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Biochemical mechanisms of endothelial and smooth muscle cell apoptosis induced by photodynamic therapy Granville, David James 2001

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BIOCHEMICAL MECHANISMS OF ENDOTHELIAL AND SMOOTH M U S C L E C E L L APOPTOSIS INDUCED BY PIJOTODYNAMIC THERAPY by D A V I D J A M E S G R A N V I L L E B . S c , Simon Fraser University, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S Department of Pathology and Laboratory Medicine; Faculty of Medicine We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A March 1,2001 © David James Granville, 2001 UBC Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her re p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of P A T H O L O G Y A)\/E> L A B O R g T O R V n E ^ i o / f J f c The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date MARC 1-1 \C .2QO\ 1 of 1 12/20/00 10:36 A M Abstract Photodynamic therapy (PDT) is a clinically approved therapeutic modality for several types of cancer and age-related macular degeneration. P D T is under investigation for the treatment of cardiovascular conditions such as restenosis, transplant rejection and atherosclerosis. Although the vasculature is a major target of P D T in the treatment of these ailments, the cellular and biochemical effects of P D T on endothelial cells (EC) and smooth muscle cells ( S M C ) are largely unknown. Thus, my thesis work was focused on defining the biochemical processes underlying E C and S M C apoptosis in response to PDT. We hypothesized that P D T acts directly upon mitochondria to elicit rapid apoptotic cell death and involves the release of mitochondrial proteins and activation of caspases, and represents a balance of effects conveyed by members of the Bcl-2 family of proteins. P D T was shown to trigger mitochondrial release of cytochrome c and apoptosis, inducing factor (AIF), involving pro-apoptotic Bcl-2 homologues such as Bax and B i d , and activate caspases -2,-3, -6, -7, -8 and -9 during E C and S M C apoptosis. Furthermore, A I F was shown to migrate from mitochondria to the nucleus, whereupon it is believed to initiate chromatin condensation and early (Stage I) D N A fragmentation. The effect of P D T on intracellular C a regulation in normal and Bcl-2-overexpressing cells was examined. Overexpression of the anti-apoptotic Be l -2 proto-oncogene did not protect cells against PDT-mediated intracellular C a release. On the contrary, increased intracellular Ca release was observed in Bcl -2 overexpressing cells. Additional studies showed that Bcl-2 overexpression was associated with increased endoplasmic reticular and mitochondrial C a 2 + load. PDT-induced C a 2 + release was due to the release of C a 2 + from E R and mitochondrial stores and was not due to an influx of intracellular C a 2 + from bathing culture media. Furthermore, extrusion of C a 2 + via plasma membrane C a 2 + ATPases was not hindered, while ER-mediated sequestration of Ca was impaired. Impairment o f ER-mediated sequestration of C a 2 + was shown to be due to caspase-independent degradation of sarco/endoplasmic reticulum C a ATPases ( S E R C A ) . In summary, our results offer key insights into the anti-tumour effects of P D T as well as its effects on E C and S M C . P D T was shown to induce rapid changes to both mitochondria and endoplasmic reticuli. Results pertaining to biochemical mechanisms of E C and S M C may eventually lead to the discovery of novel therapeutic modalities for the treatment of common vascular disorders. T A B L E O F C O N T E N T S Abstract 1 List of Tables v i i List of Figures v i i i Abbreviations x List o f Publications, Abstracts and Presentations x i i Acknowledgements xv i i i C H A P T E R I B A C K G R O U N D .1 1.1 Apoptosis 1 1.1.1 Historical Perspective 1 1.1.2 Apoptosis versus Necrosis 3 1.1.2.1 Necrosis 3 1.1.2.2 Apoptosis 3 1.1.3 Regulation of Apoptosis 7 1.1.3.1 Receptor-mediated Apoptosis 7 1.1.3.2 Caspase Family 11 1.1.3.3 Bcl-2 Family 15 1.1.3.4 Nuclear Factor-KB 20 1.1.3.5 Calcium 21 1.2 Biological Significance 27 1.2.1 Cardiovascular 27 1.2.1.1 Transplant Vascular Disease ( T V D ) 27 1.2.1.2 Animal Models of T V D 28 1.2.1.3 Apoptosis in Human T V D 29 1.2.1.4 Cardiac Ischemia/Reperfusion Injury 30 1.2.2 Cancer 37 1.2.3 Development 40 1.2.4 Aging 41 1.2.5 Inj ury and Repair 44 1.2.6 Vi ra l Pathogenesis 45 1.3 Photodynamic Therapy (PDT) 49 1.3.1 Overview 49 1.3.2 Introduction 52 1.3.3 Mitochondrial Regulation of PDT-Induced Apoptosis 57 1.3.4 Role of Caspases in PDT-Induced Apoptosis 58 1.3.5 Role of Bcl-2-Related Proteins in the Regulation of Apoptosis 60 1.3.6 Effects of P D T on Intracellular Calcium Regulation 62 1.3.7 P D T , Cel l Signalling and Apoptosis 63 1.3.8 Transcriptional Regulators 64 iv 1.3.9 Conclusions 66 CHAPTER II RATIONALE, HYPOTHESIS, AND EXPERIMENTAL AIMS 67 CHAPTER III MATERIALS AND METHODS 70 3.1 Cell Culture 70 3.1.1 Transformed Cell Lines 70 3.1.1.1 HL-60 Cells 70 3.1.1.2 HeLa Cells 71 3.1.2 Primary Cells 72 3.1.2.1 Endothelial Cells 72 3.1.2.2 Smooth Muscle Cells 72 3.2 Photodynamic Therapy In vitro 73 3.2.1 Photosensitizer and Light Source 73 3.2.2 Induction of PDT 76 3.2.2.1 HL-60 Cells 76 3.2.2.2 HeLa Cells 76 3.2.2.3 HUVEC 77 3.2.2.4 HASMC 77 3.3 Cell Viability Assays 77 3.3.1 MTT Assay 77 3.3.2 Propidium Iodide Uptake 78 3.3.3 Trypan Blue Exclusion Assay 79 3.3.4 Cell Morphology 79 3.4 Protein Analysis 79 3.4.1 Preparation of Cell Extracts 79 3.4.1.1 Whole Cell Extracts 79 3.4.1.2 Cytosolic Extracts 80 3.4.2 Protein Quantification 81 3.4.3 Protein Analysis 81 3.4.3.1 Antibodies 81 3.4.3.2 SDS-Polyacrylamide Gel Electrophoresis 82 3.4.3.3 Western Immunoblotting 84 3.4.3.4 Fluorometric Quantitation of Caspase Activity 85 3.5 Flow Cytometry 86 3.5.1 DNA Fragmentation (PI staining) 86 3.5.2 7A6 Antigen Detection 87 3.6 Direct and Indirect Immunofluorescent Microscopy 87 3.6.1 Mitotracker Staining ., 87 3.6.2 Immunocytochemistry 88 3.6.3 Calcium Imaging 90 CHAPTER IV RESULTS 92 4.1 Aim I Effects of PDT on Transformed Cells 92 4.1.1 HL-60 Cells 92 4.1.1.1 Protease Activi ty and D N A Fragmentation 92 4.1.1.2 Bcl-2 Blocks Caspase-3 and Caspase-6 Processing 101 4.1.1.3 Bcl-2 Inhibits Caspase-3-like Protease Activi ty 101 4.1.1.4 Bcl-2 and B c l - X L Inhibit PDT-Induced D N A Fragmentation 105 4.1.1.5 Bcl-2 Provides Partial Protection Against PDT-Induced Ce l l Death 107 4.1.1.6 D F F : A Link Between B c l - X L , Caspase-3 and D N A Fragmentation I l l 4.1.2 H e L a Cells 116 4.1.2.1 Ce l l Shrinkage and Membrane Blebbing in PDT-Treated H e L a Cells 116 4.1.2.2 Caspases and Ce l l Morphology 116 4.1.2.3 P D T Induces Activation of Caspases-3, -6, -7, -8 and - 9 in H e L a Cells 120 4.1.2.4 Mitochondrial Cyt c Release Occurs Prior to Caspase Activation in PDT-Treated H e L a Cells 124 4.1.2.5 Role of Bcl-2 and B c l - x L in PDT-Induced HeLa Cel l Apoptosis 126 4.1.2.6 Intracellular Calcium Regulation 128 4.2 A i m II Effects of P D T on Primary Human Endothelial Cells 142 4.2.1 Cel l Shrinkage and Membrane Blebbing 142 4.2.2 Altered Cytosolic Levels of Bax and Cyt c 145 4.2.3 Activation of Caspases-2, -3, -6, -7, -8 and - 9 During P D T -Induced H U V E C Apoptosis 145 4.3 A i m III Effects of P D T on Primary Smooth Muscle Cells 151 4.3.1 PDT-Induced Apoptosis in H A S M C 151 4.3.2 Cellular Redistribution of Bax, Cyt c and A I F During H A S M C Apoptosis 158 4.3.3 Caspase-3, -6, -7, -8 and - 9 Activation During PDT-Induced H A S M C Apoptosis 159 C H A P T E R V DISCUSSION , 164 5.1 Mechanisms of PDT-Induced Tumour Cel l Apoptosis 165 5.2 Endothelial Cel l and Smooth Muscle Cel l Apoptosis 173 C H A P T E R VI CONCLUSIONS AND F U T U R E PROSPECTS 178 R E F E R E N C E S 185 VI List of Tables Table 1 A comparison of apoptosis versus necrosis 6 Table 2 Caspase substrates that are cleaved during PDT-induced apoptosis 60 v i i List of Figures Figure 1 Molecular pathways involved in FasL, T N F , and TRAIL-induced apoptosis 8 Figure 2 Pro-apoptotic signalling between E R and mitochondria 19 Figure 3 Roles of calcium during ischemia/reperfusion-induced apoptosis 22 Figure 4 Calcium and A k t in apoptosis 26 Figure 5 Causes of E C and S M C apoptosis in T V D 36 Figure 6 Role of defective apoptosis in tumorogenesis 38 Figure 7 Manipulation of apoptosis by Adenovirus 48 Figure 8 Understanding of PDT-induced apoptosis prior to May , 1997 50 Figure 9 Current knowledge of PDT-induced apoptosis 51 Figure 10 P D T procedure 54 Figure 11 Chemical structure of verteporfin 74 Figure 12 Absorption spectrum of verteporfin and emission spectrum of light box 75 Figure 13 Intracellular calcium imaging 91 Figure 14 Effects of different protease inhibitors on PDT-induced D N A fragmentation 93 Figure 15 Caspase-1 and caspase-3 activity in HL-60 cells after P D T 95 Figure 16 P D T induces processing of caspase-3 but not caspase-1 96 Figure 17 Inhibition of caspase-3 and P A R P cleavage by Z - A s p - D C B 97 Figure 18 Inhibition of caspase-3 and P A R P cleavage by serine protease inhibitors 98 Figure 19 Caspase-6 activation occurs downstream of caspase-3 activation 100 Figure 20 Bcl-2 prevents PDT-induced caspase-3 and - 6 processing 102 Figure 21 Bcl-2 inhibits caspase-3/7-like activity in HL-60 cells 103 Figure 22 B C 1 - X L inhibits caspase-3/7-like activity and D N A fragmentation 104 Figure 23 Bcl-2 inhibits PDT-induced D N A fragmentation in H L - 6 0 cells 106 Figure 24 Bcl-2 partially protects against cell death in H L - 6 0 ce l l s -MTT assay 108 Figure 25 Bcl-2 partially protects against cell death in H L - 6 0 cells-PI uptake 109 Figure 26 Bcl-2 partially protects against cell death in HL-60 cells-Trypan blue assay .... 110 Figure 27 Rapid caspase-3, D F F and P A R P cleavage in PDT-treated H L - 6 0 cells 113 Figure 28 D F F is cleaved by a caspase-3-like protease 114 Figure 29 B c l - X L inhibits PDT-induced D F F cleavage 115 Figure 30 Apoptotic morphological changes of HeLa cells in response to P D T 117 Figure 31 Caspase-3/7-like activity in HeLa cells after P D T 118 Figure 32 Inhibition of caspases prevents morphological changes associated with PDT-induced H e L a cell apoptosis 119 Figure 33 Activation of caspases-3, -6, -7 and -8 during H e L a cell apoptosis 121 Figure 34 Caspase-8 activation occurs downstream of caspase-3 during P D T -induced apoptosis 122 Figure 35 Cleavage of D F F , P A R P and Ras -GAP during H e L a cell apoptosis 123 Figure 36 Caspase-8 activation occurs downstream of cytochrome c release during PDT-induced apoptosis 125 v i i i Figure 37 Cytochrome c release is not inhibited by Bcl-2 during PDT-induced apoptosis. 127 Figure 38 Light dose-dependent intracellular calcium release in HeLa/neo and HeLa/Bcl-2 cells 129 Figure 39 Verteporfin dose-dependent intracellular calcium release in HeLa/neo and HeLa/Bcl-2 cells 131 Figure 40 Bcl -2 increases intracellular calcium load 132 Figure 41 Intracellular calcium release in HeLa/neo and HeLa/Bcl-2 cells depleted of E R calcium stores 134 Figure 42 Incomplete depletion of cell calcium stores in Bcl-2-transfected H e L a cells 135 Figure 43 C P A induces an immediate rise in intracellular calcium levels 136 Figure 44 S E R C A 2 degradation in HeLa/neo and HeLa/Bcl-2 cells 138 Figure 45 S E R C A 2 degradation is not mediated by caspases 140 Figure 46 Intracellular calcium levels do not influence cytochrome c release during PDT-induced apoptosis 141 Figure 47 Effects of P D T on endothelial cell morphology 143 Figure 48 Caspase-3 activation in apoptotic endothelial cells 144 Figure 49 Cellular redistribution of Bax and cytochrome c during endothelial cell Apoptosis 146 Figure 50 Activation of caspases-2, -3, -6, -7, -8 and - 9 during endothelial cell apoptosis 147 Figure 51 Increased caspase-3/7-, caspase-6- and caspase-9-like protease activity during endothelial cell apoptosis 148 Figure 52 Cleavage of cell cycle inhibitory proteins during endothelial cell apoptosis 150 Figure 53 Effects of P D T on smooth muscle cell morphology 152 Figure 54 P D T induced D N A fragmentation in smooth muscle cells 153 Figure 55 Effects of P D T on 7A6 antigen unmasking 154 Figure 56 Unmasking of the mitochondrial 7A6 antigen is not caspase-dependent .155 Figure 57 Caspase-3 activation and degradation of nuclear lamins during P D T -induced smooth muscle cell apoptosis 157 Figure 58 Cellular redistribution of Bax and cytochrome c during PDT-induced smooth muscle cell apoptosis 160 Figure 59 Cellular redistribution of A I F from mitochondria to nuclei during smooth muscle cell apoptosis 161 Figure 60 Activation o f caspases-3, -6, -7, -8, and - 9 during smooth muscle cell apoptosis 162 Figure 61 Z V A D - f m k inhibits morphological alterations associated with P D T -induced apoptosis 163 ix Abbreviations Abbreviation Definition A I F Apoptosis inducing factor Apaf-1 Apoptotic protease activating factor-1 Bax Bcl-2-associated protein-x B B E Bovine brain extract Bcl-2 B cell lymphoma-2 B c l - X B cell lymphoma protein-x bFGF Basic fibroblast growth factor BI-1 Bax inhibitor-1 C A D Caspase activated deoxyribonuclease C A R D Caspase recruitment domain Caspase-1 Cysteinyl aspartate-specific