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Pathways responsible for apoptosis in chick cardiomyocytes Kong, Jennifer Y. 1996

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PATHWAYS RESPONSIBLE FOR APOPTOSIS IN CHICK CARDIOMYOCYTES by Jennifer Y. Kong Bachelor of Science, University of B r i t i s h 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 A p r i l , 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. Department of E X p e n / v i g*^ 0-^  ^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 calcium. [Ca+2]i increased by thapsigargin exposure yielded greater DNA fragmentation, as assessed by ELISA, than camptothecin alone suggesting that increased [Ca+2]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. i v Table of Contents Page ABSTRACT i i Table of Contents i v L i s t of Figures v i L i s t of abbreviations v i i i Acknowledgements ix CHAPTER I Introduction I) Morphology of C e l l death 3 A. Apoptosis 3 B. Necrosis 4 C. Non-apoptotic programmed c e l l death 5 D. B i o l o g i c a l 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) I n t r a c e l l u l a r pathways to apoptosis 8 A. Protein kinase i n h i b i t i o n , .., 9 1. Staurosporine .". 9 B. Protein kinase C 10 1. Protein kinase C a c t i v a t i o n 11 2. PKC involvement i n apoptosis 12 IV) The r o l e of calcium (Ca+2) i n apoptosis 13 V) Growth factor and c e l l s u r v i v a l 15 VI) de novo protein synthesis and apoptosis 15 VII) Genes and t h e i r products i n apoptosis 16 A. c-Fos and c-Jun 16 B. Bcl-2 17 C. c-Myc 17 D. p53 17 VIII) Potential s i g n i f i c a n c e of cardiac apoptosis 18 A. Chick embryo v e n t r i c u l a r myocytes culture 18 B. Histogenesis of the embryonic chick v e n t r i c u l a r myocardium 19 C. Ventricular myocytes of seven day old embryo...19 IX) Objectives and hypotheses with t h e i r rationale 2 0 CHAPTER II Materials and Methods 1. Is o l a t i o n of Chick Embryonic Cardiac C e l l s 24 2. Microscopy 25 a) C e l l v i a b i l i t y assay - Trypan Blue 25 b) C e l l morphology - Acridine orange 25 c) C e l l morphology - Hoescht dye 25 d) C e l l morphology - NBD-phallacidin 26 3. DNA Fragmentation 26 4. DNA Fragmentation (Enzyme-linked immunosorbent assay) 27 5. [35S] Methionine incorporation and preparation of c e l l 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 s t a t i s t i c a l 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 V I L i s t of Figures Figure Number Figure T i t l e Page 1. The ef f e c t of camptothecin on c e l l 34 v i a b i l i t y assessed by trypan blue 2. The c e l l morphology of cardiomyocytes 35 aft e r camptothecin treatment 3. DNA fragmentation i n cardiomyocytes 37 exposed to camptothecin or DMSO 4. DNA fragmentation quantitated by ELISA 3 8 5. EFfect of EGTA on camptothecin-induced 39 c e l l death 6. C e l l morphology of cardiomyocytes exposed 41 to camptothecin and pretreatment with EGTA or taurine 7. DNA fragmentation i n cardiomyocytes exposed 43 to taurine and camptothecin 8. DNA fragmentation (ELISA) i n cardiomyocytes 44 exposed to taurine, thapsigargin, and camptothecin 9. de novo protein synthesis during 45 camptothecin exposure 10. E f f e c t of staurosporine on c e l l v i a b i l i t y 47 11. Staurosporine-induced DNA fragmentation 48 (ELISA) 12. Dose response of staurosporine-induced DNA 49 fragmentation 13. C e l l morphology of cardiomyocytes exposed 51 to staurosporine 14. E f f e c t of EGTA and BAPTA on staurosporine- 52 induced c e l l death 15. E f f e c t of EGTA and BAPTA on staurosporine- 53 induced DNA fragmentation 16. C e l l morphology of cardiomyocytes 54 exposed to staurosporine, BAPTA, and PMA 17. E f f e c t of PMA on staurosporine-induced 56 c e l l death and DNA fragmentation. V I 1 18. E f f e c t of i n s u l i n and EGF on staurosporine- 57 induced c e l l death. 19. E f f e c t of i n s u l i n and EGF on staurosporine- 58 induced DNA fragmentation. 20. de novo protein synthesis during 60 staurosporine-induced apoptosis 21. E f f e c t of chelerythrine on c e l l v i a b i l i t y 61 22. E f f e c t of chelerythrine on DNA 62 fragmentation 23. C e l l morphology of cardiomyocytes exposed 64 to chelerythrine 24. E f f e c t of Ca + 2 on chelerythrine-induced 65 DNA fragmentation. 25. E f f e c t of growth factor on chelerythrine- 66 induced DNA fragmentation. v i i i L i s t of Abbreviations BAPTA-AM 1,2-bis-(o-Aminophenoxy)ethane-N,N,N',N'-tet r a a c e t i c acid Ca + 2 Calcium [Ca + 2]i I n t r a c e l l u l a r calcium CAMP adenosine-3',5'-monophosphate or c y c l i c AMP campto Camptothecin DAG Diacylglycerol DMSO Dimethylsulfoxide EGF Epidermal growth factor EGTA Ethyleneglycol-bis(S-aminoethyl)-N,N,N',N'-te t r a a c e t i c acid Hoe Hoescht 33258 dye PIP 2 Phosphatidylinositol-4,5-biphosphate PKC Protein Kinase C, or Ca+2 and lipid-dependent kinase PKA Protein Kinase A, or cAMP-dependent kinase PMA Phorbol-12 myristate 13-acetate SDS Sodium dodecyl sulfate thap Thapsigargin i x Acknowledgement s This work would not have been possible without Dr. Simon Rabkin, whose support and guidance throughout t h i s project i s greatly appreciated. It i s my hope that some of the work presented w i l l be useful as the basis for further experimental i n q u i r i e s i n his lab. I thank Carol Smythe for her technical assistance and support. Others i n Dr. Rabkin's lab who helped me were V a l e r i Goutsouliak, Danny V i l l a c r u s i s , Ramin Mehin, and Irene Bremsak. I thank them for t h e i r experiences and assistance. During t h i s research, I had the opportunity to work i n Dr. Frank J i r i k ' s laboratory at the Biomedical Research Centre. This added an ex c i t i n g and educational dimension to my studies. I am grate f u l to Dr. Frank J i r i k and Nicole Janzen for advice and p r a c t i c a l help. I would e s p e c i a l l y l i k e to thank Mike Scheid and Dr. Vince Duronio for helping me develop some techniques. This study was supported i n part by a grant from the Heart and Stroke Foundation of B r i t i s h Columbia and the Yukon. Lastly, I wish to thank my family and Jon for t h e i r enduring support and patience. 1 CHAPTER I INTRODUCTION M u l t i c e l l u l a r organisms have evolved from simpler organisms by having mechanisms to regulate p r o l i f e r a t i o n , d i f f e r e n t i a t i o n , aging and death. During the l i f e of an organism, i t becomes necessary for c e l l s to be eliminated. Injured c e l l s , must be removed to prevent secondary damage that might occur to other c e l l s when a c e l l break down. C e l l death may come to targeted redundant c e l l s to maintain a constant c e l l number. Simi l a r l y , transformed or infected c e l l s must be removed to prevent tumorigenesis or subsequent infections of neighbouring c e l l s . C e l l s no longer needed in embryonic development must also be eliminated at the correct developmental stage, such as i n limb bud formation. Three classes of c e l l death are currently recognized: programmed c e l l death (or apoptosis), necrosis, and non-apoptotic programmed c e l l death. Apoptosis i s a functional type of c e l l death which i s programmed ge n e t i c a l l y into every c e l l (Schwartz and Osborne 1993a). Necrosis i s the form of death associated with s t r u c t u r a l and biochemical degradation of c e l l i n t e g r i t y due to accidental c e l l u l a r damage. A type of c e l l death which doesn't f a l l into either category has been l a b e l l e d non-apoptotic programmed cell death (Schwartz and Osborne 1993a) . Hence, a c e l l has d i f f e r e n t modalities of death. The question a r i s e s : why do c e l l s undergo 3 (or more) d i f f e r e n t types of death? The v a r i e t y of types of death types may have evolved to meet demands other than simply to eliminate a c e l l . C e l l u l a r breakdown, seen i n necrosis, encourages phagocytosis and r e u t i l i z a t i o n of the c e l l constituents by other c e l l s . 2 Unfortunately, t h i s c e l l u l a r r e c y c l i n g might also a i d the spread of p o t e n t i a l l y i n f e c t i v e v i r us or inflammatory agents, i f the disassembled c e l l had been infected (Clem and M i l l e r 1993, Takizawa et a l 1993) . Extensive DNA and RNA fragmentation seen i n apoptosis may deactivate v i r a l polynucleotides thus i n h i b i t i n g the machinery needed for v i r a l r e p l i c a t i o n . However, th i s fragmentation may lead to the accumulation of these p o t e n t i a l l y harmful v i r a l oligonucleotides (Clem and M i l l e r 1993, Takizawa et a l 1 9 9 3 ) . The early fragmentation of DNA i n apoptosis may be p a r t i c u l a r l y important i n v i r a l containment, since i t could h a l t v i r a l r e p l i c a t i o n and possibly also inactivate already assembled DNA virus (Martz and Howell 1 9 8 9 ) . Thus, d i f f e r e n t types of death may function to deal with the containment and proper disposal of the injured/infected c e l l . The most complicated mode of c e l l death i s apoptosis. Apoptosis i s an active process i n which the c e l l p a r t i c i p a t e s i n i t s own destruction u t i l i z i n g a set of genetic factors and a complex network of enzymes (Schwartzman et a l 1993, Wyllie 1 9 9 2 ) . Apoptosis may be considered to be a functional form of c e l l death and i s important to the c e l l as an es s e n t i a l regulator i n ontogenesis and tissue homeostasis (Binder and Hiddemann 1 9 9 4 ) . It occurs during embryogenesis, i n p a r a l l e l with the deletion of autoreactive T c e l l s during thymic maturation, i n senescence of neutrophil polymorphs following removal of s p e c i f i c growth factors, and i n the presence of s t i m u l i l i k e tumour necrosis factor and glucocorticoids (Arends et a l 1990, Sen 1992) . Apoptosis can be induced by various chemicals, radiation, or c e l l u l a r i n j u r i e s , dependent on the c e l l type and dosage. Apoptosis i s also induced 3 by cytotoxic T lymphocytes and natural k i l l e r c e l l s (Sanderson 1981), i o n i z i n g r a d i a t i o n (Yamada and Ohyama 1988), and monoclonal antibodies l i k e anti-Fas 13 (Yonehara et a l 1989) and anti-APO-1 (Trauth et a l 1989) . Apoptosis i s an important mode of c e l l death influencing the health of c e l l s , tissues, and organs. I) Morphology of c e l l death A. Apoptosis In most cases, apoptosis occurs i n stages. I n i t i a l l y , an ind i v i d u a l c e l l embedded i n normal tissue loses contact with i t s neighbours. Chromatin within the nucleus condenses r e s u l t i n g i n karyohexis (nuclear d i s i n t e g r a t i o n ) . The c e l l shrinks due to loss of cytoplasmic volume and condensation of cytoplasmic proteins occur. Most of the i n t r a c e l l u l a r organelles remain intact, although there i s condensation of the cytoplasm (Wyllie et a l 1980, Gerschenson and Rotello 1992, Arends and Wyllie 1991). The second stage i s characterized by membrane r u f f l i n g and blebbing (zeiosis) leading to c e l l u l a r fragmentation and the formation of apoptotic bodies (blebs). The formation of blebs appears to involve disruption of cytoskeletal-membrane interactions (Orrenius et a l 1992) . Apoptotic bodies frequently contain whole organelles and nuclear remnants (Arends and Wyllie 1991, Williams et a l 1974, Wyllie 1980). In the f i n a l stage, the neighbouring c e l l s and macrophages phagocytose the c e l l u l a r fragments for complete degradation of the c e l l . Apoptosis occurs without leakage of i n t r a c e l l u l a r macromolecules therefore, i t does not e l i c i t any inflammatory response. Chromatin condensation i s associated with the a c t i v a t i o n of an endogenous, Ca + 2 and Mg+2-dependent endonuclease which cleaves double 4 stranded DNA at the most accessible internucleosomal inner region, generating mono- and oligonucleosomes (Burgoyne et a l 1 9 7 4 ) . In contrast, the DNA of the nucleosomes i s t i g h t l y complexed to core histones H2A, H2B, H3 and H4 and i s therefore protected from i cleavage by the endonuclease (Stach et a l 197:9) . The DNA fragments produced by endonucleases are discrete multiples of a 180 bp subunit, which are detected as a "DNA ladder" on agarose gels. Because DNA degradation precedes plasma membrane breakdown, there i s an accumulation of mono- and oligonucleosomes i n the cytoplasm (Duke and Cohen 1 9 8 6 ) . An in t a c t membrane i s maintained i n apoptotic development u n t i l i t s l a t e stages. Even a f t e r the breakdown of apoptotic bodies, the membrane i n t e g r i t y i s preserved. The difference i n membrane function i s considered to discriminate between necrosis and apoptosis (Wyllie 1 9 8 0 ) . In addition, there i s no inflammatory reaction and organelle swelling i n apoptosis, i n contrast to necrosis (Wyllie 1 9 8 0 ) . B. Necrosis Necrosis i s most commonly produced by an interruption of blood flow to an organ with r e s u l t i n g changes i n c e l l u l a r ion permeability leading to osmotic swelling and l y s i s (Jennings et a l 1 9 9 5 ) . Necrosis i s i n i t i a t e d by c e l l u l a r damage that disrupts osmotic balance (Farber 1 9 9 0 ) . Ions, e s p e c i a l l y Ca + 2, then passively enter the c e l l r e s u l t i n g i n swelling as water enters i n response to the ion i n f l u x . Necrosis i s characterized by d i l a t i o n of the endoplasmic reticulum, increases i n mitochondrial volume, amassing of nuclear chromatin, early membrane breakdown, and c e l l disintegration- r e s u l t i n g i n the release of lysosomal enzymes 5 (Searle et a l 1982, Kerr et a l 1972) . Necrosis i s also characterized by the early breakdown of the i n t r a c e l l u l a r energy supply (Farber 1990). This confirms that necrosis i s a passive process that does not require active p a r t i c i p a t i o n of the c e l l i n i t s own death. The release of c e l l u l a r components during c e l l l y s i s leads to an inflammatory response which causes secondary damage to neighbouring c e l l s (Searle et a l 1982, Kerr et a l 1972, Wyllie 1980) . C. Non-apoptotic programmed c e l l death Non-apoptotic programmed c e l l death encompasses c e l l death which can not f i t into either necrosis or apoptosis. Not a l l dying c e l l s display the usual changes associated with apoptosis. For example, there have been reports which show c e l l death with ze i o s i s , but no DNA fragmentation; or c e l l s which undergo DNA fragmentation but do not show the t y p i c a l morphological changes associated wi'fch> apoptosis. For example, i n the tobacco hawkmoth Manduca sexta, muscle i s l o s t during metamorphosis so a functional form of c e l l death i s needed. However, the muscle l o s t during metamorphosis does not display membrane blebbing, chromatin margination or the fragmentation of DNA (Schwartz et a l 1993b, Schwartz and Truman 1983). A s i m i l a r response i s also seen i n mammalian neurons that die during development (Chu-Wang and Oppenheim 1978, Clarke 1990). C i l i a r y neurons of the chick die during normal neurogenesis, yet the death appears to be non-apoptotic while the death induced by target removal appears to be apoptotic ( P i l a r and Landmesser 197 6). D. Biological' -implications of apoptosis Programmed c e l l death i s a functional form of elimination. 