University of Groningen Hyperthermia and protein aggregation Stege, Gerardus Johannes Jozef IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1995 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Stege, G. J. J. (1995). Hyperthermia and protein aggregation: role of heat shock proteins Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-02-2018
Rijksuniversiteit Groningen HYPERTHERMIA AND PROTEIN AGGREGATION role of heat shock proteins Proefschrift ter verkrijging van het doctoraat in de Geneeskunde aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus Dr. F. van der Woude in het openbaar te verdedigen op woensdag 14 juni 1995 des namiddags te 2.45 uur precies door Gerardus Johannes Jozef Stege geboren op 21 maart 1963 te Weerselo
Promotor Co-promotor Prof. Dr. A.W.T. Konings Dr. H.H. Kampinga
Beoordelingscommissie Prof. Dr. L. de Ley, Rijksuniversiteit Groningen Prof. Dr. W.W. de Jong, Katholieke Universiteit Nijmegen Prof. Dr. G.C. Li, Memorial Sloan Kettering Cancer Center, New York, USA Paranimfen Pieter K. Wierenga Jeanette F. Brunsting Word-processing: WordPerfect 5.1 Illustrations: Harvard Graphics 3.0 and SlideWrite Plus 4.0 Printing: CopyPrint 2000, Enschede The printing of this thesis was financially supported by Becton Dickinson Paes Nederland BV Bio-Rad bv
This study was financially supported by the Dutch Cancer Society (GUKC 89-09) and the Interuniversitair Instituut voor Radiopathologie en Stralenbescherming in the Netherlands (IRS 7.22). Part of the work (chapters 3 and 4) was also supported by the National Cancer Institute of the National Institute of Health in the USA (CA 31397 and CA 56909). ISBN 90-9008211-5
aan mijn ouders aan Anne-Marie
Contents Chapter 1: General introduction 1.1 HYPERTHERMIA 2 1.1.1 Cell survival curves 2 1.1.2 Protein denaturation 4 1.1.2.1 Heat-induced protein denaturation and aggregation in cells 6 1.1.3 Heat effects on cellular structures 7 1.1.3.1 Plasma membrane 7 1.1.3.2 Heat effects on cytosolic structures and processes 8 1.1.3.3 Heat effects on nuclear structures and processes 9 1.2 HEAT SHOCK PROTEINS 13 1.2.1 Molecular chaperones 14 1.2.2 Heat shock proteins as molecular chaperones 18 1.2.2.1 Small heat shock proteins 18 1.2.2.2 Hsp60 family 21 1.2.2.3 Hsp70 family 25 1.2.2.4 Hsp90 family 46 1.2.3 Regulation of the stress response 48 1.2.3.1 Transcriptional activation of heat shock genes 49 1.2.3.2 Translational regulation of heat shock gene expression 53 1.2.4 Thermotolerance, thermoresistance and intrinsic heat sensitivity; 54 role of heat shock proteins 1.2.4.1 Thermotolerance 55 1.2.4.2 Thermoresistance 57 1.2.4.3 Intrinsic heat sensitivity 61 1.3 HEAT AND RADIATION 62 1.3.1 The synergism of heat and radiation 62 1.3.2 Mechanisms of interaction 63 1.3.2.1 Inactivation of repair enzymes 64 1.3.2.2 Heat-induced alteration of the chromatin structure 65 1.3.3 Heat radiosensitization and thermotolerance 67 1.4 SCOPE OF THE THESIS 68 Chapter 2: Hyperthermic cell killing and calcium homeostasis J. Cell. Physiol. 155, 452-460 (1993) Int. J. Radiat. Biol. 64, 459-468 (1993) Eur. J. Cell Biol. 63, 68-76 (1994) 2.1 Introduction 73 2.1.1 Role of intracellular free calcium 74 2.1.2 Role of extracellular calcium 74
2.1.3 Intracellular calcium modifiers 74 2.1.4 Effect of intracellular calcium on heat-induced protein aggregation 75 2.2 Materials and methods 75 2.2.1 Cell cultures 75 2.2.2 Cell loading with fura-2/am 76 2.2.3 [Ca 2+ ] i assay 76 2.2.4 Hyperthermic treatments 77 2.2.5 Ionophore and Ca 2+ treatments 77 2.2.6 Determination of cell survival 78 2.2.7 Isolation of nuclei and flow cytometric analysis 78 2.3 Results 79 2.3.1 On the measurement of [Ca 2+ ] i at 37 C and 79 hyperthermic temperatures 2.3.2 Relation between heat-induced increases in [Ca 2+ ] i and 84 hyperthermic cell killing 2.3.3 Relation between effects of heat and ionomycin on [Ca 2+ ] i and 84 cell killing 2.3.4 Effect of extracellular Ca 2+ 88 2.3.5 Intracellular calcium and nuclear protein aggregation 94 2.4 Discussion 96 2.4.1 Increased [Ca 2+ ] i as cause of hyperthermic cell killing 96 2.4.2 Toxicity of ionomycin in relation to changes in [Ca 2+ ] i 98 2.4.3 Toxicity of ionomycin in relation to changes in [Ca 2+ ] e 98 2.4.4 Combined heat and ionomycin treatments 98 2.4.5 Ca 2+ and nuclear protein aggregation 99 2.4.6 Effect of intracellular calcium on hsp induction 100 2.4.7 Concluding remarks 100 2.5 Acknowledgements 101 Chapter 3: On the role of hsp72 in heat-induced intranuclear protein aggregation Int. J. Hyperthermia 10, 659-674 (1994) 3.1 Introduction 105 3.2 Material and methods 106 3.2.1 Cell cultures 106 3.2.2 Heating and cell survival 106 3.2.3 Isolation of nuclei and flow cytometric analysis 106 3.2.4 Protein gel electrophoresis and immunoblotting 107 3.3 Results 107 3.4 Discussion 113 3.5 Acknowledgements 116