protease-1 (a.k.a. ICE) Caspase-2 Cysteinyl aspartate-specific protease-2 (a.k.a. ICH-1) Caspase-3 Cysteinyl aspartate-specific protease-3 (a.k.a. CPP32, Y A M A , Apopain) Caspase-4 Cysteinyl aspartate-specific protease (a.k.a. T X , ICH-2 , I C E R E L - H ) Caspase-5 Cysteinyl aspartate-specific protease (a.k.a. I C E R E L - I H , T Y ) Caspase-6 Cysteinyl aspartate-specific protease (a.k.a. Mch2) Caspase-7 Cysteinyl aspartate-specific protease (a.k.a. Mch3 , I C E - L A P 3 , C M H - 1 ) Caspase-8 Cysteinyl aspartate-specific protease (a.k.a. F L I C E , M A C H , Mch5) Caspase-9 Cysteinyl aspartate-specific protease (a.k.a. I C E - L A P 6 , Mch6) Caspase-10 Cysteinyl aspartate-specific protease (a.k.a. Mch4) C a M Calmodulin C D K 2 Cyc l in dependent kinase-2 C H U K Conserved helix-loop-helix ubiquitous kinase C P A Cyclopiazonic acid C P A N Caspase activated nuclease C R A D D Caspase and RIP adapter with death domain D C I Dichloroisocoumarin D D Death domain D E D Death effector domain D F F D N A fragmentation factor DISC Death inducing signaling complex D c R l Decoy receptor-1 (a.k.a. TRID) DcR2 Decoy receptor-2 (a.k.a. T R U N D D ) D M S O Dimethyl sulfoxide D R 4 Death receptor 4 (a.k.a. T R A I L - R 1 ) DR5 Death receptor 5 (a.k.a. T R A I L - R 2 ) E C Endothelial cells E N O S Endothelial nitric oxide synthase E R K 1 Extracellularly regulated kinase-1 F A D D Fas-associated death domain FAP-1 Fas-associated phosphatase-1 FasL Fas ligand FLICE FADD-like ice (a.k.a. caspase-8) FLIP Flice inhibitory protein (a.k.a. I-FLICE, Usurpin, CASH, FLAME, Casper, MRIT GraB Granzyme B HASMC Human aortic smooth muscle cells HUVEC Human umbilical venous endothelial cells IAP Inhibitor of apoptosis protein ICAD Inhibitor of CAD ICE Interleukin-ip converting enzyme (a.k.a. caspase-1) IKB Inhibitor kappaB IL Interleukin INK c-jun N-terminal kinase LDL Low density lipoprotein MAPK Mitogen activated protein kinase MHC Major histocompatibility complex NCX *j i Sodium Ca exchanger NHE i  proton exchanger NFAT Nuclear factor of activated T cells N F - K B Nuclear factor-kappaB NIK N F - K B inducing kinase NO Nitric oxide NOS2 Nitric oxide synthase-2 (a.k.a. iNOS) PARP Poly (ADP-ribose) polymerase PCD Programmed cell death (a.k.a. apoptosis which required de novo gene expression) PDGF Platelet derived growth factor PDT Photodynamic therapy PMCA Plasma membrane Ca 2 + ATPases PMSF Phenylmethylsulfonyl fluoride PS Phosphatidylserine RAIDD Receptor interacting protein ICH-1 /CED-3 homologous protein RIP Receptor interacting protein ROS Reactive oxygen species RyR Ryanodine receptor SAPK Stress activated protein kinase SERCA2 Sarcaplasmic/endoplasmic reticulum Ca 2 + ATPase-2 SMC Smooth muscle cells TGF Transforming growth factor TNF Tumour necrosis factor TRAF TNF receptor associated factor TRAIL TNF related apoptosis inducing ligand (a.k.a. Apo2L) TVD Transplant vascular disease List of Publications, Abstracts and Oral Presentations (a) Peer-reviewed publications 1. Hunt, D .W.C. , Jiang, H.J., Granville, D.J., King , D .E . and Levy, J .G. (1996) Monoclonal antibody LR-1 recognizes murine heat-stable antigen, a marker of antigen-presenting cells and developing hematopoietic cells. Int. Arch. Allergy Immunol. I l l :218-229. 2. Granville, D.J., Levy, J.G. and Hunt, D . W . C . (1997) Photodynamic therapy induces caspase-3 activation in HL-60 cells. Cell Death Differ. 4:623-629. 3. Granville, D.J., Jiang, H . , A n , M . T . , Levy, J .G. , McManus, B . M . and Hunt, D . W . C . (1998) Overexpression of B C 1 - X L prevents caspase-3-mediated activation of D N A fragmentation factor (DFF) produced by treatment with the photochemotherapeutic agent BYD-UA. FEBS Lett. 422:151-154. 4. Granville, D.J., Levy, J.G. and Hunt, D . W . C . (1998) Photodynamic therapy using B P D -MA,verteporfin induces rapid cell death by apoptosis and induces tyrosine phosphorylation of multiple proteins. Photochem. Photobiol. 67:358-362. 5. Granville, D.J., Carthy, C M . , Yang, D . C , Hunt, D . W . C . and McManus , B . M . (1998) Interactions of viral proteins with host cell death machinery. Cell Death Differ. 5:653-659. 6. Carthy C M . , Granville, D.J., Watson, K . A . , Anderson, D.R. , Wilson, J.E., Yang, D . C , Hunt, D . W . C , and McManus, B . M . (1998) Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in H e L a cells. J. Virol. 72:7669-7675. 7. Granville, D.J., Carthy, C M . , Hunt, D . W . C . and McManus, B . M . (1998) Apoptosis: molecular aspects of cell death and disease. Lab. Invest. 78:893-913. 8. Granville, D.J., Carthy, C M . , Jiang, H . , N g , F .W. , Shore, G . C , McManus , B . M . and Hunt, D.W.C.(1998) Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy. FEBS Lett. 437:5-10. 9. Kogl in , J. , Granville, D . J . , Glysing-Jensen, T., Mudgett, J.S., Carthy, C M . , McManus, B . M . and Russel, M . E . (1998) Attenuated acute cardiac rejection in NOS2- / - recipients correlates with reduced apoptosis. Circulation. 99:836-842. 10. Granville, D.J., A n , M . T . , Levy, J.G., McManus, B . M . and Hunt, D . W . C . (1999) Bcl-2 overexpression blocks caspase activation and downstream apoptotic events instigated by photodynamic therapy. Br. J. Cancer 79:95-100. 11. Hunt, D . W . C , Jiang, H . , Granville, D.J., Chan, A . H . , Leong, S. and Levy, J .G. (1999) Consequences of the photodynamic treatment of resting and activated peripheral T lymphocytes. Immunopharmacol. 41:31-44. 12. Jiang, H . , Granville, D.J., McManus, B . M . , Levy, J .G. , and Hunt, D . W . C . (1999) Selective depletion of a thymocyte sub-population in vitro with an immunomodulatory porphyrin photosensitizer. Clin. Immunol. Immunopathol. 91:178-187. 13. Carthy C M . , Granville, D.J., Jiang, H . , Thompson, C . B . , McManus , B . M . and Hunt, D . W . C . (1999) Rapid release of mitochondrial cytochrome c and Apo2.7 expression by a photochemotherapeutic agent: Bcl-2 and B c l - X L do not prevent early mitochondrial events but still exert cytoprotective activity. Lab. Invest. 79:953-965. x i i 14. McDonald, P . C , Wong D. , Granville, D.J., and McManus B . M . (1999) Emerging roles of endothelial cells and smooth muscle cells in transplant vascular disease. Transplant. Rev. 13: 109-127. (Invited review) 15. Granville, D.J., Shaw, J.R., Leong, S., Carthy, C . M . , Margaron, P., Hunt, D . W . and McManus, B . M . (1999) Release of cytochrome c, Bax migration, B i d cleavage and activation of multiple caspases during endothelial cell apoptosis. Am. J. Pathol. 155:1021-1025. 16. Dong, C. , Granville, D.J., Tuffnel, C .E . , Kenyon, J. , English, D . , Wilson, J.E. and McManus, B . M . (1999) Bax and apoptosis in acute and chronic rejection of rat cardiac allografts. Lab. Invest. 79:1643-1653. 17. Granville, D.J., Carthy, C M . , Jiang, H . , Levy, J.G., McManus, B . M . , Matroule, J .Y., Piette, J. and Hunt, D . W . C . (2000) N F - K B activation by the photochemotherapeutic agent verteporfin. Blood 95:256-262. 18. Granville, D.J. and Hunt, D . W . (2000). Porphyrin-mediated photosensitisation-Taking the apoptosis fast lane. Curr. Opin. Drug. Disc. Dev. 3(2): 232-243 {Invited review). 19. Belzacq, A . , Jacotot, E . , Vieira, H . L . , Mistro, D . , Granville, D.J., X i e , Z . , Reed, J . C , Kroemer, G . and Brenner, C. (2001) Apoptosis induction by the photosensitizer verteporfin. Identification of mitochondrial adenine nucleotide translocator as a critical target. Cancer Res. 61:1260-1264. 20. Granville, D.J., McManus, B . M . , and Hunt, D . W . (2001). Photodynamic Therapy: Shedding light on the biochemical pathways regulating porphyrin-mediated cell death. Histol. Histopathol. 16:309-317. {Invited re view) 21. McGarrity, T.J. , Peiffer, L .P . , Granville, D.J., Carthy, C M . , Levy, J .G. Khandelwal, M . , and Hunt, D . W . C . (2001) Apoptosis associated with esophageal adenocarcinoma: influence of photodynamic therapy. Cancer Lett. 10;163(1):33-41. 22. Mi l le r , L . , Granville, D.J., and McManus, B . M . (2000) Apoptosis in Cardiac Transplant Rejection. Clin. Cardiol. (Invited review)(/« press) 23. Matroule, J Y , Carthy, C M . , Granville, D.J., Hunt, D . W . , and Piette, J. (2000) Mechanism of colon cancer cell apoptosis by pyropheneophorbide-a methylester photosensitization. Oncogene (In press) 24. Granville, D.J., Cassidy, B . A . , Ruehlmann, D . , Choy, J. , Margaron, P., Kroemer, G . , van Breemen, C , Hunt, D . W . and McManus, B . M . (2000) Smooth muscle cell apoptosis: cellular redistribution of Bax, A I F and cytochrome c followed by the activation of multiple caspases. Am. J. Pathol. (Submitted). 25. Granville, D.J., Jiang, H . , McManus, B . M . and Hunt, D . W . (2000) Jurkat lymphoma cells expressing low levels of mitochondrial peripheral benzodiazepine receptors (PBR) are highly sensitive to PDT-induced apoptosis. FEBS Lett. (Submitted). 26. Granville, D.J., Jiang, H . , Bruce M . McManus and Hunt, D . W . (2000) Photodynamic therapy and death receptor ligation have an additive effect upon the induction of apoptosis in Jurkat cells. International Immunopharmacol. (Submitted). 27. Carthy, C M . , Yanagawa, B . , Yang, D . , Granville, D.J., Cheung, P., Rudin, C M . , Thompson, C . B . , Hunt, D . W . and McManus, B . M . (2001) Cytochrome c release and activation of multiple caspases following picornavirus infection: mediators for loss of host cell viability and progeny virus release. J. Virol. (Submitted). 28. L i , R , Bounds, D . , Granville, D.J., Ip, S., Jiang, H . , Margaron, P. and Hunt, D .W. (2001) A new porphyrin-derived photosensitizer induces rapid apoptosis with early release of x i i i mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 in human keratinocytes. Investigative Dermatol. (Submitted). 29. Choy, J., Granville, D.J., Hunt, D . W . and McManus, B . M . (2001) Endothelial Cel l Apoptosis and Its Role in Coronary Artery Disease. J. Mol. Cell Cardiol. (Submitted) 30. Granville, D.J., Ruehlmann, D . , Choy, J., Thompson, C B , V a n Breemen, C , Hunt, D .W. , and McManus, B . M . (2000) Rapid impairment of sarcoplasmic/endoplasmic reticulum ATPases ( S E R C A ) and emptying of E R calcium stores during photodynamic therapy-induced apoptosis. (In preparation). (b) Abstracts 1. Granville, D.J., Jiang, H . , A n , M . T . , Levy, J .G. , McManus, B . M . , and Hunt, D . W . C . (1997) Photodynamic therapy- Involvement of caspases, Bcl-2 and N F K B . IBC 4 t h Annual Conference on Apoptosis. San Diego, C A , U S A 2. Granville, D.J., Jiang, H . , A n , M . T . , Levy, J . G , McManus, B . M . , and Hunt, D . W . C . (1998) Overexpression of B C 1 - X L prevents caspase-3-mediated activation of D N A Fragmentation Factor (DFF) produced by treatment with the photochemotherapeutic agent B P D - M A . American Association of Cancer Research Conference on Apoptosis. Palm Springs, C A , U S A . 3. Carthy C M . , Granville, D.J., Watson, K . A . , Anderson, D.R. , Wilson, J.E., Yang, D . C , Hunt, D . W . C , and McManus, B . M . (1998) Caspase activation and cleavage of substrates following coxsackievirus B3 infection: A n important role in virus-induced cytopathic effect. United States and Canadian Academy of Pathology. Boston, M A , U S A 4. Granville, D.J., Jiang, H . , A n , M . T . , Levy, J .G. , McManus , B . M . and Hunt, D . W . C . (1998). Overexpression of B C 1 - X L prevents caspase-3-mediated activation of D N A fragmentation factor (DFF) produced by the photochemotherapeutic agent B P D - M A . American Society for Photobiology. Snowbird, Utah, U S A . 5. Carthy, C M . , Granville, D.J., Watson, K . A . , Anderson, D.R. , Wilson, J.E., Yang, D . C . and McManus , B . M . (1998) Caspase activation and cleavage o f D N A fragmentation factor following infection: effect of inhibition of viral progeny release. F A S E B . San Francisco, C A , U S A . 6. Hunt, D . W . C , Jiang, H . , Levy, J .G. and Granville, D.J.(1998). Impact of P D T and Fas-mediated apoptosis on cells of the immune system. American Society for Photobiology. Snowbird, Utah, U S A . 7. Carthy, C M . , Granville, D.J., Watson, K . A . , Anderson, D.R. , Wilson, J.E., Yang, D . C . and McManus, B . M . (1998) Cytochrome c release followed by caspase activation and cleavage of D N A fragmentation factor following coxsackievirus infection. American Society for Virology. 17 t h Annual Meeting. Vancouver, B C , Canada. 8. Granville, D.J., Kainth, J .K. , Leong, S., McDonald, P., Hunt, D . W . , Margaron, P. and McManus, B . M . (1999). Endothelial Cel l Apoptosis Involves the Activation of Multiple -Caspases. Keystone Symposium. Santa Fe, N M , U S A . 9. Granville, D.J., Shaw, J., Leong, S., Carthy, C M . , Margaron, P., Hunt, D . W . and McManus, B . M . (1999). Release of Cytochrome c, Bax migration, B i d cleavage and activation of multiple caspases during endothelial cell apoptosis. Keystone Symposium. Breckenridge, C O , U S A . 10. Carthy, C M . , Granville, D.J., Jiang, H . , McManus, B . M . and Hunt, D . W . (1999). Rapid release of mitochondrial cytochrome c and promotion of Apo2.7 expression with a xiv porphyrin photosensitizer: Bcl-2 and B c l - X L do not prevent early mitochondrial events but still exert cytoprotective activity . Keystone Symposium. Breckenridge, C O , U S A . 