6 Only the c e l l that i s selected for death i s eliminated by i t s own c e l l u l a r system responsible for the induction and control of apoptosis. Apoptosis may be a defense mechanism that i s d i s t i n c t from more t r a d i t i o n a l l y accepted defense mechanisms such as DNA repair systems, antioxidant systems, heat shock proteins, and nuclear enzymes such as poly(ADP)ribose polymerase. It may also be the key mechanism for removal of damaged cells. Apoptosis i s also involved i n the removal of embryonic cells during embryonic morphogenesis. The correct functioning of apoptosis i s c r i t i c a l to the phenotypic f i d e l i t y of m u l t i c e l l u l a r organisms. Defective controls of apoptosis maybe the etiology for some diseases. Apoptosis may lead to the cumulative breakdown of tissue and organ i n t e g r i t y seen with age, AIDS, and autoimmune diseases (Tomei et a l 1994). Conversely, an i n h i b i t i o n of apoptotic mechanisms may be involved i n the pathogenesis of cancers and degenerative diseases of the central nervous system (Carson and Ribeiro 1993, C o l l i n s and Lopez-Rivas 1993, Fesus et a l 1991). Where apoptosis i s a protective measure preventing malignant transformation and v i r a l i n f e c t i o n , a defective apoptotic mechanism can allow the uninhibited p r o l i f e r a t i o n of these abnormal c e l l s . Thus, the regulation of apoptosis i s c r u c i a l . II) Nuclear pathways to apoptosis Many pathways can lead to or control apoptosis. However, since apoptosis i s a genetic program, i t i s r a t i o n a l to st a r t investigation at the nuclear l e v e l . During apoptosis, the a c t i v i t y of nuclear enzymes may be altered so as to allow the induction or down-regulation of apoptotic death genes or c e l l s u r v i v a l genes such as p53 and b c l - 2 , respectively. Hence nuclear enzymes 7 responsible for gene a c t i v i t y would be an i d e a l locale to i n i t i a t e the investigation into apoptosis. A. Topoisomerases Topoisomerases are the enzymes responsible for r e l i e v i n g the to r s i o n a l stress caused by DNA unwinding i n DNA synthesis and tr a n s c r i p t i o n . There are two major types: DNA topoisomerase I (topo I) and topoisomerase II (topo I I ) . Topo I produces changes i n DNA supercoiling by catalyzing the single-strand cutting of the DNA, thus allowing the rotation of a free end ( r e l i e v i n g a supercoil) followed by the resealing of the nick (Mathews and van Holde 1990) . Topo I i s more active with negative than p o s i t i v e supercoils. Because the relaxation of DNA supercoils i s energet i c a l l y favourable, topo I does not require high energy cofactors. Topo II a l t e r s the supercoiling i n order to f a c i l i t a t e chromosome folding and twisting and cuts both strands of one DNA double h e l i x so that a neighbouring region of h e l i x can pass through the cut ends, and eventually reseals the cut (Darnell et a l 1986) . Topoisomerases are reportedly involved i n producing apoptosis (Tritton 1991, Walker et a l 1991, Forbes et a l 1992). 1. Inhibitors of topoisomerases I and II Inhibitors of topo I and II were found to induce apoptosis i n thymocytes (Onishi et a l 1993), mature unstimulated lymphocytes (Roy et a l 1992) and human lymphocytic leukemia c e l l s (Rajotte et a l 1992). Topo i n h i b i t o r s were able to i n h i b i t DNA fragmentation i n target c e l l s exposed to cytotoxic lymphocytes (Nishioka and Welsh 1992). 2. Camptothecin The topo I i n h i b i t o r , camptothecin, i s an a l k a l o i d i s o l a t e d 8 from the stem wood of Camptotheca acuminata (family Nyssaceae) . Camptothecin (campto) has been reported to i n h i b i t growth of experimental tumours i n rodents (Hartwell and Abbott 1969) and to i n h i b i t the synthesis of DNA and RNA i n various c e l l l i n e s (Horwitz et a l 1971, Kessel 1971). Campto "poisons" topo I when the enzyme i s associated with DNA during cleavage, increasing the steady-state concentration of topo I-DNA cleaved complexes, thus introducing high levels of transient enzyme-associated breaks i n the genome (Froelich-Ammon and Osheroff 1995). I l l ) I n t r a c e l l u l a r pathways to apoptosis An i n t r a c e l l u l a r pathway must be i n place' to transduce signals from e x t r a c e l l u l a r s t i m u l i to the nucleus. In addition, the control for the c e l l u l a r decision of which form of death program to implement (ie. apoptosis vs. necrosis vs non-apoptotic programmed c e l l death) must be f l e x i b l e enough to respond to a wide range of ex t r a c e l l u l a r s t i m u l i . Hence, protein kinases have been postulated as the mechanism, not only for signal transduction, but also for a c e l l u l a r "rheostat control" that can respond to the degree of c e l l u l a r damage and can implement the d i f f e r e n t forms of c e l l death. Protein phosphorylation i s performed by protein kinases. Protein kinases have been s u b c l a s s i f i e d into i d e n t i f i a b l e groups, depending on the target amino acid residue where the phosphorylation occurs. Certain classes of kinases are predominant i n d i f f e r e n t c e l l u l a r e f f e c t s . The serine/threonine kinase pathway i s involved i n the regulation of many processes p i v o t a l for growth and d i f f e r e n t i a t i o n (Nishizuka 1986). Hence, the investi g a t i o n of serine/threonine kinases i s an ideal pathway to perform primary 9 investigations into signal transduction pathways responsible for apoptosis. The a c t i v i t y of protein kinases i s regulated a l l o s t e r i c a l l y . In serine/threonine kinases, kinases are composed of regulatory and c a t a l y t i c domains. In the kinase resting state, the regulatory domain keeps the c a t a l y t i c part of the enzyme inactive. This i n h i b i t i o n i s reversed when a second messenger or activator (eg. cAMP, cGMP, Ca + 2, DAG) binds to the regulatory domain (Tamaoki and Nakano 1990). ,Hence, there are regulators of kinase pathways which may control apoptosis. A. Protein Kinase I n h i b i t i o n Apoptosis, l i k e c e l l cycle and d i f f e r e n t i a t i o n , i s a complex process under dynamic regulation v i a a feedback control system. Thus, apoptosis may be controlled by proteins that mediate both the induction and i n h i b i t i o n processes. In addition, the i n h i b i t o r y pathway may involve proteins that i n h i b i t apoptosis, not only within the c e l l , but also i n other c e l l s through d i f f u s i b l e products and c e l l surface events. 1. Staurosporine To f i r s t investigate the role of protein kinases i n apoptosis, i n h i b i t o r s are often used. Staurosporine was f i r s t i d e n t i f i e d from Streptomyces culture as an a l k a l o i d with weak a n t i b a c t e r i a l and antifungal a b i l i t i e s (Omura et a l 1977). However, staurosporine i s a potent broad spectrum i n h i b i t o r of protein kinases. It potently i n h i b i t s PKA, PKC, cGMP dependent kinases, PTK, MLCK, and cdc2 kinase. Staurosporine i n h i b i t s the ATP binding s i t e within the c a t a l y t i c domain (C-terminal) of PKC while having no e f f e c t on the binding of phorbol esters to the regulatory domain (Ruegg and 10 Burgess 1989) . Staurosporine both antagonizes (Cotter et a l 1992) and i n i t i a t e s apoptosis i n HL-60 (Bertrand et a l 1993). Staurosporine induces apoptosis i n human malignant glioma c e l l l i n e s (Couldwell et a l 1994) and i n lymphoma and mammary carcinoma c e l l l i n e s (Shi et a l 1994) . Staurosporine induced a dose-dependent increase i n DNA fragmentation i n various c e l l l i n e s such as HL-60, lymphoma, lung carcinoma, and lung f i b r o b l a s t c e l l l i n e s (Bertrand et a l 1994) . In addition, staurosporine arrests c e l l s i n GI and G2 of the c e l l cycle (Abe et a l 1991, Crissman et a l 1991, Bruno et a l 1992), which i n i t s e l f , can induce apoptosis (Abe et a l 1991, Crissman et a l 1991, Bruno et a l 1992). B. Protein Kinase C Protein kinase C (PKC) plays an important r o l e as mediator between e x t r a c e l l u l a r growth signals and gene expression and can be regarded as a decision point which switches c e l l s into p r o l i f e r a t i o n or s e l f - e l i m i n a t i o n . It i s instrumental i n promoting gene expression d i r e c t l y or i n d i r e c t l y v i a d i f f e r e n t t r a n s c r i p t i o n factors. The PKC family comprises at least 12 mammalian isoforms which d i f f e r i n structure and enzymatic properties. PKC isoforms show d i f f e r e n t biochemical properties which occur at d i f f e r e n t proportions i n d i f f e r e n t tissues, suggesting that the PKC isoforms can mediate d i f f e r e n t c e l l u l a r functions. The members of the PKC family can be c l a s s i f i e d into three groups: c l a s s i c a l isoforms (cPKCs a, &I/X1, and y) ; novel isoforms [nPKCs 5? £ , T|(L), 0/ and | I ] ; and a t y p i c a l isoforms (aPKCs l , and X) (Hug and Sarre 1993) . The c l a s s i c a l isoforms meet the o r i g i n a l d e f i n i t i o n of PKC as a Ca + 2-and phospholipid-dependent protein kinase. The novel isoforms lack 11 the region responsible for Ca + 2 dependence, r e s u l t i n g i n Ca + 2 independence. The a t y p i c a l forms according to Buchner (1994) are not able to bind and cannot be activated by phorbol esters, and are independent of Ca + 2. In cardiac myocytes, PKCa, and ^ are located i n the cytosol i n non-stimulated c e l l s and are translocated (hence activated) to the nuclear envelope a f t e r stimulation with PMA or norepinephrine (Disatnik et a l 1994). In contrast, PKC8 and e are l o c a l i z e d i n the intranuclear area i n resting c e l l s according to Disatnik (1994). Upon stimulation PKC8 i s translocated to the nuclear envelope and PKCe i s translocated to myofibrils for further signal transduction effects (Disatnik et a l 1994). 1. PKC a c t i v a t i o n Upon c e l l receptor activation, phosphatidylinositol-4,5-biphosphate (PIP2) i s hydrolysed by phospholipase C into i n o s i t o l -1, 4,5-triphosphate (IP3) and d i a c y l g l y c e r o l (DAG). DAG activates PKC causing PKC to transduce the signal toward and within the c e l l nucleus. Signal transduction toward and within the c e l l nucleus can be mediated d i r e c t l y by PKC i n several ways: a) PKC i t s e l f translocates to the c e l l nucleus; b) nuclear PKC i s activated by a messenger from the cytoplasm reaching the c e l l nucleus; and c) nuclear PKC i s activated by an activator generated i n the nucleus, i n the course of another signal cascade. Activated PKC can also translocate to the plasma membrane where PKC modulates ion channel a c t i v i t y (Dolphin 1990) . Activated PKC phosphorylates components of the signal transduction system, including G-proteins and receptors (Dolphin 1990). Physiological messengers reported to t r i g g e r nuclear translocation of PKC include i n s u l i n - l i k e growth factor-1 (IGF-1), 12 IL-3, PDGF, a-thrombin, angiotensin II and vitamin D3 (Divecha et a l 1991, Fields et a l 1989, Fi e l d s et a l 1990, Leach et a l 1992, Haller et a l 1994, Simboli-Campbell et a l 1994). This suggests a rol e for translocated PKC i n the mitogenic response. The physiological s t i m u l i inducing nuclear translocation of PKC compromise agonist binding to phosphotyrosine kinase receptors (IGF-1, PDGF) and to G-protein-coupled receptors (a-thrombin, angiotensin II, type 1) (Divecha et a l 1991, Fi e l d s et a l 1989, Fields et a l 1990, Leach et a l 1992, Haller et a l 1994). Activated PKC phosphorylates components of the signal transduction system. PKC can phosphorylate a number of nuclear substrates (Buchner 1995), including the tumour suppressor protein p53 (Baudier et a l 1992) . Hence, PKC i s activated by various agonists and during i t s activation, i t translocates to other regions within the c e l l . Activated PKC can then phosphorylate components of signal transduction systems and nuclear substrates. 2. PKC involvement i n apoptosis To date, there are c o n f l i c t i n g data on the ro l e of PKC i n apoptosis. Studies have shown PKC to both activate and block apoptosis (Lucas and Sanchez-Margalet 1995) . The a c t i v a t i o n of PKC appears to induce apoptosis i n HL-60 (Cotter et a l 1992) and T - c e l l hybridoma c e l l s (Jin et a l 1992). Conversely, stimulation of the PKC pathway was able to prevent apoptosis induced by col c h i c i n e i n chronic lymphocytic leukemia c e l l s (Forbes et a l 1992) or by glucocorticoids (Kanter et a l 1984), Ca + 2 ionophores (McConkey et a l 1989a), and growth factor deprivation i n immature thymocytes (Rodriguez-Tarduchy and Lopez-Rivas 1989). The use of PKC i n h i b i t o r s , i n concentrations that are below t h e i r t o x i c i t y l i m i t s , 13 reverts the suppression of apoptosis by IL-3 and GM-CSF, i n hemopoietic c e l l s (Rajotte et a l 1992). Phorbol esters are known tumour promoters which activate PKC, through the phosphorylation of c e l l u l a r proteins and the induction of gene t r a n s c r i p t i o n (Greenberg and Z i f f 1984). Phorbol esters induce as well as i n h i b i t apoptosis (Cotter et a l 1990, McConkey and Orrenius 1991b, Suzuki et a l 1991, T r i t t o n 1991, Forbes et a l 1992, Terai et a l 1991). In vascular endothelial c e l l s , the phorbol ester TPA suppressed apoptosis without a requirement of any cooperative factors (Araki et a l 1990b). In IL-2 dependent T lymphocytes, removal of IL-2 activates apoptosis, but a c t i v a t i o n of PKC by phorbol esters blocks the i n i t i a t i o n of t h i s death program (Rodriguez-Tarduchy and Lopez-Rivas 1989). Phorbol esters s p e c i f i c a l l y i n h i b i t e d the normal phy s i o l o g i c a l process of apoptosis i n cultures of C3H-10T1/2 (CL8) mouse f i b r o b l a s t s (Tomei et a l 1988) . Thus, i t has been speculated that phorbol esters i n h i b i t the process of programmed c e l l death which may be i n i t i a t e d following cytotoxic stress. The use of PMA i s problematic because short time courses activate PKC whereas long time courses down-regulate PKC a c t i v i t y . Within each c e l l type, t h i s can be complex and may i n part r e f l e c t the nature of the findings above. IV) The role of calcium (Ca+2) i n apoptosis Because apoptosis involves Ca + 2 dependent kinases and endonucleases, calcium may have an important regulatory function i n apoptosis (Trump and Berezesky 1992) . Not only can increased levels of c y t o s o l i c calcium a f f e c t a signal transduction pathway leading to apoptosis, but i t also can trigger secondary events r e s u l t i n g 14 from the a c t i v a t i o n of other Ca + 2-dependent enzymes culminating i n c e l l death (El Alaoui et a l 1992). Nuclear Ca + 2 transport and the regulation of nuclear Ca+2-dependent enzymes are involved during apoptosis i n many c e l l types (Nicotera et a l 1994, Nicotera et a l 1992). An increase i n c y t o s o l i c calcium occurs at a l a t e r stage of apoptosis i n c e l l s (Lennon et a l 1992) suggesting that calcium i n f l u x might play d i f f e r e n t roles at d i f f e r e n t times during programmed c e l l death. Calcium has been implicated i n apoptosis induced i n s p e c i f i c c e l l systems by glucocorticoids (Cohen and Duke 1984, McConkey et a l 1989a, McConkey et a l 1989b), calcium ionophores (Ojcius et a l 1991, Rodriguez-Tarduchy et a l 1992, Rodriguez-Tarduchy G et a l 1990), y - i r r a d i a t i o n (Story et a l 1992), growth factors (Rodriguez-Tarduchy et' a l 1990), and hormone deprivation (Kyprianou et a l 1988). C e l l death was countered by application of Ca + 2 chelating agents (Lockshin and Zakeri 1991, Bellomo et a l 1992). As well, the transfections of genes encoding Ca + 2 buffering proteins such as calbindin-D28K blocked c e l l death (Dowd et a l 1992). However, apoptosis can be produced by factors which are independent of calcium, suggesting that calcium i s involved i n one of the many pathways leading to apoptosis (Bansal et a l 1990, Alnemri and Litwack 1990). There are c o n f l i c t i n g data on the exact role of Ca + 2 i n the development of apoptosis. It has been proposed that loss of calcium homeostasis, rather than a sustained r i s e i n [Ca* 2]^ i s a determining factor i n c e l l death by apoptosis (Kluck et a l 1994). However, because [Ca + 2] i i s a major second-messenger molecule, increased [Ca + 2] i may also promote c e l l s u r v i v a l by stimulating