Pathways Responsible for Apoptosis in Chick
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PATHWAYS RESPONSIBLE FOR APOPTOSIS IN CHICK CARDIOMYOCYTES by Jennifer Y. Kong Bachelor of Science, University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine) We accept this thesis as conforming to the required standard THE .UNIVERSITY OF BRITISH COLUMBIA April, 1996 © Jennifer Kong, 1996 In presenting this thesis in partial fulfilment of the requirements for an ach/anced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or i publication of this thesis for financial gain shall not be allowed without my written permission. 0 Department of E Xpen/vi g*^-^ ^Cet'cirzc^ The University of British Columbia Vancouver, Canada Date Ji)HS^ 2$. ^% DE-6 (2/88) 11 ABSTRACT The mechanisms responsible for apoptosis in the heart are currently being defined. The present study was designed to determine the roles of nuclear enzymes and signal transduction protein kinases in the development of apoptosis in chick embryo cardiomyocytes. Topoisomerase I was chosen as an example of a nuclear enzyme involved in apoptosis. Topoisomerase I is the enzyme responsible for relieving torsional stress in DNA replication and transcription. To determine whether inhibition of topoisomerase I would produce apoptosis in cardiomyocytes, the inhibitor camptothecin was used. Cardiomyocytes, obtained from 7 day old embryonic chick hearts, were treated with camptothecin and examined microscopically or their DNA was examined for fragmentation. Apoptotic cell death was produced by camptothecin as fluorescent microscopy with acridine orange demonstrated cardiomyocytes that were shrunken with cytoplasmic blebs and nuclear fragmentation. In contrast, untreated cells did not manifest these cellular alterations. Apoptosis was further substantiated by Hoescht 33258 dye stained cardiomyocytes that showed a strongly fluorescent nucleus which was undergoing disintegration. Cell death as quantitated by trypan blue exclusion showed that camptothecin, 10 [IM, significantly increased cell death by 25.1±1.4% (+SEM) . Cardiomyocytes were lysed and the DNA isolated and run on a 2% agarose gel. DNA laddering, indicated by fragments of approximately 200 bp or multiples, were found in camptothecin treated cells. DNA fragmentation was also observed quantitatively in camptothecin treated cells, as assessed by an enzyme linked immunosorbent assay (ELISA). Fragmented DNA was isolated from lysed cells and adsorbed onto a microtitre plate. Primary antibody specific for DNA histones was then added and subsequently treated with a horse-radish peroxidase linked- secondary antibody specific for DNA. The colorimetric results were reported relative to control. Camptothecin exposure (10\iM) induced 1.5±0.5 fold more DNA fragmentation than control cells. Alterations in intracellular calcium appeared to be a component of the mechanism of action of camptothecin-induced apoptosis. Ca+Z levels that can be decreased by the chelator EGTA reduced cell death induced by camptothecin, as demonstrated by membrane bleb formation, DNA fragmentation on agarose gel electrophoresis, and DNA fragmentation on the ELISA. Taurine, a free amino acid in many tissues which affects L-type and T-type Ca+2 channels, also reduced camptothecin-induced apoptotic morphology and DNA specific fragmentation, determined by ELISA. To further substantiate the role of calcium and to investigate the source of Ca+2 mediated topoisomerase-induced apoptosis, cardiomyocytes were exposed to thapsigargin, an inhibitor of sarcoplasmic and endoplasmic reticulum Ca+2-ATPases which increases intracellular +2 calcium. [Ca ]i increased by thapsigargin exposure yielded greater DNA fragmentation, as assessed by ELISA, than camptothecin alone +2 suggesting that increased [Ca ]i induced apoptosis itself. With the caveat our use of agents that indirectly implicate the mechanism, these data show that apoptosis in cardiomyocytes is under regulatory control by DNA topoisomerase I and intracellular calcium modulates the pathway whereby topoisomerase I inhibition causes apoptosis. To investigate the signal transduction mechanisms responsible for apoptosis, I investigated the role of serine/threonine kinases. Staurosporine, a potent serine/threonine kinase inhibitor, was used to investigate the role of kinase inhibition on the development of apoptosis. Staurosporine induced cell death in a dose and time dependent response to a maximal death of 40. 9±6.3% at 1\IM for 6h. DNA fragmentation, 2. 