11. McGarrity, T . , Peiffer, L . , Khandelwal, M . , Granville, D.J., Levy, J. and Hunt, D . (1999) Changes in procaspase 3, caspase 3 cleavage activity and apoptosis during photodynamic therapy of esophageal adenocarcinoma. American Gastroenterological Association. 12. Granville, D.J., Cassidy, B . A . , Ruehlmann, D . , Ip, S., McDonald , P., Carthy, C M . , Margaron, P., V a n Breemen, C , Hunt, D . W . and McManus, B . M . (1999) Endothelial cell and smooth muscle cell apoptosis: cellular redistribution of cytochrome c and Bax followed by the activation of multiple caspases. Canadian Society for Clinical Investigation. Vancouver, B C , Canada. 13. Granville, D.J., Cassidy, B . A . , Ruehlmann, D . , Ip, S., McDonald , P., Carthy, C M . , Margaron, P., V a n Breemen, C , Hunt, D . W . and McManus , B . M . (1999) Endothelial cell and smooth muscle cell apoptosis: cellular redistribution of cytochrome c and Bax followed by the activation of multiple caspases. C l in . Invest. Med . 14. Matroule, J .Y . , Carthy, C M . , Granville, D.J., Henin, A . C . , Hunt, D . W . and Piette, J. (1999) Role of N F - K B in colon cancer cell apoptosis mediated by photodynamic therapy. 8 t h Congress European Society of Photobiology. Granada, Spain. 15. Granville, D.J., Cassidy, B . A , Ruehlmann, D , Ip, S., Choy, J., Carthy, C M , Margaron, P., V a n Breemen, C , Hunt, D . W . and McManus, B . M . (2000) Mapping the mechanisms of endothelial cell and smooth muscle cell death: cellular redistribution of cytochrome c and Bax followed by the activation of multiple caspases. United States-Canadian Academy of Pathology. Lab Invest. 80(1): 51 A . 16. Taylor, L . A . , Carthy, C M . , Yang, D . , Stanton, L . W . , Saad, K . , Wong, D . , Granville, D.J., Cheng, P., Luo, Z.S. , Shreiner, G . and McManus, B . M . (2000) Host gene regulation during coxsackievirus b3 infection in mice using R N A microarray analysis. Cardiovascular Genomics Meeting. Miami , F A , U S A . 17. Belzacq A . S . , Jacotot, E . , L A Vieira H . , Granville, D.J., Kroemer, G . , and Brenner, C. (2000) Verteporfin, a photochemotherapeutic agent, acts on A N T to induce a pro-apoptotic permeabilization of mitochondrial membranes. E U R E S C O Meeting, A p r i l 7-14, 2000. 18. Hunt, D . W . , Jiang, H . , Granville, D.J.. Richter, A . M . , and North, J.R. (2000) Amplification of PDT-mediated apoptosis by death receptor ligation. 13 t h International Congress on Photobiology/28 t h Annual Meeting of the American Society for Photobiology. San Francisco, C A , U S A . 19. Granville, D.J., Cassidy, B . A . , Choy, J., Ruehlmann, D . , Jiang, H . , Ip, S., Margaron, P , Hunt, D . W . C . and McManus, B . M . (2000). Defining the mechanisms of photodynamic therapy-induced endothelial and smooth muscle cell apoptosis. 13 t h International Congress on Photobiology^S 1 1 1 Annual Meeting of the American Society for Photobiology. San Francisco, C A , U S A . 20. Matroule, J Y , Carthy, C M . , Granville, D.J., Hunt, D . W . , and Piette, J. (2000) Mechanism of colon cancer cell apoptosis by pyropheneophorbide-a methylester photosensitization. 13 t h International Congress on Photobiology/28 t h Annual Meeting of the American Society for Photobiology. San Francisco, C A , U S A . 21. Granville, D.J., Cassidy, B . A . , Ruehlmann, D . , Ip, S., Choy, J. , Carthy, C M . , Margaron, P., V a n Breemen, C , Hunt, D . W . and McManus, B . M . (2000) Mapping the mechanisms of endothelial cell and smooth muscle cell death: cellular redistribution of cytochrome c and Bax followed by the activation of multiple caspases. University of British Columbia xv Department of Pathology and Laboratory Medicine 1 s t Annual Research G A L A . 22. Carthy, C M . , Cheung, P .K . , Granvi l le , D.J., Hunt, D .W. , Yang, D . and McManus , B . M . (2000) Cytochrome c release and activation of multiple caspases following picornavirus infection: Mediators of loss of host cell viability and progeny virus release. University of British Columbia Department of Pathology and Laboratory Medicine 1 s t Annual Research G A L A . 23. Hunt, D . W . , Jiang, H . , Granvi l le , D.J., Chan, A . , Richter, A . , and North, J. (2001) T cells and photodynamic therapy. International Society for Optical Engineering. San Jose, C A , U S A 24. Granvi l le , D.J., Ruehlmann, D. , Choy, L , Van Breemen, C , Hunt, D .W. , and McManus, B . M . (2001) Rapid impairment of sarcoplasmic/endoplasmic reticulum ATPases ( S E R C A ) and emptying of E R calcium stores during photodynamic therapy-induced apoptosis is not impaired by Bcl-2 . Keystone Symposium. Keystone, C O , U S A . (c) Oral Presentations 1. "Anti-cancer therapy with a light-activated drug promotes apoptosis" IBC ' s 3 r d Annual International Conference on Apoptosis: Practical Applications and Novel Therapies. Boston, M A , U S A , November 1996. Invited Speaker. 2. "Role of Bcl-2 Family and Caspases in Photodynamic Therapy" 25 t h Annual Meeting for the American Society for Photobiology. St. Louis, Missouri, U S A , July 10-15, 1997. 3. "Apoptosis: an overview" Vancouver Vascular Biology Research Centre. St. Paul's Hospital, Vancouver, B . C . Canada, October 17,1997. Invited Speaker. 4. Keynote address, "Apoptosis and disease. Dissecting the role of caspases" IBC 4 t h Annual Conference on Apoptosis: Therapeutic Strategies for Regulating Cel l Death. San Diego, C A , U S A , October 27-28,1997. Invited Chairperson. 5. "Photodynamic therapy: involvement of caspases, Bcl-2 and N F K B " IBC 4 t h Annual Conference on Apoptosis. San Diego, C A , U S A , October 27-28,1997. Invited Speaker. 6. "Apoptosis: Why all the fuss?" Canadian Association of Laboratory Animal Science. Jack Bel l Research Centre, Vancouver, B . C . Canada, December 3, 1997. Invited Speaker. 7. "Molecular mechanisms of coxsackievirus-induced apoptosis" Research in Progress Meeting. Gourlay Conference Room. St. Paul's Hospital, Vancouver, B . C . , Canada , February 16, 1998. Invited Speaker. 8. "Role of calcium in apoptosis" Vancouver Vascular Biology Research Centre. St. Paul's Hospital, Vancouver, B . C . , Canada, February 25, 1998. Invited Speaker. 9. "Activation of multiple caspases following coxsakievirus B3 infection" Gross-to-Nano Departmental Rounds. Eye Care Centre Auditorium, Vancouver General Hospital, Vancouver, B . C . , Canada, February 26, 1998 10. "Overexpression of B C 1 - X L prevents caspase-3-mediated cleavage of D N A fragmentation factor (DFF) and D N A fragmentation produced by the photochemotherapeutic agent B P D - M A " . American Society for Photobiology. Snowbird, Utah, U S A . July 11-15, 1998 11. "Photodynamic therapy: mitochondrial disruption and caspase activation" IBC 5 t h Annual Conference on Apoptosis. San Fransisco, C A , U S A , December 10-11, 1998. Invited Speaker. xv i 12. "Apoptosis in endothelial and smooth muscle cells" Gross-to-Nano Departmental Rounds. Eye Care Centre Auditorium, Vancouver General Hospital, Vancouver, B . C . , Canada, February 26, 1999. 13. "Apoptosis: The role of cell damage, cell death and removal of apoptotic debris in the pathogenesis of transplant vascular disease" Research in Progress Meeting. Gourlay Conference Room. St. Paul's Hospital, Vancouver, B . C . , Canada, May 3, 1999. 14. "Role of endothelial cell and smooth muscle cell apoptosis in transplant vascular disease" Vancouver Vascular Biology Research Centre. St. Paul's Hospital, Vancouver, B .C . , Canada, March 4,1999. 15. "Endothelial cell and smooth muscle cell apoptosis: Cellular redistribution of cytochrome c and Bax followed by the activation of multiple caspases" Canadian Society for Clinical Investigation, Vancouver, B . C . , Canada, June 26, 1999 16. "Effects of Oxidative Stress on Endothelial and Smooth Muscle Cells: Implications for Transplant Vascular Disease" Gross-to-Nano Departmental Rounds. Eye Care Centre Auditorium, Vancouver General Hospital, Vancouver, B . C . , Canada, January 27, 2000. 17. "Rapid impairment of sarcoplasmic/endoplasmic reticulum ATPases ( S E R C A ) and emptying of E R calcium stores during photodynamic therapy-induced apoptosis is not impaired by B c l - 2 " Gross-to-Nano Departmental Rounds. Eye Care Centre Auditorium, Vancouver General Hospital, Vancouver, B . C . , Canada, November 2, 2000. x v i i Acknowledgements Over the past few years, the field of apoptosis has grown enormously. The average number of publications has ranged between 600-800 articles per month over the past five years. Thus, keeping up to date with the literature, as well as the research pertaining to my dissertation would have never been possible without the help and input of others. I am grateful to many people for their help. In particular, I thank my supervisor Dr. Bruce McManus for accepting myself as a graduate student and providing me with far more opportunities than I had ever envisioned at the start of my term. M y goals entering graduate studies were to build not only upon the experimental/technical expertise that I had already attained, but to excel in the areas of experimental planning, leadership, writing and speaking. Working with Bruce has given me tremendous experience in all o f these areas. In addition, Bruce has been a great mentor and has always encouraged and challenged me to be creative and strive to reach my goals. I thank him for all the extra time he has put in on weekends and evenings reading and correcting manuscripts as well as meeting with me at his house to discuss my research. To this extent, I am also thankful to Bruce's wife Janet, who has also been a source of support not only for someone to talk to, but also for enduring Bruce's busy schedule and being a great host. I am also deeply indebted to Dr. David Hunt. Over the past few yearsj Dave has been a co-supervisor and advisory member of my research committee. Dave has been a good friend and mentor. He has always taken the time to discuss and support new ideas as well as to listen to and cope with my occasional problems and frustrations. Additionally, I thank Dave for initially encouraging me to seek further education and making it possible for me to retain my connection to Q L T until I completed my studies. This unique opportunity allowed me to learn and gain expertise in both industrial and academic research environments. Additionally, this allowed access to equipment not currently available at St. Paul's Hospital. I am also greatly thankful to the other members of my committee, Drs. A l y Karsan and Mladen Korbelik, for their support and intellectual input over the past few years. I would also like to thank all the people that I had the opportunity to work with in both the McDonald Research Wing and Q L T . In particular, I thank the co-op students (Janet Shaw, Brighid Cassidy and Jonathan Choy) for all their hard work. I thank Huijun Jiang for her help with the flow xv i i i cytometry and Drs. Casey van Breemen and Dietrich Reuhlmann and for their help and expertise with the calcium studies. I thank the sponsors of my research, Q L T Inc, St. Paul's hospital Hospital foundation and the Heart and Stroke Foundation of Canada. Without these sources of support, my research would not have been possible. I would also like to thank our numerous collaborators: Drs. Craig Thompson, Jacques Piette, Gordon Shore, Mary Russell, Joerg Kogl in , Guido Kroemer, Catherine Brenner, and Xiaodong Wang for various reagents and scientific input. Last, but not least, I would like to thank my family for their continued support during my studies. I 'd especially like to thank my wife, Karen, for all o f her support, understanding and putting up with me working at home almost every night and many weekends for the past few years. I would also like to thank my newborn son, Ky le , that has not only been able to cheer me up on the worst of days, but has also been a reliable 'alarm clock' every morning to ensure that I wake up for work. M y parents have also been a crucial source of support. They have always sacrificed their own lives and financial well being to make sure that we had everything we needed to succeed in whatever we pursued. I also thank my in-laws for their support. In particular, my father-in-law for his interest pertaining to my research over the past few years. xix CHAPTER I BACKGROUND Apoptosis is as fundamental to cellular and tissue physiology as are cell division and differentiation. For this reason, apoptosis has become the focus of intense scientific inquiry over the past decade. This surge of attention towards this form of cell death is due to the pivotal role played by apoptosis in normal organ development, deletion of vestigial structures during embryogenesis, control of cell numbers, elimination of nonfunctional, harmful, abnormal or misplaced cells, as well as its role in many genetic and acquired diseases. Significant advances have been made in defining the regulatory mechanisms of apoptosis in the etiology of certain viral infections, neurodegenerative disorders, autoimmune diseases, immunologic deficiencies and malignancies. This section w i l l highlight recent developments in the molecular biology of apoptosis and new insights into the role played by apoptosis in disease processes. 