a signal transduction pathway, possibly the same one stimulated by 15 trophic factors (Franklin and Johnson 1994) . Hence, calcium may be involved i n the development of apoptosis, either directly by a f f e c t i n g the a c t i v i t y of Ca + 2 dependent enzymes or indirectly v i a second messengers. V) Growth factor and c e l l s u r v i v a l Growth factors stimulate c e l l p r o l i f e r a t i o n , involving new gene expression and protein synthesis (Pardee 1989). However, some factors may trigger s p e c i f i c s i g n a l l i n g pathways which stimulate c e l l s u r v i v a l rather than c e l l p r o l i f e r a t i o n . A pathway may be a control mechanism for apoptosis. For example, i n s u l i n - l i k e growth factor I (IGF-I) promotes sur v i v a l , but not p r o l i f e r a t i o n i n g l i a l c e l l s and bone marrow-derived IL3-dependent c e l l l i n e s (Barres et a l 1992, Rodriguez-Tarduchy et a l 1992). In vascular endothelial c e l l s , apoptosis i s suppressed by f i b r o b l a s t growth factor involving PKC a c t i v a t i o n (Araki et a l 1990b). In addition, growth factor receptor (IL-3) occupancy can also control the decision of a c e l l to survive or p r o l i f e r a t e i n the presence of a given growth factor, as iri the case of the IL-3 dependent c e l l l i n e BAF3 (Collins et a l 1994). - On the other hand, trophic factor removal causes a c e l l to undergo apoptosis, as i n the case of IL-3 withdrawal from BAF-3 c e l l s (Rodriguez-Tarduchy et a l 1990) . Thus, growth factors can stimulate pathways responsible for c e l l p r o l i f e r a t i o n and c e l l s u r v i v a l . VI) de novo protein synthesis and apoptosis Apoptosis requires de novo protein synthesis to i n i t i a t e and regulate the suicide program. A current hypothesis suggests that the c e l l s committed to undergo apoptosis promote the suicide cascade by synthesizing messenger/effector molecules of c e l l death 16 (Wright et a l 1*92, Walker et a l 1991, Rung et a l 1990, Barry et a l 1990, Thakkar and Potten 1992) . The opposite has also been postulated (Robertson et a l 1993, Manchester et a l 1993): the death program i s c o n s t i t u t i v e l y expressed and has to be continuously counterbalanced by the synthesis of suppressor molecules. Cycloheximide and actinomycin D, i n h i b i t o r s of protein and RNA synthesis, can both induce and i n h i b i t apoptosis (Gong et a l 1993, Perreault and Lemieux 1993) suggesting that de novo protein synthesis i s i n t e g r a l to the i n d u c t i o n / i n h i b i t i o n of apoptosis. VII) Genes and t h e i r products i n apoptosis Apoptosis, irequires the induction of a novel genetic programme and nuclear oncoproteins with t r a n s c r i p t i o n a l regulatory a c t i v i t y . Products of the c-fos, c-jun, c-myc, bcl-2, and p53 genes may work independently or cooperatively to determine the fate of the c e l l . A. c-Fos and c-Jun c-Fos and c-jun are nuclear proteins which intera c t to form the t r a n s c r i p t i o n a l complex AP-1. Once dimerized, they can interact with polymerase II and i n i t i a t e t r a n s c r i p t i o n . In normal conditions, when i t s partner i s present, overexpression of either gene i s s u f f i c i e n t to increase c e l l d i v i s i o n (Kato et a l 1992). However, i n lymphoid c e l l s , the expression of proto-oncogenes c-fos and c-jun i s J-induced during apoptosis (Colotta et a l 1992). EGF treatment of human breast cancer c e l l s , overexpressed with EGF receptors, was associated with an 18 f o l d induction of c-fos and a 16 f o l d induction of c-jun during the development of apoptosis (Armstrong et a l 1994). This suggests an active r o l e for fos and jun i n programmed c e l l death. 17 B. Bcl-2 : Bcl-2 i s a family of gene products also including bax, bad, and bcl-x. Bcl-2 gene product has been implicated i n apoptosis as a s u r v i v a l factor. Bcl-2 expressed i n transgenic mice enhanced c e l l s u r v i v a l (McDonnell et a l 1989) . Bcl-2 protects a wide range of c e l l s from triggers which would otherwise induce apoptosis, such as camptothecin (Zhong et a l 1993, Mah et a l 1993) . However, bcl-2 does not i n h i b i t T - c e l l receptor mediated apoptosis (Vaux et a l 1992) . Bax, a protein with extensive homology to bcl-2, counteracts the death-repressing a c t i v i t y of bcl-2 (Oltvai et a l 1993) . The two proteins are able to compete with one another i n forming homo- or heterodimers. The actual s u s c e p t i b i l i t y to death might be determined by the r a t i o of Bax to bcl-2. C. c-Myc Being an early response gene, c-myc i s normally down-regulated by conditions inducing growth arrest ( S h i c h i r i et a l 1993). In transformed c e l l s deprived of growth factor, the myc protein i s c o n s t i t u t i v e l y overexpressed which leads to apoptosis (Evans 1993, Arends et a l 1993). EGF treatment of human breast cancer c e l l s , overexpressed with EGF receptors, was associated with an 11 f o l d induction of c-myc during the development of apoptosis (Armstrong et a l 1994). Antisense i n h i b i t i o n of c-myc expression, i n a T c e l l hybridoma c e l l l i n e , prevents myc-induced apoptosis (Shi et a l 1992) . D. p53 p53 tumour suppressor gene product p a r t i c i p a t e s i n the induction of apoptosis i n myeloid leukemic c e l l s and colon cancer 18 c e l l l i n e s (Yonisch-Rouach et a l 1991, Lowe et: a l 1993a, Lowe et a l 1993b) . Increased p53 expression has been demonstrated i n apoptotic rat prostate e p i t h e l i a l c e l l s (Colombel et a l 1992) . In addition, transgenic mice with the p53 gene "knocked out" become re s i s t a n t to radi a t i o n induced apoptosis (Clarke et a l 1993, Hengartner et a l 1992). VIII) Potential s i g n i f i c a n c e of cardiac apoptosis C e l l growth and death i n many c e l l types are i n equilibrium during the normal c e l l state, but under diseased conditions, t h i s equilibrium i s s h i f t e d (Bright and Khar 1994). Apoptosis p a r a l l e l s c e l l s undergoing p r o l i f e r a t i o n or d i f f e r e n t i a t i o n . Cardiac hypertrophy i s often considered analogous to c e l l growth i n non-cardiac c e l l s because cardiomyocytes are terminally d i f f e r e n t i a t e d and cannot divide; thus they respond to various s t i m u l i by increases i n c e l l s i z e . Hypertension-induced cardiac hypertrophy i s often followed by the development of heart f a i l u r e . I t i s suspected that apoptosis may be involved i n the development of heart f a i l u r e a f t e r the development of cardiac hypertrophy (Bing 1995). A. Chick embryo v e n t r i c u l a r myocytes culture model The use of a chick embryo culture allows us to observe the biochemistry of the cardiomyocyte without the influence of systemic vasculature and c i r c u l a t i n g hormones. Furthermore, use of thi s cardiomyocyte culture eliminates the po t e n t i a l action of stimulation of c e l l surface receptors on the coronary and systemic vasculature. In addition, the use of a c e l l culture permits the observation of apoptosis without phagocytosis, i n contrast to tissue studies. 19 B. Histogenesis of the Embryonic Chick Ventricular Myocardium During heart development i n the chick embryo, prospective heart-forming mesoderm c e l l s migrate on an endoderm substratum, eventually forming two v e s i c l e s on either side of the developing foregut. The two heart rudiments are brought together i n the ventral midline and they fuse i n an anterior-to-posterior d i r e c t i o n . The inner l i n i n g becomes the endocardium and the outer layer becomes the myocardium. This occurs at the 2 5th and 3 0th hour of incubation i n the chick embryo. The newly formed heart tube twists into a looped structure with s p e c i f i c chambers. This twisting may be caused by the migration of sheets of c e l l s and by changes i n c e l l shape. The f i r s t region formed a f t e r fusion i s the truncus, or conus arteriosus, which leads to the v e n t r i c l e . Next to form i s the atrium and the l a s t part to form i s the sinus venosus, the heart chamber that receives venous blood. The heart tube bends to form an S shape. The heart begins to beat just a f t e r the paired heart rudiments begin to fuse (Oppenheimer and Lefevre 1989). C. Ventricular myocytes of the seven day old embryo Ventricular myocytes of the seven day old embryo contain many myofibrils, but they often s t i l l lack alignment. Whereas myofibrils i n s e r t into i n t e r c a l a t e d discs nearly at r i g h t angles i n the mature heart, the random f i b r i l s of these embryonic myocytes often in s e r t at an acute angle. Similar configurations have been seen as late as the tenth day of development (Noble and Cocchi 1990) . The monolayer culture of beating seven day embryonic chick myocytes i s an appropriate model for the inves t i g a t i o n of 20 s i g n a l l i n g i n cardiomyocytes during apoptosis. Our studies were performed on 4th to 7th incubation day using confluent monolayers of spontaneously beating myocytes. Previous studies by t h i s lab showed that the proportion of myocytes at t h i s time was at least 90% as v e r i f i e d by the proportion of c e l l s showing spontaneous contraction or displaying muscle s p e c i f i c markers on immunohistologic examination. IX) Objectives and hypotheses with t h e i r rationale I hypothesize that cardiomyocytes are capable of undergoing apoptosis. There are multiple p o t e n t i a l factors and pathways that might be involved. Because apoptosis involves a genetic mechanism, an i d e a l location for apoptotic control i s the nucleus. The control point might be associated with gene t r a n s c r i p t i o n since "death genes" must be activated during apoptosis. Topoisomerase I i s an i d e a l example of a nuclear control point for apoptosis. Some mechanism must be i n place to transmit the signal from the e x t r a c e l l u l a r environment to the nucleus. Hence, I hypothesize that some s i g n a l l i n g mechanisms must be i n place to allow the c e l l to respond to e x t r a c e l l u l a r s t i m u l i . I hypothesize that the PKC s i g n a l l i n g pathway may be a part of apoptotic control, because PKC i s a mediator between e x t r a c e l l u l a r growth signals and gene expression and can be regarded as a decision, point which switches c e l l s into p r o l i f e r a t i o n or s e l f - e l i m i n a t i o n . Because apoptosis involves Ca + 2-dependent enzymes i n i t s program, I hypothesize that Ca + 2 can modify the development of apoptosis. I speculate that a decrease i n Ca + 2 delays or abolishes the apoptotic program. 21 Growth factors may stimulate a pathway which can cause either c e l l p r o l i f e r a t i o n or c e l l s u r v i v a l . I hypothesize that growth factors w i l l be able to modulate the development of apoptosis. De novo protein synthesis i s required for apoptosis, either to produce "death" or cytoprotective proteins. I hypothesize that de novo protein synthesis w i l l be observed during treatment with d i f f e r e n t agents inducing apoptosis. My s p e c i f i c objectives to test my hypothesis and t h e i r r ationale are summarized as follows: 1. To establish that apoptosis occurs in cardiomyocytes Because the death of a cardiomyocyte would compromise the terminally d i f f e r e n t i a t e d heart, one might speculate that cardiomyocytes would not have a g e n e t i c a l l y programmed machinery for c e l l suicide. However, one must consider the need for programmed c e l l death should a s i t u a t i o n a r i s e when the elimination of one c e l l i s necessary for the good of the heart. Hence, I w i l l attempt to e s t a b l i s h whether apoptosis occurs i n cardiomyocytes. 2. To establish the role of topoisomerase I in the development of apoptosis The nucleus i s a prime location to e s t a b l i s h the control mechanisms of apoptosis. Topo I i s an e s s e n t i a l enzyme involved i n DNA r e p l i c a t i o n and t r a n s c r i p t i o n . It i s l o g i c a l that topo I i n h i b i t i o n would put the c e l l ' s genomic DNA i n a damaged state, since the machinery for r e p l i c a t i o n and protein synthesis are halted. Hence, the role of topo I w i l l be investigated to observe the e f f e c t of nuclear enzyme a c t i v i t y on the genetic program of apoptosis, recognizing that conclusions based on topo I are from the use of the topo I i n h i b i t o r camptothecin. 22 3. To establish the role of protein kinases in apoptosis Protein kinases are postulated to be part of the transduction pathway responsible for apoptosis. Staurosporine has been documented as a broad i n h i b i t o r of protein kinases and has been shown to induce apoptosis i n some c e l l l i n e s . I propose to implicate kinases i n the development of apoptosis by using staurosporine. Conclusions of the ro l e of protein kinases are based on the i n d i r e c t evidence provided by staurosporine usage. 4. To implicate protein kinase C in apoptosis A s p e c i f i c activator (PMA) and i n h i b i t o r (chelerythrine) for PKC w i l l be used to examine the ro l e of PKC i n apoptosis. The resu l t s from PKC w i l l then be compared with staurosporine to determine whether PKC i s the kinase responsible for staurosporine's r e s u l t s . Conclusions on the ro l e of PKC i s based on the used of the PKC i n h i b i t o r chelerythrine and the PKC activator PMA. 5. To establish the role of calcium in apoptosis Based on the Ca + 2 dependency of enzymes involved i n apoptosis, I w i l l explore the po t e n t i a l role of Ca + 2 i n apoptosis. The role of Ca + 2 i n apoptosis induced by either i n h i b i t i o n of topo I or protein kinase signal transduction w i l l be determined using Ca + 2 chelators and ionophores to a l t e r the l e v e l of c e l l u l a r Ca + 2. 6. To establish the role of growth factors in apoptosis Growth factors have been implicated i n the stimulation of c e l l p r o l i f e r a t i o n and c e l l s u r v i v a l i n other c e l l u l a r models. I w i l l explore the po t e n t i a l pathway stimulated by growth factor addition ( i n s u l i n and EGF) as a means of delaying or abolishing apoptosis. 7. To establish de novo protein synthesis in apoptosis in cardi omyocytes Apoptosis requires de novo protein synthesis. It i s unclear whether th i s protein synthesis would produce either death proteins or cytoprotecti.ve'proteins. I w i l l determine the l e v e l of protein synthesis that occurs during apoptosis i n cardiomyocytes, as well as any s p e c i f i c protein synthesized i n apoptotic c e l l s . 24 CHAPTER II MATERIALS AND METHODS 1. I s o l a t i o n of Chick Embryonic Cardiac C e l l s Monolayer culture of beating 7 day embryonic chick v e n t r i c u l a r c e l l s were prepared using previously described methods (Rabkin and Sunga 1987). F e r t i l i z e d White Leghorn eggs were incubated i n an automatic incubator for 7 days at 37.8°C and 87% humidity. Hearts were then removed from the 7 day chick embryos under s t e r i l e conditions i n a tissue culture hood. Vent r i c l e s were i s o l a t e d from a t r i a and used for further culturing. V e n t r i c l e s were cut into 0.5 mm fragments under dissecting microscope and disaggregation was car r i e d out by 5 minute digestions i n 0.005% trypsin, 0.1% bovine serum albumin, and DNAse (lxl0~ 7 Dornase units/mL) i n the balanced s a l t solution DMS8 at 37.8°C. DMS8 i s composed of the following (in mM) : NaCl 170, KC1 5.4, NaH2P04 4.3, Na2HP04 1, dextrose 5.6. This procedure was repeated 5 times, each time with replacement of disaggregation solution. After 5 digestions, the digests were d i l u t e d 5 f o l d i n culture medium 818A. 818A was composed of Medium 199 (20%), f e t a l c a l f serum (6%), antibiotic-antimycotic (10 000 units/mL p e n i c i l l i n G sodium, 10 000 |lg/mL streptomycin sulfate, and 25 pxr/mL amphotericin B)(1%), and the balanced s a l t solution DBSK (73%). DBSK contains (in g/L) : NaCl 6.8; MgS047H20 0.2; NaH2P04H20 0.13; dextrose 1; NaHC03 2.2. The disaggregated c e l l s were centrifuged for 3 minutes at 1 OOOxg. C e l l s were plated into either 35 or 100-mm culture plates at 1x10s c e l l s per culture dish or 4.5xl0 6 c e l l s per culture dish, respectively. Cultures were incubated i n a humidified 5% C02-95% a i r atmosphere at 37°C. Studies were performed on the 4th to 7th incubation day using 25 confluent monolayers of spontaneously beating myocytes (Rabkin 1993, Rabkin 1989) . The proportion of myocytes at t h i s time was at least 90% as v e r i f i e d by the proportion of c e l l s showing spontaneous contraction or displaying muscle s p e c i f i c markers on immunohistologic examination. 