University of Groningen. Hyperthermia and protein aggregation Stege, Gerardus Johannes Jozef

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1 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): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 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

3 Promotor Co-promotor Prof. Dr. A.W.T. Konings Dr. H.H. Kampinga

4 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

5 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 and CA 56909). ISBN

6 aan mijn ouders aan Anne-Marie

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8 Contents Chapter 1: General introduction 1.1 HYPERTHERMIA Cell survival curves Protein denaturation Heat-induced protein denaturation and aggregation in cells Heat effects on cellular structures Plasma membrane Heat effects on cytosolic structures and processes Heat effects on nuclear structures and processes HEAT SHOCK PROTEINS Molecular chaperones Heat shock proteins as molecular chaperones Small heat shock proteins Hsp60 family Hsp70 family Hsp90 family Regulation of the stress response Transcriptional activation of heat shock genes Translational regulation of heat shock gene expression Thermotolerance, thermoresistance and intrinsic heat sensitivity; 54 role of heat shock proteins Thermotolerance Thermoresistance Intrinsic heat sensitivity HEAT AND RADIATION The synergism of heat and radiation Mechanisms of interaction Inactivation of repair enzymes Heat-induced alteration of the chromatin structure Heat radiosensitization and thermotolerance SCOPE OF THE THESIS 68 Chapter 2: Hyperthermic cell killing and calcium homeostasis J. Cell. Physiol. 155, (1993) Int. J. Radiat. Biol. 64, (1993) Eur. J. Cell Biol. 63, (1994) 2.1 Introduction Role of intracellular free calcium Role of extracellular calcium 74

9 2.1.3 Intracellular calcium modifiers Effect of intracellular calcium on heat-induced protein aggregation Materials and methods Cell cultures Cell loading with fura-2/am [Ca 2+ ] i assay Hyperthermic treatments Ionophore and Ca 2+ treatments Determination of cell survival Isolation of nuclei and flow cytometric analysis Results On the measurement of [Ca 2+ ] i at 37 C and 79 hyperthermic temperatures Relation between heat-induced increases in [Ca 2+ ] i and 84 hyperthermic cell killing Relation between effects of heat and ionomycin on [Ca 2+ ] i and 84 cell killing Effect of extracellular Ca Intracellular calcium and nuclear protein aggregation Discussion Increased [Ca 2+ ] i as cause of hyperthermic cell killing Toxicity of ionomycin in relation to changes in [Ca 2+ ] i Toxicity of ionomycin in relation to changes in [Ca 2+ ] e Combined heat and ionomycin treatments Ca 2+ and nuclear protein aggregation Effect of intracellular calcium on hsp induction Concluding remarks Acknowledgements 101 Chapter 3: On the role of hsp72 in heat-induced intranuclear protein aggregation Int. J. Hyperthermia 10, (1994) 3.1 Introduction Material and methods Cell cultures Heating and cell survival Isolation of nuclei and flow cytometric analysis Protein gel electrophoresis and immunoblotting Results Discussion Acknowledgements 116

10 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, (1994) 4.1 Introduction Material and methods Cell cultures Heating and cell survival Isolation of nuclei and flow cytometric analysis Protein gel electrophoresis and immunoblotting Preparation of cell extracts and gel mobility-shift assay Results Discussion Nucleolar localization domain is essential for hsp72 function ATP binding domain of minor importance for protective function of hsp72? Nuclear protein disaggregation Reduced thermotolerant levels in Sma and Bgl cells Acknowledgements 128 Chapter 5: Cells overexpressing hsp27 show accelerated recovery from heat-induced nuclear protein aggregation Bioch. Biophys. Res. Commun. 204, (1994) 5.1 Introduction Material and methods Results and discussion 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 Material and methods Cell culture, heat treatment and cell survival Isolation of nuclei and flow cytometry analysis Protein gel electrophoresis and immunoblotting Results Discussion Thermotolerance: protection against nuclear protein aggregates Thermotolerance: enhanced disaggregation of nuclear protein aggregates Acknowledgements 154

11 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 Material and methods Cells and culture conditions Incubation conditions Determination of cell survival Isolation of sub-cellular fractions Electron spin resonance (ESR) Thermal gel analysis (TGA) Flow cytometric determination of nuclear protein content Protein gel electrophoresis and immunoblotting Results Discussion Cross-resistance and target-tolerance concept Target-resistance and heat shock proteins Acknowledgements 173 Chapter 8: Heat-induced intranuclear protein aggregation and thermal radiosensitization Int. J. Radiat. Biol. 67, , (1995) 8.1 Introduction Material and methods Cell cultures Heating, irradiation and cell survival Isolation of nuclei and flow cytometry analysis Results Discussion Thermotolerance and TER Intranuclear protein aggregates and TER: role of HSP s Intranuclear protein aggregates predictive for thermal radiosensitization? Acknowledgements 184 Chapter 9: General discussion 9.1 Hyperthermia and intracellular free calcium Heat-induced protein denaturation and aggregation: role of heat shock proteins Hsp Functional domains of hsp70 190

12 Hsp70 versus hsc Model for hsp70 action; role of cofactors Hsp Protection against aggregation Accelerated disaggregation of nuclear proteins Phosphorylation of hsp Effects of thermotolerance Nuclear protein aggregation and disaggregation: role of heat shock proteins Protein denaturation and aggregation in isolated membrane fractions Possible protective action of hsp70 and hsp27 against protein denaturation and aggregation; a model Role of heat sensitive proteins in thermotolerant cells; the "threshold concept Heat radiosensitization 208 References 213 Summary 241 Nederlandse samenvatting 245 List of publications 249 Dankwoord (Acknowledgements) 250