1.1 Apoptosis 1.1.1 Historical Perspective Cel l death has been a topic of scientific interest for well over a century. Virchow suggested the importance of cell death in atheromas, and at the gross level, the process was described as degeneration, mortification, softening and necrosis of cells '. Walther Flemming may have been the first to describe apoptosis when he observed the gradual degradation and disappearance of nuclei in epithelial lining cells of regressive ovarian follicles. He also observed the condensation of chromatin forming "half-moons" a process which he termed "chromatolysis" 3. In 1914, chromatolysis was further characterized by Ludwig Graper who 1 proposed the existence of an amitotic mechanism to counteract cell death. Graper also hypothesized that the physiological removal of cells occurs by chromatolysis, followed by 2 engulfment by a neighboring cell . Glucksmann first emphasized the concept of programmed cell death in embryology in 1951, wherein he postulated that cell death occurred at specific times during the development of an organism 3 . However, it was not until 1965 that criteria for two morphologically distinct forms 4 of cell death were presented . Following the induction of hepatic ischemia by compromising the hepatic portal vein, Kerr noted the phenomenon of parenchymal shrinkage in which individual hepatocytes were constantly being converted into small round cytoplasmic masses containing 4 traces of pyknotic chromatin . In subsequent studies he demonstrated that their histology was different from that of necrotic cells and that the tissue surrounding these cells did not elicit inflammation. Furthermore, staining for acid phosphatase showed that lysosomes in necrotic 4 cells ruptured, whereas in ischemic hepatocytes the lysosomes remained intact . In 1969, Ken-described the formation of membrane-enclosed vesicles containing preserved organelles and condensed chromatin 5 . Kerr initially referred to this physiological phenomenon as 'shrinkage necrosis' 6. The following year Kerr, Wyl l ie and Currie proposed the term 'apoptosis', a Greek word describing the process of leaves falling from a tree or petals from a flower to depict this specific form of cell death 6 ' ? . However, it has since been forwarded that the term apoptosis was originally coined in medical writings approximately 2400 years ago by Hippocrates, the father of g Western medicine . The word was first used by Hippocrates (460-370 B C ) to describe the 'falling off of the bones' in the context of bone fractures. Galen (129-201 A D ) then later adopted g the word to also describe the 'dropping of the scabs' . 2 1.1.2 Apoptosis Versus Necrosis 1.1.2.1 Necrosis Necrosis does not require energy, typically affects groups of contiguous cells, and an inflammatory reaction usually develops in the adjacent viable tissue in response to the released 9 10 cellular debris ' . One of the most prominent alterations that occurs during necrosis is cellular swelling, which may be attributable to the loss of selective permeability of membranes in dying 11 . 1 2 cells . Membrane ion-pump activity ceases in cells prior to the onset o f necrotic cell death . The disturbance o f the plasma membrane is believed to be a major factor leading to secondary damage of membranes of cytoplasmic organelles such as mitochondria Dilation of the endoplasmic reticulum (ER) and disaggregation of polysomes are also observed during necrosis' 3. In the late stages of necrosis, prominent mitochondrial swelling and rupture of the plasma membrane are observed, followed by the passive release of lysosomal hydrolases into the 7 14 extracellular matrix ' . The release of hydrolases from ruptured lysosomes accelerates cellular 9 disintegration resulting in protein, R N A and D N A degradation . Although there is a marked dissolution of most cytoplasmic organelles during necrosis, the nucleus swells but remains relatively intact During the later phases of necrosis, nuclear D N A is degraded into a heterogeneous mixture which can be visualized as a smear when analyzed using agarose gel electrophoresis, suggesting nonspecific D N A degradation 6 ' 5 . 1.1.2.2 Apoptosis In contrast to necrosis, apoptosis may affect scattered individual cells rather than cell groups or a whole tissue or organ compartment '°. One of the key characteristics of apoptosis is cell shrinkage. A s cell shrinkage occurs, the cytoplasm condenses and nuclear chromatin appears 3 pyknotic and compacts into dense masses against the nuclear membrane. A s the process continues, the nucleus becomes fragmented. During the final stages of apoptosis, the cell becomes convoluted and breaks up into several membrane-bound vesicles containing a variety of intact organelles and nuclear fragments '° ' 1 6 . These apoptotic bodies are then engulfed by 9 neighboring cells or macrophages . In theory, the D N A from apoptotic bodies could integrate 17 and possibly alter the genomes of neighboring cells . However, during apoptosis, a mechanism to effectively destroy D N A (both host and viral) in dying cells is achieved by the activation of 17-19 caspase-activated deoxyribonuclease ( C A D ) , a process which occurs exclusively in apoptosis Once initiated, apoptosis can proceed quite rapidly and the morphological features of apoptosis may only be visible briefly in tissues due to rapid phagocytosis o f apoptotic debris by macrophages or neighboring cells l 0 ' 1 6 ' 2 0 . Thus, extensive levels of cell death may occur in a tissue in which only a few apoptotic bodies are v is ib le 1 0 ' 6 . One evident physiological difference in cells undergoing apoptosis versus necrosis is in intracellular levels of A T P . It has been demonstrated that treating cells with a calcium ionophore induces apoptosis under ATP-supplying conditions but induces necrotic cell death under A T P -21 depleting conditions, indicating that A T P levels are a determinant in the mode of cell death . 22 Furthermore, Leist and co-workers showed, using human T cells, that cell death caused by two apoptotic triggers (staurosporine and CD95 ligation) shifted from apoptosis to necrosis when cells were preemptied of A T P . Nuclear condensation and D N A fragmentation, characteristics of apoptosis, did not occur in cells predepleted of A T P and treated with either inducer, although the 22 ' kinetics of cell death were unaltered . Using pulsed ATP/depletion/repletion experiments, it was demonstrated that A T P produced through glycolysis or mitochondrial respiration was required for the execution of the final phase of apoptosis leading to nuclear condensation and 4 D N A degradation . Further, A T P is now known to be a key factor supporting the activation of 2 3 caspases that are triggered during apoptosis . Current understanding of the differences between apoptosis and necrosis are summarized in Table 1. 5 Table 1. Comparison of features related to apoptotic versus necrotic cell death. Features Necrosis Apoptosis Tissue distribution groups of cells single cells Tissue reaction lysis and release of cellular phagocytosis of membrane-contents resulting in enclosed vesicles by inflammation of surrounding macrophages or neighboring tissues cells; little or no inflammation Morphology cell cell swelling cell shrinkage, loss of membrane contact with neighboring cells plasma membrane loss of integrity, enhanced blebbing, formation of permeability, apoptotic bodies, PS phosphatidylserine (PS) externalization externalization organelles damaged intact nucleus disintegrated condensed then fragmented lysosomes Ruptured, release of intact hydrolytic enzymes mitochondria defective, A T P depletion, swelling, permeability swelling, ruptured, cyt c transition, may rupture, cyt c release release Biochemistry D N A non-specific degradation internucleosomal D N A cleavage Protein non-specific degradation activation of caspases, calpains Substrates non-specific hydrolysis specific substrates Anti-death molecules 24 Bcl-2 (in some cases) Bcl-2-l ike proteins, IAPs, FLIPs , crmA, caspase inhibitors A T P requirement N o Yes 6 1.1.3 Regulation of Apoptosis Research carried out over the past decade has demonstrated that apoptosis is a complex, tightly regulated process. The putative molecular pathways for Fas-, T N F - and T R A I L - mediated cell death (apoptosis) are described in the following sections and illustrated in Figure 1. 1.1.3.1 Receptor Mediated Apoptosis Members of the T N F superfamily, that includes Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL , A P O - 2 L ) and T N F bind specific cell surface receptors and activate a 25 suicide program through the involvement of intracellular adapter proteins . These ligands occur in membrane bound and soluble forms. Upon binding their respective receptors these factors initiate intracellular reactions that culminate in caspase activation and apoptotic cell death. The receptors for FasL, T N F and T R A I L are Fas ( A P O l , CD95), TNF-R1 and T R A I L - R 1 (DR4) and T R A I L - R 2 25 (DR5), respectively . These receptors are characterised by the presence of cysteine-rich extracellular domains and a homologous cytoplasmic sequence motif termed a "death domain" 25 which permits an engagement with the apoptotic machinery of the cell . Fas is expressed by many different cell types and its presence signifies that these cells 25 may be receptive to apoptosis-inducing signals from FasL-bearing cells . In the periphery, Fas-FasL interactions serve to limit the proliferation of activated T cells, promote the lysis of virally-infected cells by cytotoxic T cells, and contribute to the maintenance of a state of immune privilege in different tissues by imperiling the survival of activated inflammatory cells 2 6 . Normal and malignant cells may express Fas and/or FasL. The administration of agonistic anti-Fas 27 28 antibodies to mice produces widespread tissue effects leading to death from liver damage ' . 7 Figure 1. Molecular pathways involved in the execution of Fas-, T N F - and TRAIL-mediated apoptosis. Binding o f T N F - a , FasL or T R A I L to their specific receptors initiates receptor trimerization followed by the recruitment o f adapter proteins to form the death-inducing signaling complex (DISC). DISC formation initiates the recruitment and autocatalytic activation of caspase-8 leading to subsequent activation of downstream 'executioner' caspases. Caspase-8 may instigate the activation of downstream caspases directly or indirectly through the cleavage of B i d into a truncated form capable of initiating cyt c release. Cyt c, ATP, Apaf-1 and pro-caspase-9 form a complex known as the apoptosome which catalyzes caspase-9 activation. Many pro-apoptotic stimuli induce mitochondria (MT) to release A I F and cyt c during apoptosis which may be blocked by Bcl-2 or B c l -X L overexpression. Once released from mitochondria, A I F translocates to the nucleus and initiates stage I nuclear apoptosis (chromatin condensation and 50 kb D N A fragmentation). Caspase-3 induces stage II D N A fragmentation (200 kb) by cleaving I C A D / D F F which allows the translocation of C A D into the nucleus and D N A fragmentation. Caspase-3 also cleaves structural proteins and repair enzymes as well as other caspases such as caspase-6 which cleaves nuclear lamins. Caspase-3-mediated cleavage of cell cycle inhibitory proteins p21 and p27 may initiate premature entry into the cell cycle via C D K 2 activation. T N F / T N F - R 1 binding can also elicit an anti-apoptotic response via T R A F - 2 activation, ultimately resulting in NF-KB-mediated transcription o f anti-apoptotic genes such as c-I AP , A20 , B c l - X L and A 1 . T R A I L may also bind to "decoy" receptors that do not transmit pro-apoptotic signals to the cell. Increased intracellular C a 2 + also contributes to apoptosis and is outlined in detail in figures 2 and 3. The A k t / P K B anti-apoptotic pathway is outlined in figure 4. 