2. Microscopy a) C e l l v i a b i l i t y assay - Trypan Blue To assess c e l l v i a b i l i t y , cardiomyocytes were grown on a cover s l i p i n a 35mm p e t r i dish. After 72 h i n culture, drug agents or t h e i r diluent was added to the media. At predetermined times coverslips were removed, stained with 0.4% trypan blue and examined microscopically i n a haemocytometer to determine the number of c e l l s that were or were not stained by trypan blue. Dead c e l l s r e t a i n the blue dye whereas l i v e c e l l s do not. Trypan blue i s a standard assay for measuring c e l l death, including c e l l death (Lee et a l 1993) . b) C e l l morphology - acridine orange To examine c e l l morphology, cardiomyocytes, grown on cover s l i p s , were treated with drug agents or t h e i r diluent. The medium was removed and c e l l s were stained with acridine orange (100|J,g/mL) and dried. C e l l s were examined microscopically with a Zeiss (Standard 16) microscope using a fluorescent l i g h t source as previously described (Rabkin and Sunga 1987). Acridine orange i s pH-sensitive fluorescent dye which fluoresces green at neutral pH and red at an a c i d i c pH (approx. pH 5) (Nairn and Rolland 1980). c) C e l l morphology - Hoescht dye Hoescht--.dyes are s p e c i f i c for DNA and ' therefore i d e a l for observing nuclear changes (Bester et a l 1994, Yamamoto et a l 1990). 26 To provide further examination of nuclear changes, cardiomyocytes grown on coverslips were fix e d washed i n cold PBS and then fi x e d i n 3.7% paraformaldehyde for 10 min. The coverslips were b r i e f l y washed i n cold PBS and then extracted with cold acetone for 4 min. The coverslips were then stained with Hoescht 33258 (Hoe 33258) for 20 min, washed b r i e f l y , and then mounted on PBS/glycerol (1:1). C e l l s were examined microscopically with a Zeiss (Standard 16) microscope using a fluorescent l i g h t source as previously described (Rabkin and Sunga 1987). d) C e l l morphology - NBD p h a l l a c i d i n 7-nitrobehz-2-oxa-l,3-diazole-(NBD) p h a l l a c i d i n i s a fluorescent dye s p e c i f i c for F-actin and therefore i d e a l for observing cytoskeletal changes (Packman and Lichtman 1990). To examine for changes i n actin, cardiomyocytes grown on, coverslips were fixed washed i n cold PBS and then fi x e d i n 3.7% paraformaldehyde for 10 min. The coverslips were b r i e f l y washed i n cold PBS and 'then extracted with cold acetone for 4 min. The coverslips were then stained with 1:10 d i l u t i o n of NBD p h a l l a c i d i n i n PBS for lh, washed b r i e f l y , and then mounted on PBS/glycerol (1:1) . C e l l s were examined microscopically with a Zeiss (Standard 16) microscope using a fluorescent l i g h t source as previously described (Rabkin and Sunga 1987). 3. DNA Fragmentation The method described i s a modification of the method described by Scheid (et a l . 1995). Cardiomyocytes from the media and dish were i s o l a t e d because dead c e l l s become non-adherent and f l o a t into the medium. The medium was centrifuged at 200g and the r e s u l t i n g p e l l e t processed concomitantly with the adherent cardiomyocytes 27 is o l a t e d from the dish. Cardiomyocytes were washed with PBS, lysed (0.6% SDS, 10 mM EDTA), and the c e l l s scraped from the dish with a rubber policeman. 5M NaCl was added, mixed gently, and incubated at 4°C for 16 h. Samples were centrifuged at 16 OOOg for 20 min. The supernatant was removed, treated with RNAse A, and incubated at 37°C for 3 0 min. Proteins were removed by phenol-chloroform-isoamyl alcohol (25:24:1) extraction and the layers separated by centrifugation. The upper aqueous layer was removed and transferred to a fresh tube. 3M sodium acetate and 100% ethanol were added and the samples were incubated at -2 0°C for 10 min. The DNA was p e l l e t e d by 16 OOOg spin for 10 min and washed with 70% ethanol. The DNA was electrophoresed on a 2% agarose gel, stained with ethidium bromide, and destained with d i s t i l l e d water for 90 min. 4. DNA Fragmentation (Enzyme-linked immunosorbent assay, ELISA) C e l l s plated into 35 mm culture dishes at 1x10s c e l l s per culture dish were grown for 4 days. C e l l s were washed twice with l x PBS and then lysed. Meanwhile, antibodies s p e c i f i c for histones were coated onto a mic r o t i t r e plate and washed. Lysates from lysed cardiomyocytes were centrifuged at 15 OOOg to p e l l e t down whole c e l l s , c e l l debris, and inta c t DNA. The supernatant containing fragmented DNA was added to the coated m i c r o t i t r e plate and allowed to react with the immobilized primary antibody. After several washings, the secondary antibody, anti-DNA-peroxidase, was added and incubated for 90 min. The excess antibodies were washed off and the substrate reaction solution was added. The colorimetric substrate (2,2'-azino-di-[3-ethylbenzthiazoline sulfonate]) was measured at i t s absorbance wavelength of 405 nM. 28 5 . [ 3 5S]Methionine Incorporation and Preparation of C e l l lysates The cardiomyocytes were cultured on 100 mm dishes i n 818A medium containing 6% f e t a l c a l f serum for 4 days p r i o r to the experiment. Samples of l x l O 6 i s o l a t e d myocytes were rinsed twice i n methionine-free DMEM and incubated i n t h i s medium for 1 h. C e l l s were l a b e l l e d with 50 |J,Ci/mL trans [35S] methionine i n DMEM for 2 h. Drugs were added for s p e c i f i c times and the reactions were stopped by removal of the radioactive media was removed and the c e l l s washed twice with PBS. • C e l l s were lysed with l y s i s buffer containing 137 mM NaCl, 20mM T r i s pH'7.4, ImM MgCl2, ImM CaCl 2, 1% Nonidet P-40, ImM phenylmethylsulphonylfluoride (PMSF), and 2mM sodium orthovanadate. One m i l l i l i t r e of the l y s i s buffer was added to each dish, and the plates agitated for 2 0 minutes at 4°C. Thereafter, the c e l l s and debris were scraped from the plates with a rubber policeman. The l y s i s buffer and c e l l debris were transferred to s t e r i l e 1.5mL eppendorf tubes. At t h i s point, the samples are known as whole c e l l lysates. The samples were centrifuged at 600g for 10 min. The r e s u l t i n g supernatant and p e l l e t are soluble protein and nuclear proteins, respectively. Some cytoskeletal proteins may p e l l e t with the nuclear proteins. The supernatant was transferred to another eppendorf tube and the p e l l e t resuspended i n fresh l y s i s buffer! Aliquots of the supernatant and resuspended p e l l e t were retained for protein quantitation by the Bradford assay. Aliquots reserved for polyacrylamide gel electrophoresis were resuspended i n 4x electrophoresis sample loading buffer (125 mM Tris/HCl 6.8, 4% SDS, 10% S-mercaptoethanol, 20% g l y c e r o l , 0.01% bromophenol blue), b o i l e d for 4 minutes, and stored overnight at 4°C. 2 9 6. [35S] Methionine Incorporation Assay Five m i c r o l i t r e aliquots reserved from whole c e l l lysates were applied onto two 1 cm2 squares of Whatman No. 3 f i l t e r s and allowed to a i r dry. F i l t e r s were baked at 50°C for 10 min and then b o i l e d i n 10% t r i c h l o r o a c e t i c acid for 10 min. F i l t e r s were washed twice i n water, ethanol, and acetone and then l e f t to a i r dry. F i l t e r s were then placed i n s c i n t i l l a t i o n v i a l s and 250 uL of IN NaOH were added. The v i a l s were l e f t to incubate at 55°C for 30 min. Acetic acid and ACS counting solution were then added and l e f t overnight i n the dark. V i a l s were then counted on a program s p e c i f i c for 1 4C/ 3 5S energy l e v e l s . 7. Protein Determination Protein content was assessed using the method of Bradford (Bradford 1976). Bradford reagent was purchased from Bio-Rad Canada Ltd (Mississauga, Ont) . Bovine serum albumin was used as a standard. 8. SDS Polyacrylamide Gel Electrophoresis (SDS PAGE) SDS PAGE of extracts was performed according to the method of Laemmli (197 0) on 10% separating gels containing 5.6 mL resolving buffer, 16.9 mL 30% acrylamide/Bis, 150 |IL 10% ammonium persulfate (freshly prepared) , and 3 0 |XL TEMED. Samples were dissolved i n SDS gel loading buffer, b o i l e d for 4 min, and equal amounts of protein loaded onto 4% stacking gels (6mL stacking buffer, 1.5mL 3 0% acrylamide/Bis, 50|XL 10% ammonium persulfate, and 10|IL TEMED) overlying the 10% polyacrylamide resolving g el. Following electrophoresis, gels were fixed with isopropanol: water : acetic acid (25:65:10) for 3 0 min and then fixed with AMPLIFY™ (Amersham Canada Ltd, Oakville Ont) for another 3 0 min. Gels were mounted onto 30 Whatman No 3 f i l t e r paper and dried. 9. Radiolabelled Proteins [ 3 5S]Methionine l a b e l l e d protein gels were mounted, dried and autoradiographed on Kodak X-AR5 X-ray f i l m using i n t e n s i f i e r screens at -70°C. Gels were exposed to Kodak X-AR5 f i l m from 1 to 5 days. 1 0 . Drug pretreatment Experiments which require drug pretreatment before exposure to the inducting agent was performed by a 30 min. preincubation of the pretreatment'-'a^ent before exposure to the in'ducing agent. 1 1 . Materials a) Biochemicals A l l c e l l culture components were from Gibco/BRL L i f e Sciences (Burlington, Canada). DNAse I was from Worthington Biochemicals (Freehold, New Jersey). Camptothecin, taurine, EGTA, BAPTA-AM, staurosporine, acridine orange, and Hoescht 33258 were from Sigma Chemicals Ltd (St. Louis,USA). Chelerythrine, and PMA, were from Calbiochem Ltd (La J o l l a , Ca, USA). NBD p h a l l a c i d i n was purchased from Molecular Probes (Eugene, Oregon). Trypan blue was purchased from BDH Chemicals Ltd (Toronto, Canada). Components of the ELISA were from Boerhinger Mannheim (Laval, Canada)";.. A l l chemicals were purchased from Fischer S c i e n t i f i c (Ottawa, Canada). b) Radiochemicals Tran 3 5S-Methionine c e l l l a b e l l i n g grade (1214 Ci/mmol s p e c i f i c a c t i v i t y ) was from ICN Pharmaceuticals Inc (Irvine, C a l i f o r n i a ) . [14C] l a b e l l e d high molecular weight protein markers were from Amersham Canada (Oakville, Ont). 31 c) Pharmacology of drugs i) Camptothecin Camptothecin, i s an a l k a l o i d i s o l a t e d from the wood of Camptotheca acuminata. Camptothecin "poisons." topo I when i t i s associated with DNA during cleavage. By poisoning topo I at thi s stage, the levels of topo I-DNA cleaved complexes are increased thus introducing high lev e l s of transient enzyme-associated breaks i n the genome (Froelich-Ammon and Osheroff 1995). i i ) Staurosporine Staurosporine i s a potent broad spectrum i n h i b i t o r of protein kinases, e s p e c i a l l y PKA and PKC. It has been shown to induce apoptosis i n human malignant glioma c e l l l i n e s . i i i ) EGTA, or Ethyleneglycol-bis(S-aminoethyl)-N,N,N',N'-tetraacetic acid EGTA i s a chelating agent s e l e c t i v e for Ca + 2. It induces apoptosis i n pheochromocytoma (PC12) c e l l s '. EGTA chelates free e x t r a c e l l u l a r calcium within the media thus preventing calcium entry into the c e l l . 1 mol of EGTA can chelate 2 moles of free Ca + 2. The e f f e c t of ImM EGTA on 818A media, which contains 1. 5mM Ca + 2, should chelate a l l free e x t r a c e l l u l a r Ca + 2 r e s u l t i n g i n an ext r a c e l l u l a r , Ca + 2-free environment. iv) BAPTA-AM, or 1,2-bis-(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BAPTA i s a Ca + 2 chelator with 105x greater a f f i n i t y for Ca + 2 than for Mg+2. BAPTA can enter the c e l l and through chemical reactions can trap free Ca + 2 ions (Chiamvimonvat 1995). v) Thapsigargin Thapsigargin i s a sesquiterpene gamma-lactone which s e l e c t i v e l y i n h i b i t s the sarcoplasmic reticulum and endoplasmic 32 reticulum Ca + 2-dependent ATPase family of i n t r a c e l l u l a r "Ca+2-pumping" ATPases and produces a three to four f o l d elevation i n the levels of [Ca + 2]i (Furuya et a l 1994) . vi) PMA, or Phorbol - 1 2-myristate - 1 3-acetate PMA i s an activator of PKC which also potentiates f o r s k o l i n -induced cAMP formation. It has been shown to i n h i b i t apoptosis induced by the Fas antigen, but induces apoptosis i n HL-60 c e l l s (Jarvis et a l 1994) . v i i ) Chelerythrine chloride Chelerythrine chloride i s a potent and se l e c t i v e i n h i b i t o r of PKC. It acts at the diglyceride binding s i t e within the PKC regulatory domain and i s over lOx more potent than H-7. It has been shown to induce apoptotic DNA fragmentation and c e l l death i n HL-60 (Jarvis et a l 1994) . 12. Methods of S t a t i s t i c a l Analysis The data are presented as the mean +_ SEM within two standard deviations. Hypothesis testing used analysis of variance. The n u l l hypothesis was rejected i f the p r o b a b i l i t y of a Type I error was less than 5% (p<0.05). 33 CHAPTER III A. RESULTS OF TOPO I INHIBITION-MEDIATED APOPTOSIS 1. C e l l V i a b i l i t y a f t e r exposure to camptothecin To investigate the poten t i a l c y t o t o x i c i t y of camptothecin i n cardiomyocytes, c e l l v i a b i l i t y was assayed by trypan blue exclusion (Fig 1) . Camptothecin-induced c e l l death was apparent and occurred i n a dose-dependent and time-dependent manner. After 6h of treatment, higher (50 (IM) and lower 10 (IM, camptothecin appeared to exert s i m i l a r e ffects (Panel A). This time-dose was point chosen for further experiments. At 6 h of treatment, camptothecin produced dose dependent c e l l death with a plateau apparent for doses greater than lOflM. Camptothecin, 10|i.M, s i g n i f i c a n t l y (p<0.05) increased c e l l death by 25.1+1.4% (N=10) compared to control. Camptothecin concentrations higher than 50 (IM could not be used as the drug was not very solub'le. 2. C e l l morphology a f t e r exposure to camptothecin To investigate the morphological changes associated with apoptosis, cardiomyocytes were exposed to camptothecin and stained with Hoe 33258 or acridine orange (Fig 2) . Hoe 33258 i s a DNA minor groove-binding ligand and s p e c i f i c a l l y binds to nuclei as i t relaxes the suprahelical organization of DNA, leading to the formation of a B-like structure (Krishna et a l 1993). Control c e l l s , exposed to the diluent, showed a weakly stained, in t a c t nucleus [Panel A). In contrast, camptothecin treated c e l l s showed a strongly stained nucleus with evidence of nuclear d i s i n t e g r a t i o n (Panel B). c e I I % 6 h 25 i 20 3 4 15 d a 10 t h 5H • - Camptothec in 10uM •—Camptothec in 50uM ^25n i i B T ime (h) 0.1 1 10 100 Camptothec in (uM) FIG 1. The effect of camptothecin on cell viability assessed by trypan blue exclusion assay. Results are reported as the difference in the percent of dead cells in camptothecin treated cardiomyocytes compared to control (diluent treated) cardiomyocytes. Panel A - Cardiomyocytes were treated with either 10 or 50 \lM camptothecin for various times. The data are the mean+SEM of 3 to 13 experiments. Panel B - Cardiomyocytes were exposed to camptothecin, 1 or 10 or 50 ]XM for 6 hours. The data are the mean±SEM of 8 to 10 experiments. 