cell Staining 298 5.1. Reagents 298 5.2. Staining Procedures 299 5.3. Fluorescence Measurements 300 6. Discrimination of Different Compartments of the Cell Cycle 301 7. Results of the Stathmokinetic Experiment 305 7.1. Cell Entrance to M or G2 + M 305 7.2. Cell Exit from Gl and GIA 307 7.3. Cell Progression Through S-Phase 309 8. Cell Cycle Perturbations Induced by Various Drugs .. 310 8.1. Drug Effects on Gl, S-, and G2-Phases 310 8.2. Terminal Point of Drug Action 313 9. Detection of Chromatin Changes 314 10. Mathematical Analysis of the Stathmokinetic Experiment 317 10.1. General Assumptions 317 10.2. Exponential and Asynchronous Growth 319 10.3. Stathmokinetic Experiment 321 10.4. Extensions of the Model 327 11. Conclusions: Novel Elements of the Technique 328 References 332 Contents xv CHAPTER 11. Flow Cytometric Studies on Intracellular Drug Fluorescence 337 Awtar Krishan 1. Introduction 337 2. Instrumentation and Sample Considerations 338 3. Anthracyclines 341 3.1. Anthracycline Fluorescence and Flow Cytometry . 341 3.2. Drug Transport Studies 345 3.3. Drug Resistance 349 3.4. Transport and Cell Cycle 352 3.5. Drug Interactions 354 3.6. Conclusions 355 4. Methotrexate 356 4.1. Fluorescence Labeling 357 4.2. DHFR Levels and MTX Resistance 358 5. Conclusions 361 References 363 CHAPTER 12. Cell Synchrony Techniques: A Comparison of Methods 367 Daoid J. Grdina, Marvin L. Melstrich, Raymond E. Meyn, Tod S. Johnson, and R. Allen White 1. Introduction 367 2. Materials and Methods 369 2.1. Cell System and Methods of Flow Cytometry Analysis 369 2.2. Mitotic Selection and HU Synchronization 369 2.3. Centrifugal Elutriation 370 2.4. Cell Sorting 371 2.5. Cell Progression Analysis 371 3. Results 372 3.1. Empirical Determinations of Cell Progression Parameters 372 3.2. Mathematical Analysis and Comparison of Cell Progression Parameters 382 4. Discussion 392 4.1. Comparison of Cell Synchronization Methods ... 392 4.2. Advantages and Disadvantages of Each Synchronization Method 396 xvi Contents

5. Conclusions 397 Appendix: Mathematical Methods 398 References 399 Index 403 Contributors

PAUL G. BRAUNSCHWEIGER • Department of Experimental Therapeutics, AMC Cancer Research Center and Hospital, Lakewood, Colorado HARRY A. CRISSMAN • Life Sciences Division, Los Alamos Na­ tional Laboratory, Los Alamos, New Mexico ZBIGNIEW DARZYNKIEWICZ • Sloan-Kettering Institute of Cancer Research, Walker Laboratory, Rye, New York PHILLIP N. DEAN • Biomedical Sciences Division, Lawrence Liver- more National Laboratory, University of California, Livermore, California FRANK DOLBEARE • Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, Liver- more, California JOE W. GRAY • Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, Livermore, California DAVID J. GRDINA • Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois TOD S. JOHNSON • Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois MAREK KIMMEL • Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York AWTAR KRISHAN • Comprehensive Cancer Center and Depart­ ment of Oncology, University of Miami Medical School, Miami, Florida MARVIN L. MEISTRICH • Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois RAYMOND E. MEYN • Division of Biological and Medical Re­ search, Argonne National Laboratory, Argonne, Illinois

xvii xuiii Contributors

MARIA G. PALLAVICINI • Biomedical Sciences Division, Law­ rence Livermore National Laboratory, University of Califor­ nia, Livermore, California JANET S. RASEY • Department of Radiation Oncology, School of Medicine, University of Washington, Seattle, Washington PAUL S. RITCH • Department of Medicine, The Medical College of Wisconsin, Milwaukee, Wisconsin STANLEY E. SHACKNEY • Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland LINDA SIMPSON-HERREN • Biochemistry Research Division, Southern Research Institute, Birmingham, Alabama JOHN A. STEINKAMP • Life Sciences Division, Los Alamos Na­ tional Laboratory, Los Alamos, California FRANK TRAGANOS • Sloan-Kettering Institute for Cancer Re­ search, Walker Laboratory, Rye, New York MARTIN VANDERLAAN • Biomedical Sciences Division, Law­ rence Livermore National Laboratory, University of Califor­ nia, Livermore, California R. ALLEN WHITE • Department of Biomathematics, University of Texas, Houston, Texas