8 Fas (Apo-1 or CD95) is a glycosylated 45 k D a type I transmembrane receptor belonging 29 to the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family . Fas ligand 29 (FasL) is a 40 kDa type II transmembrane protein which induces apoptosis by binding to Fas . Fas, and its counter ligand, FasL, as mediators of apoptosis, are implicated in peripheral deletion of autoreactive cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells 3°. Although initially thought to be restricted to lymphoid cells, studies of mouse embryogenesis and examination of adult tissues has revealed that FasL is constitutively expressed in a wide array of non lymphoid tissues. FasL does not appear to be an important factor in murine embryonic programmed cell death but, in adult mice, FasL m R N A is detectable in numerous tissues apart from heart and pancreas 3 ' . Thus, several tissues, including thymus, lung, endothelium, spleen, small intestine, liver, seminal vesicle, prostate and uterus co-express both Fas and FasL 3 1 T i s s u e s with constitutive Fas and FasL co-expression may be characterized by continuous apoptotic cell turnover, or, conversely may be resistant to FasL-induced apoptosis whereas tissues expressing only FasL are known to be immune-privileged 3 1 3 3 . A s such, FasL plays a key role in the physiological regulation of cell turnover, and in the protection of tissues against potential lymphocyte-mediated damage Several proteins that form complexes with the intracellular domain of Fas have been identified. Extracellular ligation of Fas results in the recruitment of proteins to form a death-inducing signaling complex (DISC). The binding of Fas-L to Fas results in the recruitment of an adapter protein, Fas-associated death domain ( F A D D ) , which binds to a conserved amino acid sequence known as the "death domain" on the cytoplasmic domain of the Fas receptor. In turn, F A D D associates with the proenzyme form of caspase-8 ( F L I C E / M A C H ) through dimerization of a domain known as the death-effector domain (DED). A protein tyrosine phosphatase, Fas-9 i 34 associated phosphatase-1 (FAP-1) may also have a role in DISC formation . Receptor-interacting protein (RIP) may also be recruited, but is not necessary for the DISC complex formation 3 5 . Interestingly, another death domain containing adaptor/signaling molecule, RJP-associated I C H -l/CED-3-homologous protein with death domain ( R A I D D ) , also known as caspase and RIP adapter with death domain ( C R A D D ) , has recently been identified and was shown to induce apoptosis 3 6 , 3 1 . R A I D D / C R A D D has a dual-domain structure similar to that of F A D D except it has an NH2-terminal domain that interacts with caspase-2 and a COOH-terminal death domain that interacts with RIP. These results suggest that RIP may be involved in triggering caspase-2 36 activation . Similar to the Fas-mediated death signaling cascade, binding of T N F to TNF-R1 induces DISC formation. TNF-induced DISC formation involves the recruitment of caspase-8, RIP and F A D D , but also recruits T N F receptor associated death domain ( T R A D D ) which is an adapter protein that binds to TNF-R1 and F A D D 3 5 . T R A I L and its receptors are widely expressed in human tissues. Importantly, T R A I L induces apoptosis in many transformed cell lines but not in normal cell types, even though both 25 forms express D R 4 and DR5 . It was initially believed that normal cells were protected from TRAIL-mediated apoptosis through their expression of "decoy" cell surface receptors (decoy 25 receptor 1, D c R l ) that bind T R A I L but do not transduce an apoptotic response . However, transformed cells may also express T R A I L decoy receptors. It is now evident that responses to 38 39 T R A I L are regulated by specific intracellular factors ' . T R A I L may play a role in the normal immune system by acting against virally infected or transformed cells. Similar to FasL and T N F -cc, T R A I L ligation to its cognate receptors causes the recruitment of F A D D and recruitment of 10 procaspase-8 molecules in close proximity of each other, which results in the autocatalytic r- n  40.41 activation of caspase-8 In all systems (Fas, T N F and T R A I L ) , formation of the DISC results in caspase-8 activation, which, in turn, activates other downstream caspases, ultimately resulting in apoptosis. The role of caspases in the execution of apoptosis is discussed in the following section. Certain viruses and cell types possess proteins, F L I C E (caspase-8) inhibitory proteins (FLIPs), which block formation of the DISC and subsequent recruitment and/or activation of caspase-8 thereby blocking 42 43 apoptosis ' . Although we wi l l refer to these proteins as FLIPs, it should be noted that cellular 44 45 FLIP was discovered by several groups and consequently is also referred to as F L A M E , C A S H , Casper"6 and I - F L I C E 4 7 . Northern blotting has revealed that four FLIP m R N A species exist with the highest levels expressed in the heart, followed by skeletal muscle, and peripheral blood leukocytes4 3. These results offer a possible explanation as to why mice injected with anti-Fas antibodies die from liver damage despite high levels of Fas expression in the heart 4 8. Furthermore, in E C , downregulation of cellular FLIP by exposure to oxidized lipoproteins renders these cells, 32 which are otherwise resistant to FasL-induced apoptosis, susceptible to FasL-induced apoptosis . Thus, the susceptibility of a particular cell type to receptor-mediated apoptosis appears to be intrinsically regulated by cellular proteins capable of inhibiting DISC formation and/or caspase activation. 1.1.3.2 Caspase Family Many genes involved in the executioner phase of apoptotic cell death were discovered in the nematode Caenorhabditis elegans 4 9 ' 5 °. Among the 1090 cells generated during C. elegans development, 131 undergo programmed cell death. Deletion or mutation of one gene, Ced-3, 49 abolishes all 131 programmed cell deaths that occur during development . The finding that Ced-3 11 encodes a protein that is highly homologous to the mammalian interleukin-l(3 converting enzyme (ICE) strongly suggested that the biochemical events governing apoptosis in nematodes and 49 mammals was highly conserved . ICE was the first identified human member of a class of cysteine proteases with near absolute substrate-cleavage specificity for aspartic acid (Asp) residues5'. Since the initial discovery of ICE, several I C E homologues have been identified prompting the development of the 'caspase' (cysteinyl aspartate-specific proteinases) 52 nomenclature to delineate this distinct family of proteases . There are currently 14 homologues that have been, identified in the caspase family 5 3 . Caspases are present in the cytoplasm as zymogens and require proteolytic processing by other proteases, often another caspase, or by autocatalytic cleavage to produce the active form. Caspase precursors are cleaved at internally conserved sequences of amino acids residues with Asp 54 residing in the PI cleavage site . Phylogenetic analysis of caspase family members has revealed that these enzymes may be grouped into two major subfamilies; the I C E and CED-3 subfamilies5 5. It has been suggested that members of the ICE subfamily (caspases-1, -4, and -5) have subsidiary roles in pro-inflammatory events, whereas members of the CED-3 subfamily (caspase-2, -3, -6, -7, -8, -9, and -10) are primarily involved in apoptosis 5 5 . Recently, Thornberry et al. determined the substrate specificities of nine of the known caspases 5 6 . These enzymes were placed into three distinct groups. Group I members (caspase-1, -4, and -5) have a substrate preference for the tetrapeptidic W E H D amino acid sequence, group II (CED-3, caspase-2, -3, and -7) prefer the D E X D sequence, while group III (caspase-6, -8, -9 and granzyme B) recognize ( I / L / V ) E X D sequences 5 6 . Thus, different caspases may have redundant functions within each group 5 6 . The specificity of caspases 2, 3, 7 and C E D - 3 ( D E X D ) suggests that these enzymes may function to incapacitate essential homeostatic pathways during the effector phase of apoptosis 5 6 . 12 Conversely, the optimal substrate specificity for caspases 6, 8, and 9 and granzyme B ( ( I /L /V)EXD) corresponds to cleavage sites in effector caspase proenzymes, suggesting that these enzymes may play a role upstream of executioner caspases, resulting in a proteolytic cascade that amplifies the death s igna l 5 6 . However, our group as well as other groups have now demonstrated that caspase-6 is actually activated downstream of caspase-3 In summary, it appears that a cascade mechanism for the transmission of signals occurs following the induction of the apoptotic program. Caspase-3 (CPP32/Yama/apopain) is considered to be one of the central executioner molecules that is activated in many cell types following exposure to apoptotic stimuli and is responsible for cleaving various proteins, thereby disabling important cellular structural and repair processes 5 5 . Unti l recently, little was understood concerning the biochemical events leading to caspase-3 activation. The release of three apoptotic protease activating factors (Apafs) from 58 mitochondria may play a key role in the activation of caspase-3 . Apaf-1, the human homologue 58 of CED-4 , functions downstream of Bcl-2 but upstream of caspase-3 . Apaf-1 contains a conserved amino acid sequence in its prodomain, known as the caspase recruitment domain ( C A R D ) which is believed to be involved in binding to caspases that contain similar C A R D s at 58 59 their NH2 termini (Figure 2) . In the presence of cyt c (Apaf-2) and d A T P , Apaf-1 binds to and 23 activates caspase-9 which subsequently cleaves caspase-3 . In granzyme B (GraB)-mediated apoptosis, caspase-10 is processed by GraB 6°. Caspase-10 then cleaves caspase-3 and 7 6°. However, in cases in which caspase-10 is absent or dysfunctional, GraB is capable of activating other caspases or directly kill ing the cell by cleavage of non-caspase as well as caspase substrates 6°. These secondary mechanisms provide the host with overlapping 13 safeguards to combat against abnormal or virus-infected cells . Caspase-3 can also be activated by , 61 .62 63 caspase-1 , caspase-4 or caspase-8 . In certain cell types, procaspases may also be released from mitochondria. Mancini et al. 64 demonstrated that caspase-3 has a mitochondrial and cytosolic distribution in nonapoptotic cells . The mitochondrial caspase-3, which is located in the intermembrane space, was shown to be 64 activated by numerous pro-apoptotic stimuli . Mitochondrial caspase-3 activation could be 64 blocked by Bcl-2 . Autocatalytic processing of caspase-3 may occur in the presence of 64 increased acidification . It has also recently been shown that Bcl-2 and BC1-XL may inhibit apoptosis by enhancing H + efflux from the mitochondrial intermembrane space 65'66. It will be interesting to determine whether Bcl-2 or BC1-XL, acting as ionic channels, block apoptosis by preventing the increased intermembrane acidification that is associated with autocatalytic activation of mitochondrial caspase-3. Caspases-2 and -9 have also been reported to be released during neuronal or cardiomyocyte apoptosis It has been speculated that retention of caspases within the mitochondria may be a mechanism by which cells with low turnover rates such as cardiac myocytes or neurons prevent the occurrence of accidental apoptosis68. A growing number of caspase substrates have now been identified including nuclear and signaling proteins such as poly (ADP-ribose) polymerase (PARP) 6 9, sterol regulatory element binding proteins ™, the Ul-associated 70 kD protein11, DNA-dependent protein kinase 11, IKB-CC 1 2 , 73 74 protein kinase Cy and £, , double minute 2 protein , retinoblastoma (Rb) , Ras GTPase-activating protein,75 MEKK1 7<', focal adhesion kinase Cbl 1 5, Cbl-b 1 5, Raf-1 1 5, and Akt-175. Caspase-3 activation and DNA fragmentation may be directly linked through the cleavage 78 79 of a cytosolic protein, DNA fragmentation factor (DFF) ' . DFF is a cytosolic factor isolated from HeLa cells consisting of 40 and 45 kDa, subunits of which the 45 kDa protein is cleaved into 14 smaller polypeptides by caspase-3 . In subsequent studies by Enari et al, the long sought after endonuclease, caspase-activated deoxyribonuclease ( C A D ) , which is activated exclusively during 19 apoptosis, was isolated from murine lymphoma cells and characterized . Furthermore, the murine 19 equivalent to the D F F 45 kDa protein was isolated and termed inhibitor of C A D ( ICAD) . I C A D is bound to C A D in the cytosol, thereby blocking its D N A s e activity. During apoptosis, activated caspase-3 cleaves I C A D , allowing C A D translocation into the nucleus and degradation of 18 19 chromosomal D N A . A 40 kDa DFF-inhibitable endonuclease, caspase-activated nuclease ( C P A N ) , with high homology to C A D , has been isolated from Jurkat cells and is believed to be the 80 human equivalent to C A D . Further evidence has demonstrated that B C 1 - 2 / X L prevent D N A 79 fragmentation by inhibiting caspase-3 activation and subsequent processing of D F F . Active caspases also target structural proteins. Degradation of such proteins may be responsible for the cellular morphological changes that are observed during apoptosis. Structural proteins that are cleaved in apoptosis include actin 8 1 , a-fodrin 8 2 , lamin A 8 3 , lamin B **, nuclear mitotic associated protein ( N u M A ) , focal adhesion kinase 1 1 , Rabaptin-5 8 5 , keratin 88 8 6 , and gelsol in 8 ? . In summary, the majority of caspase substrates identified thus far are involved in cell signalling, D N A repair, cell division, cell death and cell survival. Over 70 caspase substrates 53 have now been identified . Cleavage and subsequent inactivation or alteration of these proteins is a critical event contributing to the apoptotic phenotype. 