35 FIG 2. The c e l l morphology of cardiomyocytes a f t e r camptothecin treatment. Representative cardiomyocytes exposed to 10 \XM. camptothecin (Panels B & D) or i t s diluent DMSO(control) (Panels A & C) for 6 h. C e l l s were stained with either Hoe 33258 (Panels A & B) or acridine orange (Panels C & D). 35f\ 36 Acridine orange stained control c e l l s showed an o v e r a l l green appearing cytoplasm with an int a c t nucleus (Panel C) . C e l l s exposed to camptothecin, however, demonstrated extensive membrane blebbing and nuclear d i s i n t e g r a t i o n (Panel D). The nuclei often stained red, consistent with alt e r a t i o n s of c e l l u l a r pH. 3. DNA fragmentation To investigate p o t e n t i a l changes to genomic DNA, cardiomyocytes were exposed to camptothecin and the DNA was i s o l a t e d and electrophoresed on a 2% agarose gel (Fig 3). Camptothecin treated cardiomyocytes had DNA fragments of 180 bp or multiples of 180 bp (Lanes 1 and 3) . In contrast, the control c e l l s showed no DNA fragmentation or smearing (Lane 2) . To quantitate the extent of DNA fragmentation associated with apoptosis, cardiomyocytes were treated with camptothecin and processed by means of an enzyme-linked immunosorbent assay (ELISA) (Fig 4). C e l l s exposed to camptothecin had s i g n i f i c a n t l y (p<0.05) more DNA fragmentation. The amount of DNA fragmentation appeared to plateau a f t e r 10 |0,M camptothecin that produced 1.5 + 0.5 (+_SEM) (N=8) f o l d more fragmentation signal than control. 4 . Role of Ca + 2 i n camptothecin-induced c e l l death and apoptos i s:EGTA To evaluate the r o l e of Ca + 2 i n camptothecin-induced apoptosis, EGTA, a Ca + 2 chelator (Bers DM 1982), was co-incubated with camptothecin (Fig 5). Low EGTA concentrations were used to ensure c e l l v i a b i l i t y . EGTA treatment reduced camptothecin-induced c e l l death as demonstrated by trypan blue exclusion (Panel A). EGTA 3 7 F I G 3. DNA fragmentation i n cardiomyocytes exposed to camptothecin or DMSO. A representative gel of DNA i s o l a t e d from cardiomyocytes and electrophoresed on 2% agarose. Lanes 1 & 3 - camptothecin 10 (UM; Lane 2 - control; Lane 4 - 1 kb DNA ladder. A l l incubations were at 6 h. 38 FIG 4. DNA fragmentation quantitated by an enzyme-linked immunosorbent assay (ELISA). Cardiomyocytes were exposed to various concentrations of camptothecin for 6 h. Results are the sample absorbance at 405 nm and are expressed relative to control (DMSO treatment). The data are the mean±SEM (N=3 to 8). A c 30 25 d 20 t h h 10 % 5 a t o 39 0.01 0.1 EGTA (mM) EGTA + Campto (10uM) • EGTA 0.01 0.1 EGTA (mM) FIG 5. The effect of EGTA on camptothecin-induced cell death. Panel A - Effect of various concentrations of EGTA on camptotheein-induced cell death assessed by trypan blue exclusion. All cardiomyocytes were pretreated with 0.01, 0.1, or 1.0 mM EGTA before 10\iM camptothecin treatment (OmM) . The bar graph represent the mean±SEM of 3-4 experiments. Panel B - Enzyme-linked immunosorbent assay of DNA fragmentation for cardiomyocytes treated with 10 |IM camptothecin without (OmM) or with EGTA. The bar graph represent the mean±SEM of 3 to 5 separate experiments. 40 pretreatment also reduced DNA fragmentation as measured by the ELISA assay (Panel B). 5 . Role of Ca*2 In the morphologic changes of apoptosis i n the cardiomyocyte To determine whether or not Ca + 2 was involved i n the apoptotic morphology produced by camptothecin, cardiomyocytes were exposed to camptothecin with EGTA pretreatment and examined microscopically (Fig 6, Panel A) . EGTA treated c e l l s d i d not show membrane blebbing a f t e r treatment with camptothecin. C e l l death was s t i l l evident, consistent with the trypan blue data, however the absence of membrane blebbing was s t r i k i n g . To further examine the r o l e of i n t r a c e l l u l a r Ca + 2 i n production of the c e l l u l a r changes of apoptosis, cardiomyocytes exposed to camptothecin were pretreated with taurine (Panel B) . Taurine i s a free amino acid i n plasma and many tissues such as heart, muscle, brain, and blood; i t af f e c t s L-type and T-type Ca + 2 channels i n embryonic chick heart c e l l s (Kaplan et a l 1993, Satoh and Sperelakis 1993) and reduces i n t r a c e l l u l a r calcium (Kaneko and Tsukamoto 1994). Taurine reduced camptothecin-induced apoptotic changes i n c e l l u l a r morphology. Red staining, associated with a c i d i c pH and c e l l u l a r damage, was seen i n these c e l l s suggesting c e l l u l a r damage. However, the absence of membrane blebbing usually seen during apoptosis was apparent. 6 . Role of Ca + 2 i n camptothecin-induced DNA fragmentation To investigate the role of Ca + 2 l e v e l s i n camptothecin-induced DNA fragmentation, cardiomyocytes were exposed to camptothecin and PIG 6. C e l l morphology of cardiomyocytes exposed to camptothecin and_pretreatment with EGTA or taurine. C e l l s were stained with acridine orange. Representative cardiomyocytes pretreated with 10 MM EGTA (Panel A) or 10 mM taurine (Panel B) before lOuM camptothecin exposure for 6h. 42 pretreatment with taurine (Fig 7). Taurine reduced DNA fragmentation i n an apparent dose-dependent manner as the highest concentration of taurine showed the least amount of DNA laddering whereas the lower taurine concentration showed the greater camptothecin-induced DNA fragmentation. To quantitate the ef f e c t of taurine on camptothecin-induced DNA fragmentation and to compare i t to an agent that increases c e l l u l a r calcium, cardiomyocytes were exposed to camptothecin and pretreated with taurine, or thapsigargin (Fig 8) . Taurine protected the cardiomyocyte from DNA fragmentation induced by camptothecin. In contrast, thapsigargin s i g n i f i c a n t l y (p<0;05) increased DNA fragmentation to level s greater than camptothecin. 7 . de novo protein synthesis during camptothecin-induced apoptosis Cardiomyocytes were exposed to camptothecin during [ 3 5S]methionine incorporation (Fig 9) . New proteins synthesized were i s o l a t e d and run on a SDS-PAGE. Panel A exhibits a 1, 2, 4h time course of'.'ide novo protein synthesis during exposure to 10|iM camptothecin. Panel B exhibits the ef f e c t of cycloheximide, a known i n h i b i t o r of protein synthesis, on camptothecin de novo protein synthesis. B. Results of staurosporine-induced apoptosis 1. C e l l V i a b i l i t y a f t e r exposure to staurosporine To investigate the po t e n t i a l c y t o t o x i c i t y of staurosporine i n cardiomyocytes, c e l l v i a b i l i t y was assayed by trypan blue exclusion (Fig 10) . Staurosporine-induced c e l l death was apparent and occurred i n a dose-dependent (Panel A) and time-dependent manner 4 3 2036 bp 1018 FIG 7 . A r e p r e s e n t a t i v e g e l o f DNA i s o l a t e d from c a r d i o m y o c y t e s and e l e c t r o p h o r e s e d on 2% agarose. Cardiomyocytes were exposed t o c a m p t o t h e c i n and/or v a r i o u s c o n c e n t r a t i o n s o f t a u r i n e . Because a l i v e c a r d i o m y o c y t e s a r e adherent t o the d i s h and c a r d i o m y o c y t e s w h i c h had d i e d b e f o r e t h e 6 h i n c u b a t i o n would become dead and f l o a t i n t h e media, the media was examined as w e l l and termed as "media". Lane 1 - C o n t r o l ; Lane 2 - C o n t r o l media; Lane 3-10 |IM c a m p t o t h e c i n ; Lane 4 - c a m p t o t h e c i n media; Lane 5-1 mM t a u r i n e + 10 UM c a m p t o t h e c i n ; Lane 6 - media f o r l a n e 5; Lane 7 - 1 0 mM t a u r i n e + 10 [IM c a m p t o t h e c i n ; Lane 8 - media f o r l a n e 7; Lane 9 -10 mM t a u r i n e ; Lane 10 - media f o r Lane 9; Lane 11 - 1 kb DNA l a d d e r . A l l i n c u b a t i o n s were a t 6 h. 44 FIG 8. Bar graph showing DNA fragmentation quantitated by an enzyme-linked immunosorbent assay (ELISA). Cardiomyocytes were exposed to camptothecin and/or thapsigargin or taurine. Results are the sample absorbance at 405 nm and are expressed relative to control (DMSO treatment). The data are the mean±SEM (N=3). Cam = camptothecin 10\lM, 6h; Th = thapsigargin lOOnM, 6h; Th + Cam = thapsigargin + camptothecin; Ta ImM = Taurine ImM, 6h; Ta lOmM = Taurine lOmM, 6h-. Ta + cam = taurine + camptothecin. (*-p<0.05) 45 nuclear soluble B FIG 9. de novo protein synthesis during camptothecin exposure. Cardiomyocytes were exposed to camptothecin during 35S-methionine incorporation. New proteins synthesized were isolated and run on SDS-PAGE. The gel was mounted, dried, and exposed. Panel A exhibits a 1, 2, and 4 h time courses of de novo protein synthesis during exposure to camptothecin. C=Control; CAM-camptothecin 10\lM; MW reported as kDa; nuclear=nuclear protein fraction; soluble=soluble protein fraction. Panel B exhibits the effect of cycloheximide on camptothecin de novo protein synthesis during 4h. C=Control; CM-Camptothecin 10\lM; CY=cycloheximide 1\IM; MW reported as kDa. First set of C, CM,CM+CY,CY (closest to MW standards)=nuclear protein fraction. Second set=soiuble protein fraction. 46 (Panel B) . At 6 h of treatment, staurosporine produced dose dependent c e l l death. Staurosporine, 1|1M, s i g n i f i c a n t l y (p<0.05) increased c e l l death by 40.4+7.1% (N=7) compared to control. Staurosporine concentrations higher than 1 |XM could not be used as a s i g n i f i c a n t number of cardiomyocytes would die and f l o a t into the media, hence l o s t to trypan assay, before the incubation time was completed. Staurosporine exposure over time yielded a plateauing e f f e c t at times greater than 4h. Hence, the 6h time point and the ljXM dose were chosen for future experiments. 2. DNA fragmentation a f t e r exposure to staurosporine To investigate p o t e n t i a l changes to genomic DNA, cardiomyocytes were exposed to staurosporine and the DNA was i s o l a t e d and assayed for fragmentation by -ELISA (Figure 11). Staurosporine exposure induced DNA fragmentation i n a time-dependent manner. Cardiomyocytes were exposed to staurosporine and the DNA was i s o l a t e d and assayed for fragmentation either by ELISA (Figure 12 -Panel A) or by electrophoretic separation (Panel B) . Maximal DNA fragmentation yielded fragmentation 3.1+1.7 (N=8) f o l d more than control c e l l s . Staurosporine treated cardiomyocytes yielded DNA fragments of 180 bp or multiples of 180 bp i n a dose dependent manner. In contrast, the control c e l l s showed no DNA fragmentation or smearing. 3. C e l l morphology a f t e r exposure to staurosporine To investigate the effects of staurosporine exposure to 2 3 4 Time (h) of exposure to staurosporine 1uM 0.001 0.01 0.1 1 Staurosporine (uM) 10 FIG 10 - The effect of staurosporine on cell viability assessed by trypan blue exclusion assay. Results are reported as the difference in the percent of dead cells in staurosporine treated cardiomyocytes compared to control (diluent treated) cardiomyocytes. Panel A - Cardiomyocytes were exposed to staurosporine 1\IM for various times. Panel B - Cardiomyocytes were treated with various concentrations of staurosporine for 6h. The data are represented by a log dose curve and are the mean±SEM of 4 to 8 experiments. 4 8 j 0 . 5 -o n <H , , , 0 2 4 6 Time (h) of exposure to s taurospor ine 1uM FIG 11 - Staurosporine induced DNA fragmentation quantitated by an enzyme-linked immunosorbent assay (ELISA). Cardiomyocytes were exposed to staurosporine l\lM for various times. A D 3 -i N A 2.5 -f r 2 -a g m 1.5 -e n 1 J t a t i 0.5 -i o n 0 -49 0.001 0.01 0.1 Staurosporine (uM) 1 B bp 2036( I 1018 506 2 3 15 iiii 1 n i 1 FIG 12 - Dose response of staurosporine-induced DNA fragmentation. Panel A - Cardiomyocytes were exposed to various concentrations of staurosporine for 6 h . Results are the sample absorbance at 405 nm and are expressed relative to control (DMSO treatment) . The data are the mean±SEM (N-4 to 9). Panel B - Electrophoretic separation of staurosporine-induced DNA fragmentation. Lane 1 - DNA ladder; Lane 2 - staurosporine 1\1M; Lane 3 - staurosporine 0.1\lM; Lane 4 -staurosporine 0.01\lM; Lane 5 - Control; performed at 6h. 50 c e l l u l a r morphology, NBD p h a l l a c i d i n staining was performed (Fig 13) . Control c e l l (Panel A) was compared to c e l l s exposed to 1(XM staurosporine for 6h. Panels B-D demonstrate the developmental changes to c e l l u l a r morphology i n response to staurosporine exposure. Cardiomyocytes exposed to staurosporine display unique morphology, d i s s i m i l a r to the t y p i c a l apoptotic changes. During the course of staurosporine exposure, c e l l s appear to elongate due to the condensation of the cytoplasm and lose c e l l volume. NBD p h a l l a c i d i n staining exhibits an active reorganization of the cytoskeleton as the c e l l elongates and the cytoplasm condenses. 4. The e f f e c t of Ca + 2 on staurosporine-induced apoptosis The r o l e of Ca + 2 was elucidated by pretreating cardiomyocytes with EGTA or BAPTA before staurosporine addition (Fig 14) . EGTA s l i g h t l y reduced staurosporine-induced c e l l death, as assessed by trypan blue assay. BAPTA blunted staurosporine-induced c e l l death. The effect of EGTA and BAPTA on staurosporine-induced DNA fragmentation was investigated by ELISA (Fig 15) . Pretreatment with BAPTA s i g n i f i c a n t l y (p=0.02) reduced staurosporine-induced DNA fragmentation, as assayed by ELISA (Panel A). C e l l u l a r morphology was assessed by acridine orange staining. (Fig 16). Staurosporine exposure (Panel B) i s d i s t i n c t l y d i f f e r e n t than control ''cell (Panel A) . The addition of BAPTA completely abolished staurosporine-induced morphology (Panel C) . These re s u l t s suggest that Ca + 2 i s needed for staurosporine-induced c e l l death. Obviously, the more s p e c i f i c [Ca*2]! chelation with BAPTA would be more e f f e c t i v e than e x t r a c e l l u l a r Ca + 2 chelation with EGTA. 51 FIG 13 - C e l l morphology of representative cardiomyocytes exposed to staurosporine 1|IM (Panels B-D) or i t s diluent (Panel A) for 6h and stained with NBD p h a l l a c i d i n . / Ji ~ f- e f f e c t o f E G T A an<* BAPTA on staurosporine-induced cell death. Cardiomyocytes were exposed to staurosporine and/or various concentrations of either EGTA or BAPTA. Effect of EGTA or BAPTA on staurosporine-induced cell death assessed by trypan blue exclusion The bar graph represent the mean+SEM of 3 to 4 experiments 53 D 3n N A 2.5 -FIG 15 -The effect of EGTA and BAPTA on staurosporine-induced DNA fragmentation. Enzyme-linked immunosorbent assay of DNA fragmentation for cardiomyocytes pretreated with either EGTA or BAPTA before treatment with staurosporine (1[IM) . The bar graph represents the mean±SEM of 3 separate experiments. (*=p<0.05) 54 FIG 16 - C e l l morphology of representative cardiomyocytes exposed to staurosporine 1|1M (Panel B) , i t s diluent (Panel A) , BAPTA lOmM (Panel C) , or PMA 10|1M (Panel D) for 6h and stained with acridine orange. 55 5. E f f e c t of PMA on staurosporine-induced apoptosis The phorbol ester PMA was added concomitantly with staurosporine' to elucidate the e f f e c t of active PKC on staurosporine-induced apoptosis (Figure 17) . PMA prevented staurosporine-induced c e l l death (Panel A) and morphology (Fig 16, Panel D) . In addition, t h i s protection was time-dependent. However, PMA co-incubation yielded s i m i l a r l e v e l s of DNA fragmentation, assessed by ELISA, as staurosporine alone (Panel B). 