8±1.2 fold that of control, determined by ELISA and electrophoretic separation was observed in staurosporine treated cardiomyocytes. Staurosporine-induced morphology, observed by acridine orange and NBD phallacidin staining, was distinct from usual apoptotic features: staurosporine .induced cytoplasmic condensation resulting in dense vacuoles and a loss in volume. Staurosporine treatment failed to exhibit membrane blebbing and distinct nuclear disintegration. Pre treatment by the Ca+2 chelator BAPTA blunted the apoptotic response of staurosporine exposure implicating Ca+2 in staurosporine-induced apoptosis. The activation of protein kinase C (PKC) by the phorbol ester PMA blocked staurosporine-induced cell death, morphology, and DNA fragmentation suggesting that the activation of PKC can reverse staurosporine-induced apoptosis. The addition of trophic factors such as insulin and EGF demonstrated a "rescue" pathway in staurosporine-induced apoptotic cardiomyocytes. In addition, de novo protein synthesis may relate to this rescue pathway. To further investigate the signal transduction mechanisms responsible for apoptosis, the role of PKC was considered. The specific PKC inhibitor chelerythrine chloride was observed to induce cell death of 27.6±7.5% and DNA fragmentation (2.2±0.4 fold that of control) similar to staurosporine. However, chelerythrine exhibited usual apoptotic morphology contrasting staurosporine morphology. In addition, the apoptotic effects of chelerythrine are less potent than staurosporine suggesting that PKC alone is not responsible for staurosporine's apoptotic inducing abilities. iv Table of Contents Page ABSTRACT ii Table of Contents iv List of Figures vi List of abbreviations viii Acknowledgements ix CHAPTER I Introduction I) Morphology of Cell death 3 A. Apoptosis 3 B. Necrosis 4 C. Non-apoptotic programmed cell death 5 D. Biological implications of apoptosis 5 II) Nuclear pathways to apoptosis 6 A. Topoisomerases 7 . 1. Inhibitors of topoisomerases I and II 7 2 . Camptothecin 7 III) Intracellular pathways to apoptosis 8 A. Protein kinase inhibition , .., 9 1. Staurosporine .". 9 B. Protein kinase C 10 1. Protein kinase C activation 11 2. PKC involvement in apoptosis 12 IV) The role of calcium (Ca+2) in apoptosis 13 V) Growth factor and cell survival 15 VI) de novo protein synthesis and apoptosis 15 VII) Genes and their products in apoptosis 16 A. c-Fos and c-Jun 16 B. Bcl-2 17 C. c-Myc 17 D. p53 17 VIII) Potential significance of cardiac apoptosis 18 A. Chick embryo ventricular myocytes culture 18 B. Histogenesis of the embryonic chick ventricular myocardium 19 C. Ventricular myocytes of seven day old embryo...19 IX) Objectives and hypotheses with their rationale 20 CHAPTER II Materials and Methods 1. Isolation of Chick Embryonic Cardiac Cells 24 2. Microscopy 25 a) Cell viability assay - Trypan Blue 25 b) Cell morphology - Acridine orange 25 c) Cell morphology - Hoescht dye 25 d) Cell morphology - NBD-phallacidin 26 3. DNA Fragmentation 26 4. DNA Fragmentation (Enzyme-linked immunosorbent assay) 27 5. [35S] Methionine incorporation and preparation of cell lysates 28 6. [35S] Methionine incorporation assay 29 7. Protein determination 29 8. SDS Polyacrylamide gel electrophoresis (SDS PAGE)...29 9. Radiolabelled proteins 3 0 10. Drug pre treatment 3 0 11. Materials 3 0 a) Biochemicals 3 0 b) Radiochemicals 3 0 c) Pharmacology of drugs 31 12. Methods of statistical analysis 32 CHAPTER III Results A. Results of topo I inhibition-mediated apoptosis 33 B. Results of staurosporine-induced apoptosis 42 CHAPTER IV Discussion 67 CHAPTER V Summary and Conclusions 87 References 90 VI List of Figures Figure Number Figure Title Page 1. The effect of camptothecin on cell 34 viability assessed by trypan blue 2. The cell morphology of cardiomyocytes 35 after camptothecin treatment 3. DNA fragmentation in cardiomyocytes 37 exposed to camptothecin or DMSO 4. DNA fragmentation quantitated by ELISA 3 8 5. EFfect of EGTA on camptothecin-induced 39 cell death 6. Cell morphology of cardiomyocytes exposed 41 to camptothecin and pretreatment with EGTA or taurine 7. DNA fragmentation in cardiomyocytes exposed 43 to taurine and camptothecin 8. DNA fragmentation (ELISA) in cardiomyocytes 44 exposed to taurine, thapsigargin, and camptothecin 9. de novo protein synthesis during 45 camptothecin exposure 10. Effect of staurosporine on cell viability 47 11. Staurosporine-induced DNA fragmentation 48 (ELISA) 12. Dose response of staurosporine-induced DNA 49 fragmentation 13. Cell morphology of cardiomyocytes exposed 51 to staurosporine 14. Effect of EGTA and BAPTA on staurosporine- 52 induced cell death 15. Effect of EGTA and BAPTA on staurosporine- 53 induced DNA fragmentation