Chapter 4: Importance of the ATP-binding domain and nucleolar localization domain of hsp72 in the protection of nuclear proteins against aggregation Exp. Cell Res. 214, 279-284 (1994) 4.1 Introduction 119 4.2 Material and methods 120 4.2.1 Cell cultures 120 4.2.2 Heating and cell survival 120 4.2.3 Isolation of nuclei and flow cytometric analysis 120 4.2.4 Protein gel electrophoresis and immunoblotting 122 4.2.5 Preparation of cell extracts and gel mobility-shift assay 122 4.3 Results 122 4.4 Discussion 126 4.4.1 Nucleolar localization domain is essential for hsp72 function 126 4.4.2 ATP binding domain of minor importance for protective function of hsp72? 126 4.4.3 Nuclear protein disaggregation 127 4.4.4 Reduced thermotolerant levels in Sma and Bgl cells 127 4.5 Acknowledgements 128 Chapter 5: Cells overexpressing hsp27 show accelerated recovery from heat-induced nuclear protein aggregation Bioch. Biophys. Res. Commun. 204, 1170-1177 (1994) 5.1 Introduction 131 5.2 Material and methods 132 5.3 Results and discussion 133 5.4 Acknowledgements 137 Chapter 6: Thermotolerance and nuclear protein aggregation: protection against initial damage or better recovery? J. Cell. Physiol. (1995) in press 6.1 Introduction 141 6.2 Material and methods 142 6.2.1 Cell culture, heat treatment and cell survival 142 6.2.2 Isolation of nuclei and flow cytometry analysis 143 6.2.3 Protein gel electrophoresis and immunoblotting 143 6.3 Results 144 6.4 Discussion 151 6.4.1 Thermotolerance: protection against nuclear protein aggregates 151 6.4.2 Thermotolerance: enhanced disaggregation of nuclear protein aggregates 152 6.5 Acknowledgements 154
Chapter 7: Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins submitted for publication 7.1 Introduction 157 7.2 Material and methods 158 7.2.1 Cells and culture conditions 158 7.2.2 Incubation conditions 159 7.2.3 Determination of cell survival 159 7.2.4 Isolation of sub-cellular fractions 159 7.2.5 Electron spin resonance (ESR) 159 7.2.6 Thermal gel analysis (TGA) 160 7.2.7 Flow cytometric determination of nuclear protein content 160 7.2.8 Protein gel electrophoresis and immunoblotting 160 7.3 Results 161 7.4 Discussion 168 7.4.1 Cross-resistance and target-tolerance concept 168 7.4.2 Target-resistance and heat shock proteins 169 7.5 Acknowledgements 173 Chapter 8: Heat-induced intranuclear protein aggregation and thermal radiosensitization Int. J. Radiat. Biol. 67, 203-209, (1995) 8.1 Introduction 175 8.2 Material and methods 176 8.2.1 Cell cultures 176 8.2.2 Heating, irradiation and cell survival 176 8.2.3 Isolation of nuclei and flow cytometry analysis 177 8.3 Results 177 8.4 Discussion 181 8.4.1 Thermotolerance and TER 181 8.4.2 Intranuclear protein aggregates and TER: role of HSP s 182 8.4.3 Intranuclear protein aggregates predictive for thermal radiosensitization? 183 8.5 Acknowledgements 184 Chapter 9: General discussion 9.1 Hyperthermia and intracellular free calcium 186 9.2 Heat-induced protein denaturation and aggregation: role of heat shock proteins 187 9.2.1 Hsp70 189 9.2.1.1 Functional domains of hsp70 190
9.2.1.2 Hsp70 versus hsc70 192 9.2.1.3 Model for hsp70 action; role of cofactors 193 9.2.2 Hsp27 194 9.2.2.1 Protection against aggregation 195 9.2.2.2 Accelerated disaggregation of nuclear proteins 196 9.2.2.3 Phosphorylation of hsp27 196 9.3 Effects of thermotolerance 200 9.3.1 Nuclear protein aggregation and disaggregation: role of heat shock proteins 200 9.3.2 Protein denaturation and aggregation in isolated membrane fractions 202 9.3.3 Possible protective action of hsp70 and hsp27 against protein denaturation and aggregation; a model 203 9.4 Role of heat sensitive proteins in thermotolerant cells; the "threshold concept 205 9.5 Heat radiosensitization 208 References 213 Summary 241 Nederlandse samenvatting 245 List of publications 249 Dankwoord (Acknowledgements) 250