6. E f f e c t of trophic factors, i n s u l i n and EGF on staurosporine-induced apoptosis To investigate the ro l e of trophic factor addition on staurosporine-induced apoptosis, i n s u l i n and EGF were added to cardiomyocytes .preincubated with staurosporine (Fig 18). Insulin s i g n i f i c a n t l y (p=0.029) reduced staurosporine-induced death while the e f f e c t s of EGF may be s i g n i f i c a n t (p=0.057). To investigate the ro l e of growth factors oh staurosporine-induced DNA fragmentation, i n s u l i n and EGF were added to cardiomyocytes (Fig 19) . Insulin and EGF addition blunted staurosporine-induced DNA fragmentation, as assessed by ELISA (Panel A) and electrophoretic separation (Panel B). These re s u l t s suggest that the addition of EGF and i n s u l i n , to some extent, can blunt staurosporine-induced c e l l death. In addition, i n s u l i n preincubated before staurosporine treatment may activate some pathway which works i n conjunction with staurosporine-0 2 4 6 Time (h) FIG 17 - The effect of PMA on staurosporine-induced. cell death and DNA fragmentation. Cardiomyocytes were exposed to staurosporine and/or 10\lM PMA for various times. Panel A - Effect of PMA on staurosporine-induced cell death assessed by trypan blue exclusion assay. The graph represents the mean±SEM of 3-5 experiments. Panel B - Enzyme-linked immunosorbent assay of DNA fragmentation for cardiomyocytes treated with staurosporine (1[LM) and/or 10\lM PMA. The bar graph represent the mean±SEM of 3-4 separate experiments. 57 c 45 i 0 0.01 0.1 1 Staurosporine (uM) B Staurosporine • Staurosporine + Insulin 100nM • Staurosporine + EGF 50 ng/mL FIG 18 - The effect of insulin and EGF on staurosporine-induced cell death. Cardiomyocytes were exposed to l[iM staurosporine and/or lOOnM insulin or 50[lg/mL EGF. Effect of insulin or EGF on staurosporine-induced cell death assessed by trypan blue exclusion. The graph represent the mean±SEM of 5-7 experiments. N • 3 ^ l 2 n1.5 t t 1 i o n 0.5 H 0 5 8 0.01 0.1 Staurosporine (uM) at 6h • Staurospor ine • Staurospor ine + Insulin 100nM E3 Staurospor ine + E G F 50ng/mL B 1 2 3 4 5 6 7 8 bp I 2036 1018 506 FIG 19 - The effect of insulin and EGF on staurosporine-induced DNA fragmentation. Panel A - Enzyme-linked immunosorbent assay of DNA fragmentation for cardiomyocytes treated with staurosporine (l\lM) and/or insulin or EGF. The bar graph represent the mean±SEM of 3-4 separate experiments. (*=p<0.05) Panel B - Electrophoretic separation of effect of insulin on staurosporine-induced DNA fragmentation. Lane 1 - Control; Lane 2 - Insulin lOOnM; Lane 3 -media for lane 2; Lane 4 - Insulin lOOnM + Staurosporine luM; Lane 5 - media for lane 4; Lane 6 - Staurosporine luM + Insulin lOOnM; Lane 7 - media for lane 6; Lane 8 - Staurosporine luM; Lane 9 - DNA ladder; all performed at 6h. 59 induced DNA fragmentation which would account for the greater degree of DNA fragmentation seen i n Panel B. 7. de novo protein synthesis To investigate the p o s s i b i l i t y that a cytoprotective protein was synthesized during trophic factor rescue of staurosporine-induced c e l l death, [ 3 5S]methionine incorporation studies were performed (Figure 20) . New proteins were synthesized during staurosporine exposure, compared to control, as demonstrated by 3 5S-methionine incorporation assay (Panel A) and autoradiograph (Panel B). Staurosporine treatment stimulates de novo protein synthesis i n cardiomyocytes compared to control. The autoradiograph shown i s a representative of 4 separate experiments. 8. Role of PKC i n staurosporine-induced c e l l death: comparison of staurosporine and chelerythrine To elucidate whether staurosporine e f f e c t s are mediated s o l e l y by PKC, chelerythrine was used i n experiments previously performed with staurosporine and the r e s u l t s compared (Fig 21) . Chelerythrine induced c e l l death, as assessed by trypan blue exclusion assay, i n a dose-dependent manner with a maximal death of 42.9+_9.8% at 6h. This l e v e l of death was achieved at a chelerythrine concentration over lOx greater than that of staurosporine.:' DNA fragmentation following chelerythrine exposure was assessed by ELISA (Fig 22, Panel A) and electrophoretic separation (Panel B) Chelerythrine produced DNA fragmentation 2 . 2 + 0.4 f o l d more than control and yielded DNA laddering (Panel B). FIG 20 - de novo p r o t e i n s y n t h e s i s d u r i n g s t a u r o s p o r i n e i n d u c e d a p o p t o s i s . De novo p r o t e i n s y n t h e s i s was measured by i n c o r p o r a t i o n r a t e a s s a y (Panel A) or by PAGE (Panel B ) . Panel A - R e l a t i v e 3 5S-me t h i o n i n e i n c o r p o r a t i o n r a t e compared to c o n t r o l . Bar graph r e p r e s e n t s the mean+SEM of 4 s e p a r a t e e x p e r i m e n t s . Panel B - Newly s y n t h e s i z e d p r o t e i n s w i t h 3 5S-Met i n c o r p o r a t i o n were e l e c t r o p h o r e t i c a l l y s e p a r a t e d on a 10% p o l y a c r y l a m i d e g e l . The r e s u l t i n g a u t o r a d i o g r a p h i s a r e p r e s e n t a t i v e of 4 s e p a r a t e e x p e r i m e n t s . C = c o n t r o l , S=Staurosporine luM, X = I n s u l i n lOOnM, n u c l e a r = p r o t e i n s i s o l a t e d from the n u c l e a r f r a c t i o n , s o l u b l e = p r o t e i n s i s o l a t e d from the s o l u b l e f r a c t i o n . 61 FIG 21 - The effect of chelerythrine on cell viability assessed by trypan blue exclusion assay. Cardiomyocytes were exposed to various concentrations of chelerythrine for 6h and cell viability assessed by trypan blue.-. Graph represents the mean±SEM of 3 separate experiments. A 62 FIG 22 - The effect of chelerythrine on DNA fragmentation. Panel A - Enzyme-linked immunosorbent assay of DNA fragmentation for cardiomyocytes treated with various concentrations of chelerythrine for 6h. The data are the mean±SEM of 3 separate experiments. Panel B - Electrophoretic separation of DNA fragmented by chelerythrine treatment. Lane 1 - DNA ladder; Lane 2 - Control; Lane 3 - Chelerythrine 0.01\1M; Lane 4 - Chelerythrine 0. l[iM; Lane 5 - Chelerythrine l[iM; all performed at 6h. 63 Cardiomyocytes were exposed to chelerythrine and stained with either acridine orange (Fig 23-Panels A and B) or NBD p h a l l a c i d i n (Panels C and D) . Chelerythrine exposed cardiomyocytes exhibited usual apoptotic morphology (Panels A and B) and less a c t i n reorganization (Panels C and D) than staurosporine. 9 . Role of Ca + 2 i n chelerythrine-induced apoptosis The e f f e c t of Ca + 2 on chelerythrine-induced DNA fragmentation was assessed by ELISA (Fig 24). Cardiomyocytes were pre-treated with either EGTA or BAPTA before exposure to 1(0.M chelerythrine for 6h. Both EGTA and BAPTA blunted chelerythrine-induced DNA fragmentation 1 0 . Role of growth factor i n chelerythrine-induced apoptosis The e f f e c t of growth factors on chelerythrine-induced DNA fragmentation was assessed by ELISA (Fig 25 Panel A) or electrophoretic separation (Panel B). Cardiomyocytes treated with ljiM chelerythrine and either i n s u l i n (lOOnM) or EGF (50|lg/mL) for 6h. Both i n s u l i n and EGF s l i g h t l y blunted DNA fragmentation induced by chelerythrine. However, i n s u l i n decreased the i n t e n s i t y of DNA laddering, when compared to chelerythrine alone (Panel B). 64 FIG 23 - The c e l l morphology of representative cardiomyocytes treated with chelerythrine. Representative cardiomyocytes exposed to 1 {Panels A' & C) , 10 (Panel D) , or 100p:M (Panel B) chelerythrine for 6h. C e l l s were stained with either acridine orange (Panels A & B) or NBD p h a l l a c i d i n (Panels C & D). 65 irSJt Twf 5 , a g e n t S t h a t a l t e r [Ca+2]i o n chelerythrine-induced DNA fragmentation. Enzyme-linked immunosorbent assay of ^unf^lF111/ t l 0 n l n cardj-omyocytes pre-treated with either EGTA or BAPTA before exposure to chelerythrine for 6h. The bar aravh represents the mean+SEM of 3 separate experiments ' FIG 25 - Effect of the growth factors insulin or EGF on chelerythrine-induced. DNA fragmentation. Panel A - Effect of growth factors on chelerythrine-induced DNA fragmentation, as assessed by enzyme-linked immunosorbent assay of DNA fragmentation. Cardiomyocytes treated with 1\IM chelerythrine and either insulin (lOOnM) or EGF (50\Lg/mL) for 6h. The bar graph represents the mean±SEM of 3 separate experiments. Panel B - Electrophoretic separation of DNA fragments. Lane 1 - DNA ladder; Lane 2 - Control; Lane 3 - Chelerythrine luM + Insulin lOOnM; Lane 4 - Chelerythrine luM; all performed at 6h 67 CHAPTER IV DISCUSSION I) Topoisomerase i n h i b i t i o n Topo I i n h i b i t i o n had cytotoxic effects on cardiomyocytes as camptothecin, 10 (IM, increased c e l l death by 25% i n a 6 h time frame. Camptothecin produced c e l l shrinkage, membrane blebbing, and nuclear d i s i n t e g r a t i o n : a l l morphological changes c h a r a c t e r i s t i c of apoptosis. Furthermore, camptothecin induced DNA fragmentation producing small base p a i r fragments as demonstrated by DNA electrophoresis. Thus, camptothecin-induced cardiomyocyte death can be ascribed to apoptosis. Camptothecin produced apoptosis i n thymocytes, HL-60 and other c e l l l i n e s (Lee et a l 1'994) . In mouse thymocytes i n primary culture, camptothecin produced a dose-dependent internucleosomal DNA cleavage which preceded c e l l death (Onishi et a l 1993). C e l l s of the human promyelocytic HL-60 l i n e , when treated with camptothecin, exhibited DNA cleavage (Hotz et a l 1994) which correlated with electron microscopic changes i n c e l l structure t y p i c a l of apoptosis (Bertrand et a l 1993, Solary et a l 1993) . This study demonstrates the a b i l i t y of topo I i n h i b i t i o n with camptothecin to induce apoptosis i n cardiomyocytes. II) Involvement of i n t r a c e l l u l a r calcium i n camptothecin-induced apoptosis Because apoptosis i s an active process, I sought to investigate the ro l e of Ca + 2 i n apoptosis i n cardiomyocytes using d i f f e r e n t agents that have an impact on i n t r a c e l l u l a r calcium ([Ca 2 +]i). EGTA and taurine reduce [Ca2+] L l e v e l s (Bers 1982, Kaneko and Tsukamoto 1994) while thapsigargin increases [Ca2+] L (Furuya et 68 a l 1994) . EGTA chelates Ca + 2 i n the media (stoichiometrically, 1 mole of EGTA chelates 2 moles of Ca+2) , hence reducing Ca + 2 entry into the c e l l and [Ca* 2]^ A) EGTA and taurine I suggest a role for calcium i n camptothecin-induced apoptosis i n cardiomyocytes, based on the a b i l i t y of EGTA and taurine to blunt the development of apoptosis as manifested by less membrane blebbing and less DNA fragmentation i n EGTA treated c e l l s . Although EGTA produced a s l i g h t reduction i n camptothecin-induced cardiomyocyte death, we found a dramatic e f f e c t of EGTA and taurine to prevent membrane bleb formation. Indeed few cardiomyocytes treated with EGTA or taurine showed bleb formation. Thus, the eff e c t of EGTA or taurine was disproportionately greater for one of the morphologic features of apoptosis, namely bleb formation. This would support the findings i n rabbit renal tubular c e l l s that there i s a [Ca 2 +] i threshold for membrane bleb formation (Trump and Berezesky 1995). Taurine, a free amino acid i n plasma and many tissues such as heart, muscle, brain, and blood, af f e c t s calcium entry through L-type and T-type Ca + 2 channels i n embryonic chick heart c e l l s and decreases [Ca 2 +]i (Kaplan et a l 1993, Satoh and Sperelakis 1993). Taurine reduced DNA fragmentation and the morphologic changes of camptothecin-induced apoptosis i n cardiomyocytes. To my knowledge, there have been no previous studies on the e f f e c t of taurine on apoptosis. Our data suggest a ro l e for taurine i n the prevention of apoptosis i n cardiomyocytes. B) Thapsigargin 69 The proposal that [Ca 2 +]i plays a role i n camptothecin-induced apoptosis i s further strengthened by the r e s u l t s of my experiments with thapsigargin. In the present study, thapsigargin co-incubated with camptothecin consistently yielded a higher DNA fragmentation signal on the ELISA assay than either thapsigargin or camptothecin alone. These re s u l t s suggest that the accumulation of [Ca + 2] i ( by thapsigargin, plays a d e f i n i t e r o l e i n camptothecin-induced DNA fragmentation i n cardiomyocytes. Thapsigargin, a sesquiterpene gamma-lactone, s e l e c t i v e l y i n h i b i t s the sarcoplasmic reticulum and endoplasmic reticulum Ca + 2-dependent ATPase family of i n t r a c e l l u l a r "Ca+2-pumping" ATPases and produces a three to four f o l d elevation i n the level s of [Ca + 2] i (Furuya et a l 1994) . Thapsigargin induces programmed c e l l death i n androgen-independent p r o s t a t i c cancer c e l l s , which i s c r i t i c a l l y dependent upon an adequate, sustained elevation i n [Ca + 2] i (Furuya et a l 1994) . Part of the a b i l i t y of Bcl-2 to suppress apoptosis may be att r i b u t e d to i t s regulation of endoplasmic reticulum-associated Ca + 2 transport (Lam et a l 1994). Thapsigargin increased [Ca 2 +]i and subsequently induced morphologic features of apoptosis followed by internucleosomal DNA cleavage and c e l l death i n cultured human hepatoma c e l l s (Kaneko and Tsukamoto 1994), thymocytes and lymphoblastoid' c e l l l i n e s (Escargueil-Blaric et a l 1994) . In contrast, thapsigargin saved nerve growth factor-deprived rat sympathetic neurons from death (Lampe et a l 1992) . The role of calcium i n the development of apoptosis has been investigated i n other c e l l l i n e s and the re s u l t s are controversial. The re s u l t s of thi s study suggest that a decrease of [Ca + 2] i 7 by EGTA or taurine, prevents camptothecin-induced apoptosis whereas an 70 increase of [Ca+2]i induces apoptosis. My r e s u l t s are consistent with what was found i n topoisomerase i n h i b i t i o n i n HL-60 c e l l s . In HL-60 c e l l s , :."[Ca2+]i plays an essential role i n induction of apoptosis by VP-16, an epipodophyllotoxin derivative etoposide which i n h i b i t s DNA topo II. While the e x t r a c e l l u l a r Ca + 2 chelator EGTA could not block the VP-16 induced DNA fragmentation, the [Ca + 2 ] i chelator BAPTA abolished both internucleosomal DNA fragmentation and the morphologic features of apoptosis (Yoshida et a l 1993). Results from th i s study are also consistent with chick embryo spinal cord motoneuron c e l l s . In t h i s case, calcium loading with the ionophore A23187 stimulated and accelerated the p h y s i o l o g i c a l d e g e n e r a t i o n of motoneurons which w i l l undergo apoptosis (Ciutat et a l 1995). There are c o n f l i c t i n g data on the exact r o l e of Ca + 2 i n the development of apoptosis because apoptosis i s not always preceded by a r i s e i n the l e v e l of [Ca + 2 ] i and calcium chelators may produce as well as prevent apoptosis i n cultured c e l l s . For example, i n thymocytes, some studies noted that calcium chelators had no detectable e f f e c t on camptothecin-induced apoptosis (Hotz et a l 1994). In addition, i t has been suggested that [Ca + 2 ] i does not correlate with apoptosis i n thymocytes (Beaver and Waring 1994). In human leukemic c e l l s , apoptosis i s not s e n s i t i v e to Ca + 2 levels (Fernandes and^ Cotter 1993) . Hence, i t has been proposed that loss of calcium homeostasis, rather than a sustained r i s e i n [Ca + 2] i ( i s a determining factor i n c e l l death by apoptosis (Kluck et a l 1994). However, because [Ca + 2] ± i s a major second-messenger molecule, increased [Ca^li may also promote su r v i v a l by stimulating a signal transduction pathway, possibly the same one stimulated by trophic factors (Franklin and Johnson 1994) . My date., suggest a ro l e for calcium i n the apoptosis produced by topoisomerase I i n h i b i t i o n i n cardiomyocytes as agents that reduce [Ca 2 +] i decrease apoptosis, while those that increase [Ca 2 + ] i accentuate camptothecin-induced apoptosis. I l l ) Serine /threonine protein kinase i n h i b i t i o n with staurosporine Staurosporine exposure induced c e l l death and DNA fragmentation, as evidenced by an increase i n c e l l death by 41%, altered c e l l u l a r morphology, and extensive DNA fragmentation (three times that of normal c e l l s ) . Staurosporine-induced apoptosis was blunted by BAPTA and s l i g h t l y reduced by PMA, i n s u l i n and EGF. These re s u l t s suggest that serine/threonine .k'i.n a s e i n h i b i t i o n can cause apoptosis and that t h i s mechanism involves Ca + 2, PKC, and communication with signal transduction pathways stimulated by trophic factors. A) Staurosporine induces DNA fragmentation and apoptosis The data presented confirm the observations reported i n other c e l l models that staurosporine increases c e l l death and y i e l d s extensive DNA fragmentation. Staurosporine has produced apoptosis i n c e l l l i n e s from d i f f e r e n t origins, such as HL-60 and lymphoma c e l l l i n e s (Bertrand et a l 1994). Staurosporine induces apoptosis i n human malignant glioma c e l l l i n e s (Couldwell et a l 1994) and i n lymphoma and mammary carcinoma c e l l l i n e s (Shi et a l 1994) . In lymphocytes, PKC i n h i b i t i o n by staurosporine and polymyxin B induced apoptosis i n a manner which was both time and dose dependent (Lucas et a l 1994). Staurosporine induced a dose-dependent increase i n DNA fragmentation i n various c e l l l i n e s , including Burkitt lymphoma, lung f i b r o b l a s t , and small c e l l lung 72 carcinoma c e l l l i n e s (Bertrand et a l 1994). In addition, staurosporine arrested Burkitt lymphoma CA46 c e l l l i n e at G1/G2 of the c e l l cycle.