1.1.3.3 Bcl-2 Family Bcl-2 was initially discovered as an overexpressed protein in human B-cell lymphomas 88 89 arising as a result of a t(14;18) chromosomal translocation ' . Overexpression of Bcl-2 protects 15 many cell types against apoptosis in response to such diverse stimuli as viral infection, hypoxia, 90-94 ionizing radiation, or chemotherapeutic agents . A number of Bcl-2 family members have been identified in mammals: Bcl-2, B C 1 - X L , , A l / B f l - 1 , Bcl-w, N r l 3 , and Mcl-1 serve to inhibit 30 94 95 apoptosis; whereas Bax, Bik, Bak, Bad, Bid , Hrk and Bc l -Xs promote apoptosis ' ' . Members of the Bcl-2 family have been shown to homo- or heterodimerize with one another and thereby 94 96 97 antagonize or enhance the function of the other . It has been shown both in vivo and in vitro that Bcl-2 provides protection against apoptosis in the absence of new translation, suggesting that it does 98 not exert its protective effect through gene regulation . 2+ In some systems, Bcl-2 regulates mitochondrial and endoplasmic reticular C a levels and 98 the loss of mitochondrial membrane potential produced by pro-apoptotic stimuli . The effects of Bcl-2 on intracellular C a 2 + regulation wi l l be discussed in a later section. Several groups have forwarded evidence that Bcl-2 and B C 1 - X L may be pore-forming proteins similar to certain bacterial 99 toxins such as diphtheria toxin . Thus, Bcl-2 and B C 1 - X L may have a membrane transport function that regulates ion flux and protein transport across some of the intracellular membranes that Bcl-2 and its homologues are localized such as the mitochondrial membrane, endoplasmic reticulum and 99 100 the nuclear envelope ' . Recent evidence has shown that Bcl-2 can block the release of cyt c from the mitochondria, an event that may be necessary for caspase-3 activation 1 0 U 0 2 . Alternatively, the Bcl-2-related protein B C 1 - X L was also shown to block apoptosis via interaction with cyt c function by binding to it and inhibiting its availability in the cy toso l 1 0 3 . Furthermore, Kharbanda et al. demonstrated that cyt c binds directly and specifically to B C 1 - X L and not to the proapoptotic B c l - X s protein, and that B c l - X s blocks binding of cyt c to B c l - X L . These findings would support a role for B C 1 - X L in protecting cells from apoptosis by reducing the availability of cyt c in the cytosol . 16 Bcl-2 can also prevent the release of apoptosis inducing factor (AIF) from mitochondria 104 . A I F , a mitochondrial intermembrane flavoprotein, has been shown to translocate from mitochondria to nuclei during apoptosis. When added to nuclei, A I F causes larger scale (-50 kb) D N A fragmentation and peripheral chromatin condensation that resembles the first stage of nuclear apoptosis (stage I) in cells undergoing apoptosis. Complete (Stage II) D N A 104 fragmentation (-200 bp) requires the caspase-dependent activation of C A D . Recent studies have suggested that in some systems BC1-XL can prevent swelling and rupture of the outer mitochondrial membrane which offers another mechanism as to how cyt c may be released 6 6 . It has been suggested that B c l - X L may act as a channel to dissipate the increased proton accumulation from the intermembrane space and thereby prevent mitochondrial swelling, however this has been somewhat controversial since it fails to explain other observed phenomena of Bcl-2-l ike proteins such as its role in the prevention of the cyt c/Apaf-1 complex that activates caspase-9. Conversely, since the vast majority of studies have been performed with a limited number of tumour cell lines, it is possible that different cell types w i l l respond differently to a given stimuli. Since it is well understood that different cell types vary in their sensitivity to a particular stimuli, it is highly likely that the apoptotic machinery utilized to respond is also different. It has been suggested that Bcl-2 can serve as a docking proteins for Apaf-1, thereby 23 preventing caspase-9 activation . Similarly, BC1-XL has been shown to form a ternary complex with caspase-9 and Apaf-1 '° 5 . The release of cyt c along with A T P is necessary for the 23 displacement of Apaf-1 and activation of caspase-9 . However, this model is controversial since recent studies have demonstrated that Apaf-1 localizes to the cytosol and, unlike Bcl-2 or B c l -XL, does not localize to mitochondria ' ° 6 ' 1 0 7 . Thus, Bcl-2 or BC1-XL may play a role in the 17 regulation of caspase-9 activation by either directly inhibiting the cyt c/Apaf-1 complex that activates caspase-9 or by inhibiting cyt c release 2 3 ' ' ° 5 . Regardless, in either scenario, Bcl-2 and B c l - X L prevent caspase-9 activation. Although Bcl-2 localizes on the mitochondria, nucleus, and E R membranes, limited research has been done concerning the nucleus and E R (Figure 2). Several roles for Bcl-2 and E R function have been proposed 1 0 8 ' " . A n integral E R 28 k D protein, Bap31, binds to caspase-1 108 or -8, but not to caspase-3 and forms a complex with either Bcl-2 or BC1-XL . During apoptosis, p28 Bap 31 is cleaved, generating a 20 k D a product which is capable o f inducing apoptosis when expressed in otherwise normal cells '° 8 . Interestingly, Bax, which does not bind with the complex, prevents the binding of Bcl-2 to p28 Bap31, thereby blocking complex formation and 108 promoting the generation of the p20 fragment . Taken together, these results offer an explanation as to how increased levels o f Bax and subsequent heterodimerization promote apoptosis. It w i l l be interesting to determine what influence p28 Bap 31 cleavage has on Ca release from the E R and whether these events may trigger reactions that occur in the mitochondria. Although there have not been any reports connecting the two organelles in 9-4-apoptosis, mitochondria have the ability to decode ER-transmitted oscillating Ca signals to regulate other metabolic events " 2 ' " 3 . Moreover, release of C a 2 + into the cytosol has been shown 114 to induce mitochondrial permeability transition pore opening . Another ER-associated protein known as Bax Inhibitor-1 (BI-1), which localizes to the E R but not to the mitochondria, may also be involved in the regulation of these events U 5 . Although BI-1 was identified by its ability to suppress Bax-induced cell death in yeast, it does not bind or inhibit Bax directly but does associate with Bcl -2 and BC1-XL " 5 . Further experiments are necessary to determine the precise relationship between Bcl-2-l ike proteins and BI-1 in apoptosis. 18 ATP Figure 2. Putative interactive signaling model between endoplasmic reticulum (ER) and mitochondria (MT) during apoptosis. Caspase-8 cleaves Bap31 into a pro-apoptotic 20 kDa (p20) protein. This process is inhibited by Bcl-2 or Bcl -X L . Bax may promote p20 formation via heterodimerization of Bcl-2 /X L thereby facilitating Bap31 cleavage. The E R releases C a ^ during apoptosis which may be inhibited by Bcl -2 . It is not known i f the ER-associated BI-1 protein can also inhibit this process. Conversely, mitochondria can also act as a reservoir for excess Ca 2 + , and may release Ca 2 + during apoptosis. Increased intracellular C a + + induces mitochondrial PT, which has been shown to cause increased proton buildup in the mitochondrial intermembrane space. Although the role of mitochondrial caspase-3 has not been elucidated, this increased intermembrane space acidification may promote autocatalytic processing of intermembrane-localized caspase-3. Bcl-2 /X L proteins may act as pores which dissipate proton buildup. Thus, Bcl-2 /X L may prevent autocatalytic activation of caspase-3 by preventing intermembrane acidification. AIF , Caspase-2 and caspase-9 have also been shown to be released from mitochondria during apoptosis. Bcl-2 /X L block cyt c release during most forms of apoptosis. Released cyt c complexes with ATP, Apaf-1 and pro-caspase-9 to stimulate caspase-9 activation, which then processes downstream caspases. 19 Whether the primary role of Bcl-2 on the mitochondria is as an ion channel or as an adaptor/docking protein or both requires further elucidation, as do the roles of other members of the Bcl-2 family. The importance of Bcl-2 family members with respect to their function and binding capabilities to the E R and nucleus requires much greater definition. 1.1.3.4 Nuclear Factor-KB Individually, Fas-L, anti-Fas antibody and T N F induce cell death in a limited number of cell types and require the presence of cycloheximide, an inhibitor of protein synthesis, to induce apoptosis in other cell types 3 5 . Resistance to TNF-induced apoptosis is believed to be due to the activation of nuclear factor K B (NF-icB)-mediated transcription " 6 ' n 7 . T N F can elicit an anti-apoptotic pathway via the recruitment of T R A F - 2 which binds to NF-KB-inducing kinase ( N I K ) " 8 . N I K is a member of the M A P kinase kinase kinase ( M A P 3 K ) family " 8 and serves as a common mediator downstream of T R A F - 2 in the N F - K B signaling cascades triggered by stimuli such as T N F , IL-1 and other receptor-associated proteins . Unlike T R A F - 2 , which is involved in both N F - K B and the c-Jun N-terminal kinase (JNK) activation, N I K does not activate the J N K 119 pathway and is solely dedicated to N F - K B activation . However, N I K is not able to directly 120 phosphorylate I K B . Recently, it was discovered that C H U K (conserved helix-loop-helix 121 ubiquitous kinase), a previously described serine-threonine kinase (now referred to as I K B 120 kinasea ( IKKa) ) directly associates with N I K and phosphorylates I K B O I . Since I K K a is an 120 I K B O . kinase, it directly links N I K to N F - K B activation . DiDonato et al. (1997) have also 122 recently isolated a similar I K B kinase ( I K K ) . Phosphorylation o f I K B triggers proteasome-mediated degradation and subsequent removal of I K B , allowing N F - K B to translocate into the nucleus 1 2 3 . Interestingly, it has been demonstrated that caspase-3 cleaves I K B in vitro n . 20 Furthermore, phosphorylation of IKB blocks caspase-3-mediated cleavage of IKB . These results suggest a possible link between caspase activation and the N F - K B pathway through IKB. In addition to its role in immune responses, N F - K B stimulates the transcription of pro-survival genes such as A l , A20 , BC1-XL, TNFR-associated factors 1 and 2 (TRAF1 and 2) and inhibitor o f apoptosis proteins (IAPs) 1 2 4 1 2 6 . Wang et al. 1 2 7 have demonstrated that N F - K B activation can be blocked using a super-repressor form of the N F - K B inhibitor molecule IKBCC. Unlike IKBCC, the mutant form of IKBOC does not become phosphorylated and subsequently degraded, thereby N F - K B translocation and subsequent activation do not occur. Effectively blocking NF-KB-mediated transcription rendered formerly TNF-resistant cells susceptible to T N F -116 117 127 induced apoptosis ' ' . Furthermore, Chu et al. (1997) have recently shown that N F - K B is responsible for the transcription of the anti-apoptotic inhibitor of apoptosis (c-IAP-2) gene. Interestingly, when overexpressed in mammalian cells, C-IAP2 activates N F - K B and suppresses 128 T N F cytotoxicity . Both of these C-IAP2 activities were blocked in vivo by coexpressing a 128 dominant form of IKB that is resistant to T N F - induced degradation . These findings suggest that C-IAP2 could be involved in the positive feedback control on N F - K B by an IKB targeting 128 mechanism . 1.1.3.5 Calcium Increases in the levels of intracellular C a 2 + have been associated with the regulation of numerous apoptotic events (Figure 3). C a 2 + may be required for the activation of endonucleases 129 9+ associated with D N A fragmentation during apoptosis . Additionally, C a may also play a role in the activation of proteases involved in apoptosis 1 3°. 21 Figure 3. Summary of how ischemia/reperfusion injury during cardiac allograft transplantation may affect intracellular C a 2 + regulation and apoptosis. In the absence of oxygen, cells resort to anaerobic means in order to generate ATP. In the process, the intracellular milieu becomes highly acidic. Increased H + causes N a + - H + exchanger (NHE) to export FT while Na+enters the cell. Free radicals and decreased A T P levels leads to disabled Na + -K + -ATPase activity leading to elevated intracellular N a + levels. Excess N a + may then be removed via the Na + -Ca 2 + exchanger ( N C X ) resulting in an influx of Ca 2 + . C a 2 + may also enter the cell v ia voltage-gated L-type channels. Elevated C a 2 + binds to the SR ryanodine receptors (RyR) to stimulate further C a 2 + release. Free radical-mediated S E R C A 2 a damage results in the inefficient replenishment of SR C a 2 + stores. Elevated C a 2 + may contribute to apoptosis via the direct or indirect induction of mitochondrial cyt c and caspase-9 release, activation of calpains, activation of certain endonucleases, activation of calmodulin (CaM)/calcineurin or via the activation of sarcolemma scramblases and inactivation o f phospholipid translocases leading to externalization of phosphatidyl serine (PS). C a 2 + -mediated calcineurin activation can also dephosphorylate N F A T (nuclear factor o f activated T cells) resulting in its translocation into the nucleus and subsequent transcription of hypertrophic response genes in myocytes. 