(Abe et a l 1991, Crissman et a l 1991, Bruno et a l 1992) . B) Staurosporine-induced changes i n cardiomyocyte morphology Staurosporine caused cardiomyocytes to lose volume i n a time and dose dependent manner. The most dramatic change was that the cytoplasm appears to be condensing into dense vacuoles, yet the c e l l membrane appeared to remain i n t a c t . The nucleus was r e l a t i v e l y homogenized, but no d e f i n i t e membrane blebbing was observed. Because NBD p h a l l a c i d i n s p e c i f i c a l l y stains actin, the use of t h i s agent suggests that staurosporine causes cytoskeletal remodelling with dismantling of a c t i n f i b r e s . These morphologic changes were induced by staurosporine but not by the s p e c i f i c PKC i n h i b i t o r chelerythrine. The shapes and e l a s t i c properties of c e l l s are dictated by a cytoplasmic filamentous network composed la r g e l y of a c t i n (Cortese et a l 1989). Agents which 'disrupt microfilaments prevent the appearance of apoptotic bodies without a f f e c t i n g DNA fragmentation (Cotter et a l 1992). Inactivation of PKC i s known to decrease the rate of a c t i n polymerization, while activators of PKC, l i k e PMA, increase this rate (Phatak et a l 1988). Staurosporine i n h i b i t s the formation of apoptotic bodies (Cotter et a l 1992), supporting the theory that actin-containing filaments play a central role i n apoptotic process (Cotter et a l 1992). The unique morphology seen with staurosporine maybe c l o s e l y linked to i t s action on the actin-containing filamentous network. Although staurosporine exposure to cardiomyocytes did not display the c l a s s i c morphologic picture of apoptosis, one cannot conclude that staurosporine did not induce apoptosis. It i s possible that staurosporine i s unique to other apoptotic-inducing agents because staurosporine induces c y t o t o x i c i t y and DNA fragmentation, as well as a f f e c t i n g the a c t i n d i r e c t l y . Our experiments show that staurosporine produced DNA fragmentation s i m i l a r to camptothecin. As a res u l t , morphology induced by staurosporine i s unique i n cardiomyocytes compared to other apoptotic-inducing agents. In human lymphocytic leukemia MOLT-4 c e l l s , staurosporine exposure induced u l t r a s t r u c t u r a l changes, some t y p i c a l of apoptotic c e l l death and some not ( F a l c i e r i et a l 1993) . Staurosporine exposure caused the formation of numerous homogeneously electron dense micronuclei, i n addition to c l a s s i c a l signs of apoptosis ( F a l c i e r i et a l 1993). In HL-60 and Burkitt lymphoma c e l l s , staurosporine exposure caused the chromatin to condense at the periphery of the nuclei and form dense micronuclear bodies. The c e l l volume was reduced and the plasma membrane remained well defined. Some organelles such as mitochondria remained int a c t during the early stages, while others including the endoplasmic reticulum and golg i apparatus appeared d i l a t e d (Bertrand et a l 1994). In mixed mouse c o r t i c a l cultures containing both neurons and g l i a , the neurons exposed to staurosporine d i d not undergo the c e l l body swelling t y p i c a l l y induced by excitatory toxins, but rather gradual c e l l body shrinkage accompanied by chromatin condensation (Koh et a l 1995). Hence, our results support the contention that staurosporine produces apoptosis and a morphology that i s unique because of staurosporine's d i r e c t e f f e c t on a c t i n . 74 A c t i n has been implicated i n apoptotic development i n other c e l l systems. Microfilaments play an important r o l e i n the formation of apoptotic bodies. The expression patterns of S-actin declines following induction of apoptosis by actinomycin D i n HL-60 c e l l s (Naora and Naora 1995) . Similarly, i n the intersegmental muscles of the tobacco hawkmoth Manduca sexta, expression of a c t i n mRNA was greatly decreased when the c e l l s were committed to die (Schwartz et a l 1993). It i s possible that the reduction of a c t i n plays a ro l e i n the rapid d i s s o l u t i o n of c e l l s during apoptosis. The morphology induced by staurosporine showed a d i s s o l u t i o n of the cardiomyocyte cytoskeleton, confirming the theory that a c t i n reduction i s a part of apoptotic development and the formation of apoptotic bodies. C. The ro l e of Ca + 2 i n staurosporine-induced apoptosis Our study observed that a decrease i n [Ca + 2] i blunted staurosporine-induced c e l l death, DNA fragmentation, and morphology. The ro l e of Ca + 2 i n apoptosis has already been discussed i n a previous section. Agents that reduce [Ca 2 +] i decrease apoptosis, while those that increase [Ca 2 + ] i accentuate camptothecin-induced apoptosis. In a group I Burkitt lymphoma c e l l l i n e , EGTA p a r t i a l l y reduced apoptosis induced by anti-Ig or by Ca + 2 ionophore (Knox et a l 1992) . Hence, the blunting action of calcium chelators on staurosporine-induced apoptosis supports this d issertation's hypothesis that Ca + 2 i s an important regulator of apoptosis, despite the apoptotic-inducing agent. D. The ro l e of the phorbol ester PMA on staurosporine-induced apoptosis PMA blunted staurosporine-induced c y t o t o x i c i t y as demonstrated 75 by the trypan blue exclusion assay and blunted apoptotic morphology, but had l i t t l e e f f e c t on DNA fragmentation. These res u l t s w i l l be discussed separately. 1) Inhibitory action of PMA on staurosporine-induced c y t o t o x i c i t y and morphology The blunting e f f e c t of PMA on staurosporine-induced apoptosis, which I observed i n cardiomyocytes, has been reported i n other c e l l systems. Tumour-promoting phorbol esters blunted or i n h i b i t e d (time-dependent) spontaneous DNA fragmentation and c e l l death i n chronic lymphocytic leukemia c e l l s (McConkey et a l 1991a). In some Burkitt's lymphoma c e l l l i n e s , phorbol esters prevented apoptosis (Bonnefoy-Berard et a l 1994). PMA suppressed apoptosis mediated by dexamethasone or IL-2 withdrawal i n murine T c e l l s exhibiting intermediate a f f i n i t y IL-2 receptors (Gomez et a l 1994). In lymphocytes, PKC i n h i b i t i o n by staurosporine and polymyxin B each induced apoptosis i n both time and dose dependent manner, and was counteracted by PMA (Lucas et a l 1994). Similar r e s u l t s were seen i n f r e s h l y i s o l a t e d rat hepatocytes (Sanchez et a l 1992). These data suggest a r o l e for PKC i n c e l l s u r v i v a l during apoptosis. 2) E f f e c t of PMA on staurosporine-induced DNA fragmentation The r e s u l t s of t h i s study suggest that by a c t i v a t i n g PKC by PMA, staurosporine-induced c e l l death and morphology was i n h i b i t e d and that t h i s i n h i b i t i o n was time-dependent i n cardiomyocytes. However, PMA had l i t t l e or no e f f e c t on staurosporine-induced DNA fragmentation. It should be noted that the concentrations and exposure times of PMA used i n t h i s study had been previously determined (in our lab) to stimulate PKC a c t i v i t y and not down-regulate PKC a c t i v i t y i n chick embryonic cardiomyocytes.' 76 Because PMA can i n h i b i t staurosporine-induced c e l l death and morphology yet have no e f f e c t on DNA fragmentation, the res u l t s of this study suggest that either PKC acts i n a dual role during apoptotic development or PKC ac t i v a t i o n by PMA cannot reverse apoptosis past the DNA fragmentation stage. I speculate that the cardiomyocyte, responding to extensive c e l l damage and death, induces the PKC signal transduction pathways to block or delay the onset of damage or death caused by staurosporine exposure. However, i n the absence of c e l l u l a r injury, an activated PKC can also induce apoptosis, as evidenced by our DNA fragmentation (ELISA) data. Hence, PKC i s speculated to have a dual role i n apoptosis. a) The dual role of PKC i n apoptosis The c o n f l i c t i n g PKC results reported i n thi s study as well as in other c e l l l i n e s may be attr i b u t e d to a dual role for PKC i n apoptosis. This dual role may be attr i b u t e d to changes i n PKC subspecies mRNA expression during the process of apoptosis. I speculate that during i n i t i a l apoptotic induction, a s p e c i f i c PKC isoform i s predominant i n i t functions to block the development of apoptosis. However, as apoptosis i s an active mechanism requiring de novo protein synthesis, the gene for another PKC isoform may be activated during the course of apoptotic development. The re s u l t would be a s h i f t of PKC isoform dominancy and hence, a s h i f t i n the rol e of PKC (Lin et a l 1995). The newly dominant PKC isoform could t h e o r e t i c a l l y be i n e f f e c t i v e i n the la t e apoptotic stage of DNA fragmentation. This hypothesis might explain why PMA appeared to be i n e f f e c t i v e i n staurosporine-induced DNA fragmentation as seen i n t h i s study.'1 • s 77 The search for the p o t e n t i a l dominant PKC isoforms has only recently begun. Preliminary studies i n immature thymocytes suggested that glucocorticoid-induced apoptosis s e l e c t i v e l y induced an increase i n Ca + 2-independent PKC a c t i v i t y . PKCe translocated from the c y t o s o l i c f r a c t i o n to the p a r t i c u l a t e f r a c t i o n upon glu c o c o r t i c o i d treatment, suggesting the s e l e c t i v e a c t i v a t i o n of PKCe through de novo synthesis of macromolecules (Iwata et a l 1994). PMA's i n a b i l i t y to reverse DNA fragmentation observed i n th i s study may be- attributed to the d i f f e r e n t roles of the d i f f e r e n t PKC isoforms during apoptotic development. Another explanation for why PMA does not i n h i b i t staurosporine-induced DNA fragmentation i s that long exposures to phorbol esters down regulation PKC (Wu et a l 1992) . Down-regulation of PKC or i t s a c t i v i t y decreases phosphorylation of one or more substrates for PKC which are e s s e n t i a l for preventing apoptosis. In a T - c e l l hybridoma c e l l l i n e , PKCa down-regulation by phorbol ester treatment abolished activation-induced c e l l death (Young et a l 1987). The degree of PKC down-regulation, caused by increased degradation of PKC (Young et a l 1987), correlated well with the degree of c e l l death abolishment, suggesting that PKC a c t i v a t i o n represents an essential step i n the molecular mechanisms underlying c e l l death (Jin et a l 1992). IV) PKC i n h i b i t i o n and apoptosis The PKC i n h i b i t o r chelerythrine produced apoptosis. Chelerythrine i n h i b i t s PKC i n the regulatory domain, thus making chelerythrine i n h i b i t i o n s p e c i f i c for PKC, as opposed to staurosporine which i n h i b i t s at the c a t a l y t i c domain making i t a general kinase i n h i b i t o r . Our results suggest that an active PKC, 78 produced by PMA, i s involved i n c e l l s u r v i v a l . PMA (hence, activated PKC) blunting staurosporine-induced apoptosis supports th i s hypothesis. Induction of apoptosis by PKC i n h i b i t i o n with chelerythrine also supports the hypothesis that an active PKC i s involved i n the prevention of apoptosis. A. E f f e c t of PKC i n h i b i t i o n on apoptosis The data confirm the hypothesis, from other c e l l models, that i n h i b i t i o n of PKC induces apoptosis. Chelerythrine triggered apoptosis i n the Group I Burkitt's lymphoma c e l l l i n e BL60 and B104 lymphoma c e l l l i n e (Bonnefoy-Berard et a l 1994) . In non-transformed rat coronary vascular smooth muscle c e l l s , treatment with calphostin C, a PKC i n h i b i t o r , induced usual apoptotic morphology as well as a decline i n bcl-2 expression, but not c l a s s i c a l apoptotic DNA degradation into nucleosomal fragments (Leszczynski et a l 1994). Chelerythrine produced concentration-dependent increases i n DNA fragmentation and t y p i c a l apoptotic morphology i n HL-60 c e l l s (Jarvis et a l 1994). Conversely, PKC i n h i b i t i o n has also been shown to prevent apoptosis. In mouse thymocytes, PKC i n h i b i t i o n , by the i n h i b i t o r l - ( 5 -i s o q u i n o l i n y l s u l f o n y l ) - 2 - m e t h y l p i p e r a s i n e . d i h y d r o c h l o r i d e , prevented apoptosis and PMA potentiated radiation-induced apoptosis. However, the PKC activators i n i t i a t e d apoptosis i n mouse but not i n rat thymocytes suggesting that the role of PKC varies among c e l l models (Shaposhnikova et a l 1994) . B. PKC a c t i v a t i o n i n apoptosis The role of PKC i n cardiomyocytes can'be- interpreted i n two ways: a) the addition of apoptotic inducers stimulates the PKC pathway as a step for c e l l s u r v i v a l ; or b) the c e l l , i n order to survive, requires a l e v e l of PKC a c t i v a t i o n to f u l f i l the c e l l rescue process. The f i r s t a l t e r n a t i v e implies that apoptotic agents d i r e c t l y activate PKC or at least induce hydrolysis of PIP 2 to produce IP 3 and DAG which would then activate PKC; the activated PKC i n turn would then activate the s u r v i v a l pathway needed. The second al t e r n a t i v e implies that c e l l s u r v i v a l requires an independent process of c e l l stimulation at least to the point where PKC i s activated to s t a r t the program leading to c e l l s u r v i v a l . This l a s t p o s s i b i l i t y appears e s p e c i a l l y a t t r a c t i v e i n r e l a t i o n to the postulated role of trophic/mitogenic factors rescuing the c e l l from apoptosis. C. Sustained a c t i v i t y of PKC during apoptosis The sustained PKC a c t i v i t y necessary to blunt or abolish an apoptotic induction signal must be put into context. Sustained PKC a c t i v i t y i n a c e l l , minutes to hours a f t e r signal i n i t i a t i o n , i s at t r i b u t e d to the l e v e l of DAG which increases with a r e l a t i v e l y slow onset (Nishizuka 1992). A second wave of DAG appears a f t e r the f i r s t wave produced during PIP 2 hydrolysis disappears (Nishizuka 1995). Hence, active PKC can counteract apoptotic induction by i t s sustained a c t i v i t y due to waves of DAG a c t i v a t i o n of PKC. D. PKC i n h i b i t o r e f f i c a c y : a comparison between staurosporine and chelerythrine In the present study, the general kinase i n h i b i t o r staurosporine and the s p e c i f i c PKC i n h i b i t o r chelerythrine were compared. Staurosporine was more potent than chelerythrine since chelerythrine required a greater concentration than staurosporine to y i e l d i t s maximal apoptotic e f f e c t . It i s possible that 80 staurosporine)' and chelerythrine are not equipotent i n cardiomyocytes. However, these r e s u l t s suggest that staurosporine mediates i t s apoptotic e f f e c t s by i n h i b i t i n g other protein kinases i n addition to i n h i b i t i n g PKC. Furthermore, chelerythrine-induced apoptosis was s i m i l a r i n magnitude to staurosporine's re s u l t s , suggesting that PKC i s active i n apoptosis. 1. Variable staurosporine potencies Staurosporine's potency for apoptotic induction i s not seen consistently i n other c e l l models. E f f e c t i v e concentrations and times of exposure of staurosporine vary depending on the c e l l model and, as a re s u l t , staurosporine-induction of apoptosis can vary. In HL-60 c e l l s , staurosporine concentrations known to achieve maximal i n h i b i t i o n of PKC f a i l e d to induce DNA fragmentation (Jarvis et a l 1994). However, staurosporine promoted fragmentation at considerably higher concentrations (>4x K± where K^OWnM for PKC) (Jarvis et a l 1994). A long (24h) exposure of staurosporine to human glioma c e l l l i n e s f a i l e d to induce DNA fragmentation at concentrations generally used to achieve maximal i n h i b i t i o n of enzyme a c t i v i t y ; however, higher concentrations of staurosporine promoted DNA fragmentation (Ikemoto et a l 1995). 2) C a t a l y t i c vs Regulatory Domain i n PKC Considering that staurosporine i s more potent than chelerythrine, yet less potent than calphostin C, i n producing apoptosis (Jarvis et a l 1994), one must consider the differences i n these i n h i b i t o r s of PKC. As mentioned, calphostin C i n h i b i t s PKC at the regulatory domain whereas staurosporine i n h i b i t s PKC at the c a t a l y t i c domain. It i s tempting to speculate that apoptosis induced by PKC i n h i b i t i o n i s determined more dominantly at the 81 regulatory domain than the c a t a l y t i c . V) C e l l s u r v i v a l program during the development of apoptosis Ca + 2 chelators, PMA, and trophic factors such as i n s u l i n and EGF blunted apoptosis induced by kinase i n h i b i t i o n (staurosporine and chelerythrine) i n cardiomyocytes. In addition, de novo protein synthesis occurred during staurosporine exposure with and without growth factor,, rescue, fueling speculation qn the existence of a death/survival protein. This phenomenon has been reported i n other c e l l systems. Evidence that growth factor-induced c e l l s u r v ival can occur independently of p r o l i f e r a t i v e signals (Hamilton et a l 1990, Rodriguez-Tarduchy et a l 1990) supports the view that c e l l s u r v i v a l may be separately controlled by growth factors. The theory of a "second window of protection" has been postulated i n r e l a t i o n to myocardial ischemic injury. B r i e f l y , a f t e r the myocardium receives i t s i n i t i a l sublethal stress (eg. ischemia), the biochemistry of the cardiomyocyte adapts by increased synthesis of cytoprotective proteins (eg. stress proteins and/or endogenous anti-oxidants). By the time the second stress signal arrives, the myocardium i s either protected, or has at least enhanced i t s tolerance. This preconditioning can sometimes abolish or delay the res u l t s of the second injury, depending on the time course (Yellon and Baxter 1995). It i s therefore reasonable to speculate that t h i s second window of protection can be rel a t e d to apoptotic induction. Based on the PMA blunting actions on staurosporine-induced c e l l death, PKC may be involved i n a sur v i v a l program. PKC may be one of the cytoprotective kinases involved i n the second window theory. The a c t i v a t i o n of PKC isotypes, mediated by d i f f e r e n t 82 e f f e c t o r mechanisms, may function to counteract apoptotic induction. Hence, when PKC i s i n h i b i t e d by an i n h i b i t o r such as staurosporine or chelerythrine, apoptosis r e s u l t s because there i s no functioning s u r v i v a l program. The second window of protection has been reported to be i n h i b i t e d by chelerythrine given concurrently with the preconditioning i n s u l t (Baxter and Yellon 1994) supporting the theory that PKC i s involved i n c e l l protection from apoptosis. The r o l e of IGF-1 as a sur v i v a l factor has been demonstrated previously i n c e l l s derived from the nervous system where i t i s required for maintenance of cultured c e l l s and protection from d i r e c t injury (D'Mello et a l 1993). IGF i n h i b i t e d apoptosis of several IL-3 dependent c e l l l i n e s when IL-3 was removed (Rodriguez-Tarduchy et a l 1992). The addition of i n s u l i n - l i k e growth factor-I markedly i n h i b i t e d etoposide-induced apoptosis i n BALB/c3T3 c e l l s . IGF-I was not mitogenic i n the presence of etoposide. In addition, IGF-I had no e f f e c t on etoposide-induced apoptosis that had a targeted disruption of the IGF-I receptor gene (S e l l et a l 1995). These re s u l t s demonstrate an important r o l e for the IGF-I receptor as an i n h i b i t o r of apoptosis, independent of i t s mitogenic actions (Sel l et a l 1995) . IGF-I i s r e l a t i v e l y weak i n preventing apoptosis i n c e l l s with low levels of i n s u l i n receptors. IGF-I may function as a surv i v a l : f a c t o r i n response to diverse agents suggesting that i t blocks a common late i n t r a c e l l u l a r apoptosis pathway. Similar r e s u l t s with EGF were seen i n human breast cancer c e l l s which lack estrogen receptors. In thi s s i t u a t i o n , the treatment of these breast cancer c e l l s with EGF led to the i n h i b i t i o n of c e l l p r o l i f e r a t i o n , DNA fragmentation, and the 83 development of apoptotic morphology. These re s u l t s suggest that EGF can blunt apoptosis (Armstrong et a l 1994). Other growth factors may also protect against apoptosis, depending on the c e l l u l a r model. Basic f i b r o b l a s t growth factor (bFGF) was found to protect bovine a o r t i c endothelial c e l l s against radiation-induced apoptosis (Haimovitz-Friedman et a l 1991). Translocation, hence activation, of PKCOC from the cytoplasm to the membrane was observed immediately a f t e r bFGF addition, suggesting a role for activated PKC. Similarly, the phorbol ester TPA mimicked the radioprotective e f f e c t of bFGF. PKC i n h i b i t i o n counteracted the radioprotective e f f e c t of bFGF, as did the depletion of PKC by long exposure to high doses of TPA. These res u l t s suggest that active PKC prevents radiation-induced apoptosis and mimics the rescuing mechanism seen during bFGF addition (Haimovitz-Friedman et a l 1994). VI) de novo protein synthesis New protein synthesis was found during exposure to staurosporine. This data supports the observation that de novo protein synthesis must occur during the active program of apoptosis. The presence of newly synthesized proteins supports the theory of death/cytoprotective proteins. De novo synthesis of a PKC isoform could"<"'be stimulated by the induction of apoptosis, consistent with my speculation that PKC i s i n t e g r a l to the mediation of apoptosis. Based on the molecular weight of newly synthesized protein during 3 5S incorporation, I speculate that PKC-related.Jcinase protein i s synthesized during apoptotic induction due to i t s size (120 kDa) (Palmer and Parker 1995) . I speculate 8 4 that t h i s PKC 'related protein i s responsible for the i n h i b i t i o n of apoptosis seen during PMA, i n s u l i n , and EGF treatment a f t e r staurosporine exposure. I also speculate that higher molecular weight proteins seen during de novo protein synthesis may be EGF receptor (180kDa) or the insulin-receptor substrate (160kDa)(Kawase T et a l 1995). New synthesis of these growth factor receptors would support the theory of growth factor receptor occupancy mediating the cytoprotective pathway induced by i n s u l i n and EGF. Further experiments must be performed to elucidate the i d e n t i t y of these newly synthesized proteins. VII) Why "noriaal" c e l l s remain during apoptosis No matter what the c e l l l i n e or inducing agent, 100% c e l l death due to apoptosis i s never seen. Resistance to apoptosis was observed i n other c e l l l i n e s (McConkey et a l 1991a, Ido et a l 1987, Cohen and Duke 1984) as well as i n our chick embryo cardiomyocytes. These c e l l s may lack endonuclease, have al t e r a t i o n s i n PKC, lack an a u x i l i a r y protein required for a c t i v a t i o n of the endonuclease, or have enhanced e f f l u x of drugs, as i n multidrug resistance. This theory may account for the f a i l u r e of an absolute apoptotic response (100% c e l l s dead). VIII) Protein kinase signal transduction pathway i n determining the form of c e l l death To reconsider the previous question of why one c e l l l i n e has d i f f e r e n t forms of c e l l death, we must ponder the mechanism that must be responsible for choosing which death process to activate. In l i g h t of our data on signal transduction pathways i n apoptosis, we speculate that an altered protein phosphorylation may trigger d i s t i n c t pathways leading to d i f f e r e n t types of death i n one c e l l 85 l i n e . In the promyelocytic leukemia (IPC-81) c e l l l i n e , d i f f e r e n t c e l l death programs were i n i t i a t e d based on the target protein and degree of phosphorylation of cAMP dependent kinases (Gjertsen et a l 1994). In cardiomyocytes, PKC may be a kinase that i s responsible for the decision of which c e l l u l a r death program to implement i n response to a c e l l u l a r i n s u l t . IX) Why not investigate PKC i n camptothecin-induced apoptosis? The roles of protein kinase pathways i n camptothecin-induced apoptosis were not performed i n this study. The rationale for t h i s decision was based on re s u l t s of Bertrand R (et a l 1993) The authors reported that i n HL-60 c e l l s , apoptosis was induced by camptothecin but kinase activators (TPA) and i n h i b i t o r s (staurosporine) had no e f f e c t on camptothecin-induced apoptosis. However, most of the i n t r a c e l l u l a r s i g n a l l i n g modulators were able to induce DNA fragmentation i n HL-60 c e l l s by themselves, suggesting that even though modulation of s i g n a l l i n g pathways was unable to prevent camptothecin-induced apoptosis, t h e i r deregulation could induce apoptosis i n HL-60 c e l l s . Hence, we decided to investigate kinase and topoisomerase pathways i n d i v i d u a l l y and to correlate any conferring data. Given that camptothecin i s known to induce apoptosis, I was able to determine the c e l l morphology of apoptosis and compare i t to other agent-induced morphologies (staurosporine, chelerythrine). X) Limitations of the study The study was conducted i n embryonic chick cardiomyocytes i n culture, so the type of cardiomyocytes and t h e i r conditions l i m i t the implications of the study. The embryonic nature of the c e l l allows us to only extrapolate what might occur i n adult tissue. 8 6 These c e l l s iri culture allow apoptosis to be r e a d i l y and c l e a r l y demonstrated perhaps more than detection of DNA fragments in s i t u using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP nick end l a b e l l i n g (TUNEL) assay. In some organs, TUNEL f a i l e d to discriminate between apoptosis, necrosis and a u t o l y t i c c e l l death (Grasl-Kraupp et a l 1995). Another strength of the cardiomyocytes used i n the present study i s the s i m i l a r i t y of the chick and human bcl-2 gene, which protects from apoptosis (Eguchi et a l 1992, Boise et a l 1995). Furthermore, chick heart has a considerable amount of bcl-2 (Eguchi et a l 1992). 87 CHAPTER V SUMMARY AND CONCLUSIONS Topo I i n h i b i t i o n by c a m p t o t h e c i n p r o d u c e d a p o p t o s i s i n c a r d i o m y o c y t e s , e v i d e n c e d by an i n c r e a s e i n c e l l d e a t h , a p o p t o t i c c e l l morphology, DNA l a d d e r i n g , and t h e q u a n t i t a t i v e measurement o f DNA f r a g m e n t a t i o n . Decreased [ C a + 2 ] ± by t a u r i n e and EGTA p r o t e c t e d c a r d i o m y o c y t e v i a b i l i t y and re d u c e d DNA f r a g m e n t a t i o n i n d u c e d by topo I. Membrane b l e b f o r m a t i o n was alm o s t c o m p l e t e l y p r e v e n t e d by EGTA o r t a u r i n e . A r o l e f o r [Ca.2+]t i n c a m p t o t h e c i n - i n d u c e d a p o p t o s i s was f u r t h e r s u p p o r t e d by t h e f i n d i n g t h a t t h a p s i g a r g i n , w h i c h i n c r e a s e s [ C a + 2 ] i , a c c e n t u a t e d t h e DNA f r a g m e n t a t i o n p r o d u c e d by c a m p t o t h e c i n . C a l c i u m may f u n c t i o n t o r e n d e r c h r o m a t i n more s u s c e p t i b l e t o t h e a c t i o n o f an endonuclease (Evans 1993). I s p e c u l a t e t h a t topo I i s an a c t i v e and i n t e g r a l enzyme i n the normal h e a r t and t h a t changes t o topo I a c t i v i t y a r e a d e t e r m i n a n t o f a p o p t o s i s i n c a r d i o m y o c y t e s . P r o t e i n k i n a s e i n h i b i t i o n by s t a u r o s p o r i n e p r o d u c e d a p o p t o s i s i n c a r d i o m y o c y t e s , e v i d e n c e d by an i n c r e a s e i n c e l l d e a t h , u n i q u e a p o p t o t i c c e l l morphology, DNA l a d d e r i n g , and t h e q u a n t i t a t i v e measurement o f DNA f r a g m e n t a t i o n . BAPTA p r o t e c t e d c a r d i o m y o c y t e s from c e l l d e a t h and reduced s t a u r o s p o r i n e - i n d u c e d DNA f r a g m e n t a t i o n ^ Membrane b l e b f o r m a t i o n was a l m o s t c o m p l e t e l y p r e v e n t e d by BAPTA a d d i t i o n s u g g e s t i n g a r o l e f o r [ C a 2 + ] i i n s t a u r o s p o r i n e - i n d u c e d a p o p t o s i s . S t a u r o s p o r i n e - i n d u c e d a p o p t o s i s i n c a r d i o m y o c y t e s was b l u n t e d b u t n o t a b o l i s h e d by t h e a c t i v a t i o n o f PKC v i a t h e p h o r b o l e s t e r PMA. PMA a d d i t i o n b l u n t e d s t a u r o s p o r i n e - i n d u c e d a p o p t o t i c morphology, b u t not DNA f r a g m e n t a t i o n . T h i s s u g g e s t s t h a t PKC i s 88 involved i n the development of apoptosis. Staurosporine-induced unique morphologic changes i n cardiomyocytes. To my knowledge there are no s i m i l a r morphologic changes produced by agents that produce c e l l death of cardiomyocytes. The e f f e c t of staurosporine may be a t t r i b u t a b l e to i t s d i r e c t e f f e c t on a c t i n . In contrast, chelerythrine yielded morphology s i m i l a r to usual changes of apoptosis. Because the morphology yielded by staurosporine and chelerythrine are d i s t i n c t , I speculate that staurosporine i s i n h i b i t i n g other kinases besides PKC to produce i t s unique morphology. The addition of the growth factors i n s u l i n and EGF blunted staurosporine-induced apoptotic e f f e c t s . In addition, de novo protein synthesis was observed during staurosporine exposure may be affected by i n s u l i n or EGF. These re s u l t s suggest that the pathways stimulated by agonists at the i n s u l i n or EGF receptor work against the development of apoptosis, implying a possible c e l l u l a r rescue program.. Two i n h i b i t o r s of PKC, chelerythrine and staurosporine produced cardiomyocyte death implicating PKC i n the development of apoptosis. As chelerythrine did not produce the same morphologic changes as staurosporine, PKC alone i s not responsible for a l l of staurosporine's e f f e c t on apoptotic induction. The data i n t h i s thesis demonstrate that apoptosis does indeed occur i n cardiomyocytes. Apoptosis was produced by the i n h i b i t i o n of topoisomerase I, serine/threonine kinases, and protein kinase C. The induction of apoptosis was reversed by interventions that decrease i n t r a c e l l u l a r calcium. 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