22 Ca is required for the externalization of phosphatidylserine (PS) to the outerleaflet of the cell membranes of apoptotic cells 1 3 I n c r e a s e d C a 2 + activates scramblases, which translocate PS to the outerleaflet, while simultaneously inactivating the constitutively active aminoacid phospholipid translocases that act to transport PS to the inner leaflet. PS is one of many cell surface markers now identified that facilitate the recognition and uptake of apoptotic bodies by phagocytes 1 3 2 ' 3 3 . Early recognition and phagocytosis of apoptotic cells by neighboring cells is essential for the rapid removal of these cells before they lose their membrane integrity and release their contents. Although it is well-known that Bcl-2 and BC1-XL localize to the outer membranes of mitochondria, E R and nuclei, the vast majority of work on these proteins has focussed on mitochondria and very little is understood as to how these proteins affect the E R or nuclei. C a 2 + can facilitate or induce mitochondrial cyt c release 1 3 4 1 3 6 . Recent evidence in tumour cells suggests a role 137 138 for Bcl-2 in the regulation of calcium uptake by the E R ' . Previous studies have indicated that depletion of E R Ca levels with thapsigargin (TG) causes apoptosis . Bcl-2 has been shown to 0+ 9-+- 137 preserve E R C a levels by maintaining Ca uptake in the E R . In support of the latter concept, 140 i4i Bcl-2 has been shown to form cationic-selective pores in liposomes ' . Additionally, in breast epithelial cells, Bcl-2 modulates E R Ca stores via the upregulation of sarco/endoplasmic reticulum C a 2 + ATPase-2 (SERCA-2) expression and was shown to interact with S E R C A - 2 in co-138 immunoprecipitation studies . Interestingly, similar to Bcl-2, S E R C A - 2 may also localize to the 142 _+ nucleus as well as the E R . However, the effects of S E R C A - 2 or Bcl-2 on nuclear Ca are not 143 known. S E R C A - 2 has been shown to be highly sensitive to free radicals , thus, since P D T generates reactive oxygen species, damage of S E R C A - 2 and subsequent E R depletion may be a primary event involved in cell death that is instigated by this stimuli. Whether Bcl-2 can compensate for damaged S E R C A - 2 requires further elucidation. In rat fibroblasts transfected with Bcl-2 specifically targeted to 23 the E R , Bcl-2 was shown to block Myc-induced but not Etoposide-induced apoptosis , suggesting that Bcl-2 expression pertaining specifically to the E R provides a protective effect to some, but not all forms of apoptosis. C a 2 + has been shown to activate calcineurin, a calcium-dependent phosphatase, resulting in the dephosphorylation of the pro-apoptotic Bcl-2 family member termed Bad. Upon dephosphorylation, Bad has been shown to migrate to mitochondria, bind to BC1-XL and induce cyt c release 1 3 5 . Bad is retained in an inactive, anti-apoptotic state when it is phosphorylated. The calcineurin inhibitor cyclosporin can inhibit apoptosis by preventing the mitochondrial release of cyt c 134 145 into the cytosol in response to certain stimuli ' . It is attractive to speculate that a link may exist between S E R C A - 2 damage, increased intracellular C a 2 + , calcineurin activation, and apoptosis via calcineurin-mediated Bad dephosphorylation. Alternatively, increased intracellular Ca may stimulate cyt c release directly, or involve a combination of both mechanisms. Interestingly, recent studies have shown that Bcl-2 can also bind to calcineurin, preventing dephosphorylation and nuclear tanslocation of N F A T (nuclear factor of activated T lymphocytes), a transcription factor activated by 146 microtubule damage and subsequent transcription and translation of FasL . Recent studies have shown that the serine/threonine protein kinase A k t / P K B acts as an anti-apoptotic entity upon activation by phosphorylating Bad. Interestingly, E C growth factors such as vascular endothelial growth factor (VEGF) , which have been shown to provide protection to E C against apoptosis, stimulate the Akt pathway as well as upregulating Bcl-2 and A l gene expression in E C . In addition, Ak t has been shown to not only phosphorylate Bad but also caspase-9 thereby antagonizing the apoptotic pathway . Ak t has also been shown to activate eNOS in a Ca -148 149 independent manner in endothelial cells resulting in N O formation ' . During myogenesis, proliferating myoblasts withdraw from the cell cycle, acquire an apoptosis-resistant phenotype, and 24 differentiate into myotubes . It is now known that Akt is induced during myogenic differentiation with a corresponding increase in kinase activity ' 5°. In differentiating cultures, expression of dominant-negative forms of Akt increase the frequency of myocyte cell death, whereas expression of wild-type Akt protects against death, indicating that Akt is a positive modulator of myocyte survival ' 5°. Furthermore, during apoptosis, survival-promoting kinases such as Ak t have been shown to be cleaved by caspases, possibly to eliminate the elicitation of pro-survival signals in cells committed to 75 9+ death . Thus, it is likely that increased intracellular Ca may downregulate the Akt pro-survival signal via the activation of calcineurin with subsequent dephosphorylation of Bad and caspase-9. Such a chain of events would allow Bad to translocate to mitochondria, induce cyt c release and trigger the onset of apoptosis via the activation of caspase-9. These events are summarized in figure 4. 25 Survival signals (VEGF, Insulin, IGF, FGF, PDGF) Figure 4. Putative mechanism by which pro-survival growth factors stimulate the Ak t pathway and how increased C a 2 + may disable the protective effects of Ak t through calcineurin-mediated dephosphorylation of Bad. Upon binding to their specific receptors, certain growth factors can stimulate phosphorylation and activation of the kinase Akt . Ak t has been shown to prevent apoptosis by phosphorylating Bad leading to its sequestration in the cytosol to 14-3-3 proteins. Akt can also phosphorylate caspase-9 leading to its inactivation,or stimulate eNOS and/or N F - K B activity. Increased C a 2 + leads to the activation of the calcium-dependent phosphatase calcineurin. Calcineurin can promote apoptosis by dephosphorylating Bad thereby allowing it to translocate to mitochondria where it may bind to B c l - X L , displace Bax and promote cyt c release. Calcineurin also dephosphorylates N F A T which allows N F A T to translocate into the nucleus and stimulate gene transcription. Bcl-2 can prevent N F A T translocation as well as cyt c release and, in some cases, increased Ca 2 + . 26 1.2 B i o l o g i c a l S i g n i f i c a n c e Since the realization that apoptosis is a complex, integrated physiological process, this form of cell death has become one of the most intensely studied phenomena in biology. The regulation of homeostatic balance between cell proliferation and cell death is imperative for development and maintenance of multicellular organisms. Disturbance of signaling pathways that regulate apoptosis, whether by extracellular triggers, acquired or germline genetic mutations, or viral mimicry o f signaling molecules can result in a wide array of human diseases including cancers, infectious diseases, autoimmune diseases and many neurodegenerative and neurodevelopmental diseases 1 5 \ In pathologic states, resistance to cell death by apoptosis may 152 play a fundamental role in tumourigenesis . Comprehending the molecular processes that are involved w i l l lead to a better understanding of the etiology and pathogenesis of these and many other diseases, possibly one day resulting in new therapeutic modalities. The following sections demonstrate the wide diversity of diseases in which apoptosis has been shown to be involved and they are by no means meant to be a comprehensive review of such topics. 1.2.1 C a r d i o v a s c u l a r 1.2.1.1 T r a n s p l a n t V a s c u l a r Disease Cardiac transplant vascular disease (TVD) remains a troublesome long-term complication of heart transplantation. T V D is manifested by a unique and unusually accelerated form of coronary disease affecting both intramural and epicardial coronary arteries and veins ' 5 3 ' 1 5 4 . T V D is characterized by vascular injury induced by a number of factors including the immune response to the allograft, ischemia-reperfusion injury, viral infection, immunosuppressive drugs, and classic risk factors such as hyperlipidemia, insulin resistance, and hypertension 1 5 3 154_ Balloon 27 angioplasty has a limited role in the treatment of focal lesions. Experiences with coronary stenting, coronary artery bypass grafting, and transmyocardial laser revascularization have been reported to be limited *55. Furthermore, retransplantation tends to have a worse outcome than the original initial transplantation 1 5 5 . Thus, cardiac allograft vasculopathy remains a troublesome long-term complication of heart transplantation and is the major cause of death in patients surviving 1 year after transplantation 1 5 6 . T V D selectively involves the vascular bed of the allograft, including the donor aortic segment while sparing native vessels throughout the body Due to inadequate re-innervation of the cardiac allografts, most heart transplant recipients cease to experience typical anginal pain 153 associated with myocardial ischemia or infarction . Consequently, the first clinical manifestations are often ventricular arrhythmias, congestive heart failure, or sudden death Furthermore, intimal thickening is detectable, using intracoronary ultrasound, in as many as 75% of patients within one year after transplantation Therefore, T V D represents a serious concern for patients and physicians following transplantation. 1.2.1.2 Animal Models of T V D Over the past decade, several insights into the understanding of T V D have been made possible due to the development of animal models of T V D . Numerous recent experimental studies have suggested a role for apoptosis in heart transplant rejection, however, the causation, 158 159 role and mechanisms of apoptosis in T V D are poorly understood ' . In the rat T V D model, whereby a donor heart is transplanted into the abdomen of the recipient, apoptosis was observed at higher levels in allogeneic versus syngeneic grafts 1 6 0 m . Using various strains of rat T V D models, apoptosis has been observed at higher levels in E C , S M C as well as cardiac myocytes, 28 macrophages and lymphocytes in allogeneic versus syngeneic hearts In our laboratory, a Lewis-to-Fisher rat cardiac allograft model of acute and chronic cardiac rejection has been established 1 6 1 1 6 4 . In this model, apoptotic EC, lymphocytes, SMC and myocytes were detected in cardiac allografts 1 6 1. Furthermore, Bax immunopositivity was associated with TUNEL positive EC, SMC and leukocytes, but not with TUNEL-positive ischemically-damaged myocytes 1 6 1. However, differences in Bcl-2 immunopositivity were not observed between native hearts, allografts or syngrafts 1 6 1. In collaborative studies with Koglin et al. 165, the involvement of certain apoptotic proteins were assessed using a mouse heterotopic transplant model. Mouse cardiac allografts transplanted into inducible nitric oxide synthase (iNOS)-/- recipients had reduced apoptotic activity as compared with those transplanted into iNOS+/+ recipients 1 6 5. Transplants into iNOS-/- recipients were associated with lower TUNEL-positivity, reduced caspase activity and PARP cleavage, and lower levels of p53 and Bax transcript levels ' 6 5 . Thus, these studies demonstrate that iNOS pathways may be an important mediator in the promotion of acute rejection at least in part due to its role in stimulating apoptosis. We further speculated that iNOS may stimulate apoptosis via p53-mediated upregulation of Bax. Thus, Bax may play a key role in mediating apoptosis during allograft rejection. 1.2.1.3 Apoptosis in Human TVD Apoptosis has also been reported in a number of studies that examined human endomyocardial biopsies 1 5 8. Laguens et al 1 6 6 studied 63 endomyocardial biopsy specimens obtained from 6 patients, 7-to-146 days after transplantation. Myocyte apoptosis was observed in specimens exhibiting signs of rejection and appeared to correspond to the severity of rejection. 29 Apoptosis was, for the most part, adjacent to areas o f inflammation. Interstitial cell and endothelial cell apoptosis was observed in all degrees of rejection, although it was more prevalent in more severe forms m . In subsequent studies of 22 endomyocardial biopsies from cardiac allograft recipients, Jollow et al. 1 6 7 observed more prominent apoptosis of inflammatory cells as opposed to myocytes. In the latter studies, apoptosis of myocytes and inflammatory cells was seen in all biopsy specimens. A s a control for these studies, only one biopsy specimen with no inflammatory infiltrates and no myocyte damage was included. In subsequent studies, right ventricular endomyocardial biopsies from 30 cases of allograft rejection were compared with 12 biopsies with no rejection ' 6 8 . Inflammatory cell infiltration associated with myocyte damage was associated with 18 of the rejected allograft specimens. There was a 30-fold increase in apoptotic cells compared to controls. The cellular infiltrate was rich in CD68+ macrophages and CD4+ lymphocytes in the regions with myocyte death, but the infiltrates were composed predominantly of CD8+ T cells in the areas of myocardium with lesser degrees of myocyte damage. Although much of the myocyte apoptosis observed was proximally associated with inflammatory cells, cardiac myocyte apoptosis was also observed in areas without inflammation in the severely rejecting allografts. Therefore, it is likely that the cause of apoptosis following transplantation involves a complex milieu of factors including immune cell-derived factors in addition to other non-immune cell related factors. It has been proposed that apoptosis plays an important role in the slow ongoing process of chronic rejection that results in chronic left ventricular dysfunction and allograft vasculopathy 1 5 8 . In a mouse model, Bergese et al 1 8 ' observed apoptosis primarily in perivascular areas 60 days after transplantation. Conversely, others observed greater intimal thickening, apoptosis and 182 Fas-Ligand m R N A expression in allograft vessels than isograft vessels . In our laboratory, 30 apoptosis was assessed in vessels obtained from 12 cardiac allograft recipients and were compared with 10 severe atherosclerotic coronary artery disease and 14 normal coronary arteries 1 8 3 . Virtually all endothelial cells and one-third of the T cells were Fas-positive in allograft vessels. Approximately 20% of Fas-positive T cells and macrophages were apoptotic in the mild and moderate vasculopathy as compared to 80% in severe transplant vasculopathy whereas 75% of E C were apoptotic in mild-to-moderate vasculopathy as compared with 10% in the severe 183 vasculopathy group . In contrast to transplant vasculopathy, apoptosis was observed mainly in 183 macrophages of the lipid-rich cores of atherosclerotic disease . In the latter study, of the 183 normal arteries assessed, few E C were Fas-positive and even less were apoptotic . In transplanted hearts, donor antigens such as M H C class I and II are expressed on vascular E C which may lead to induction of the cellular and humoral immune responses leading to the accumulation of T lymphocytes and antibodies, respectively in the subendothelial compartment 1 8 4 . Endothelial injury may result in the secretion of P D G F , TGF(3, IL-1 and IL-6, which contribute to vascular remodeling ' 8 5 . The role of T N F - a in allograft vasculopathy is not entirely understood, but recent in vitro work may be relevant. In E C , stimulation with T N F - a may elicit either the initiation of apoptosis or the activation of N F - K B leading to the induction of an inflammatory response 1 8 6 1 8 7 . Stimulation of the N F - K B pathway also induces transcription and translation of several anti-188 189 apoptotic genes ' , whereas inhibition of the N F - K B pathway sensitizes endothelial cells to TNF-cc-induced apoptosis . Both Fas and FasL have been reported to be expressed on vascular 190 E C , although these cells are not susceptible to FasL-induced death under normal conditions However, in vitro studies have shown that under conditions of oxidative stress (increased levels of oxidized L D L ) , E C become susceptible to FasL-induced apoptosis, suggesting a role for 31 191 oxidative stress in atherogenesis . Conversely, smooth muscle cells do not express FasL, but 190 are susceptible to FasL-induced apoptosis . Additional evidence of a role for Fas in allograft rejection stems from studies demonstrating increased Fas expression on apoptotic endothelial 183 cells associated with allografts exhibiting transplant vascular disease . The latter study, combined with the notion of increased susceptibility of endothelial cells to FasL-mediated kil l ing under conditions of oxidative stress, which are likely to occur, provides an attractive mechanism by which endothelial damage may evolve. Furthermore, under normal conditions, it has been suggested that FasL expressed on vascular endothelial cells may function to inhibit leukocyte extravasation by inducing apoptosis of Fas-bearing immune cells including activated T-cells or 192 macrophages . During oxidative stress-induced E C apoptosis, mitochondrial cyt c is released 193 into the cytosol followed by the activation of caspases-2, 3, 6, 7, 8 and 9 . Interestingly, T N F - a can downregulate FasL expression resulting in decreased endothelial cell-mediated cytotoxicity 192 towards Fas-bearing cells such as activated T-cells and macrophages . Although great strides have been made in recent years regarding the involvement of Fas and T N F in vascular injury, further studies are necessary to delineate the precise role of these receptors and their respective ligands in cardiac transplant vascular disease. In summary, apoptosis is present in human cardiac allograft rejection and may occur with all degrees of rejection, and even in its absence. The prevalence and severity of apoptosis is determined predominantly by the intensity of macrophage infiltration and may be mediated by nitric oxide-related mechanisms. Apoptosis of interstitial, endothelial and inflammatory cells is also present in heart allografts and may influence the degree and extent of vascular injury contributing to allograft rejection. A summary of how various factors may contribute towards allograft vasculopathy is shown in figure 5. Ongoing apoptosis of inflammatory cells suggests an 32 immunoregulatory role. Further understanding of the role and causality of apoptosis as well as the cellular and biochemical mechanisms that are involved in cardiac myocyte death, as well as in inflammatory, endothelial and interstitial cell death, may provide insights into therapeutic modalities to suppress allograft rejection and vasculopathy. 1.2.1.4 Cardiac Ischemia/Reperfusion Injury Ischemia and reperfusion in transplantation must be considered as early contributors to endothelial as well as myocardial injury. It is likely that endothelial activation and the generation of oxygen radicals leads to subsequent activation and recruitment of host leukocytes and 153 macrophages . This post-ischemic reperfusion injury resulting early on in endothelial activation 153 and recruitment of inflammatory cells may then evolve secondary to interstitial injury . Under conditions of ischemic myocardial stress that may be associated with transplant rejection and cardiac surgery, the heart can be subjected to episodes of ischemia followed by reperfusion (IR injury). Ischemia followed by reperfusion can result in decreased myocyte viability that may lead to heart failure and ultimately to death. The characteristic pattern of ischemic cell injury involves fluid and electrolyte alterations including the loss of intracellular K + and M g + ions and the accumulation of water, N a + , CF , FT and C a 2 + ions m ' . Morphological characterization reveals the appearance of cytoplasmic, organellar, and cellular swelling with membrane blebbing and margination and clumping of nuclear chromatin ' 6 9 . Myocardium undergoing ischemic cell death exhibits both necrotic and apoptotic forms of cell death. In a rat model of myocardial infarction, apoptosis has been reported to include the majority of myocytes within the infarct during the first few hours in the evolution of the M I n ° . Interestingly, in the rat model, very little necrosis was observed during the first few hours of 33 ischemia, but was readily detectible by 24 h n ° . However, it should be noted that other 171 172 investigators have shown that apoptosis only occurs during reperfusion ' , while others still, have suggested oncosis (often used as synonymous with necrosis), cell death with swelling, as the 20 173 dominant form of cellular demise observed during ischemia ' ; Despite uncertainty, increasing evidence suggests that apoptosis does play a role in ischemia and associated myocardial injury 68,169,174,175 , , , - 172,176,177 . Furthermore, several studies have demonstrated a role lor caspases in IR injury In addition, caspase inhibition during IR injury has been shown to attenuate myocardial injury associated with reperfusion' 7 6 ' 1 7 7. In contrast to necrosis, apoptosis allows for the removal of scattered individual cells rather than cell groups or a whole tissue or organ compartment '°. However, under conditions such as IR during which larger confluent cell populations may be affected, it is plausible that many apoptotic cells may be present. In scenarios such as IR injury, during which the majority of cells within a given area may be apoptotic, these cells are not likely to be rapidly phagocytosed, since there are not enough healthy cells immediately subjacent to engulf these bodies. Thus, these cells may become secondarily necrotic resulting in membrane rupture, the release of cellular contents, exacerbation of necrosis and the induction of an inflammatory response. Based on this rationale, one may expect the inner core of the infarct to become primarily necrotic due to inefficient clearance of apoptotic debris as discussed. Conversely, on the borders of the infarcts, one may expect to observe a predominance of apoptosis, since these cells can be adequately removed on a single cell basis. In support of this concept, several investigators have observed a higher incidence of apoptotic cells on border zones of the infarcts ' 7 6 ' 7 8 . Recent studies have shown that caspase inhibition during liver ischemia can completely abrogate the cell death that 179 occurs in ischemic liver lobes . Furthermore, using a murine model to investigate the 34 involvement of inflammation in organ damage during renal ischemia, inhibition of apoptosis 180 resulted in the elimination of an inflammatory response as well as tissue injury . Further studies are required to determine the precise role of IR injury and apoptosis in transplant vascular disease. 35 Figure 5. Potential contributors to E C and S M C apoptosis in an atherosclerotic plaque. Increased endothelial permeability allows for increased levels of l ipid to cross the endothelial barrier. Monocytes/Macrophages (M) may oxidatively modify L D L ( O x L D L ) . O x L D L is consumed by macrophages and smooth muscle cells ( S M C ) which may lead to foam cell formation. Increased S M C migration and proliferation also contribute to intimal hyperplasia. Infiltrating T cells and macrophages can express FasL, T R A I L or TNF. O x L D L sensitizes E C to FasL-induced apoptosis and may also alter their response to T N F and T R A I L . These death-inducing ligands may also induce S M C apoptosis as well as induce apoptosis of other immune cells. Apoptotic E C likely detach from the endothelial lining and are shed into the bloodstream. O x L D L can interfere with the phagocytosis and clearance o f apoptotic bodies. Apoptotic bodies express phosphatidylserine (PS) on their surface. PS is a co-factor in the coagulation cascade. Thus, high levels o f apoptotic bodies due to inadequate clearance leads to increased PS which may promote coagulation and possibly thrombosis. 36 1.2.2 Cancer Cells respond to genetic insult by inducing cell cycle arrest, activating repair mechanisms 194 prior to replication or by committing suicide i f the damage is beyond repair . However, during oncogenesis, genetic alterations may occur which disable the cell 's ability to commit suicide (Figure 6) 2 6 ' 9 4 . Conversely, it has been well established that most chemotherapeutic agents 195 eliminate tumours through an apoptotic mechanism . Thus, it has been suggested that many tumours that are intrinsically resistant to chemotherapy are not capable of activating their . . 194 apoptotic machinery . Several proteins have been implicated in the inhibition of apoptosis during oncogenesis and resistance to chemotherapy. Unlike that of the multidrug resistance (MDR)-1 family of drug efflux pump proteins, these anti-apoptotic proteins do not interfere with the entry into and accumulation of drugs in tumour cells ' % . Thus, anti-apoptotic proteins constitute a novel type of multidrug resistance proteins m . The. tumour suppressor gene, p53, is not only associated with a 197 poorer prognosis in many malignancies , but also in resistance to chemotherapy and y-198 irradiation as well as in tumour relapse . Nevertheless, apoptosis can be induced by many 199-201 chemotherapeutic agents, including PDT, in p5 3-defective tumour cells . It has also been demonstrated that increased Bcl-2 or BC1-XL expression renders cells resistant to most forms of chemotherapy 1 5 2 . Thus, drugs that induce apoptosis independent or downstream of BC1-2/XL would be useful tools in the battle against cancer. Sti l l other tumours may constitutively express proteins such as FasL on their cell surface. This is often referred to as "Fas counterattack" during which Fas receptor-positive immune cells are killed off by FasL expressing tumour cells thereby „ . , i i - 202,203 allowing the tumour to evade the immune system 37 Large Intestine Progression of carcinoma -Failure of cells with DNA damage to undergo apoptosis (Bcl-2, p53 mutation, Bax, FasL, etc.) Tumor formation Liver. Metastasis -detachment of cancerous cells into bloodstream -failure of detached cells to undergo apoptosis Establishment of new tumor growth Resistance -overexpression of anti-apoptotic genes or downregulation of pro-apoptotic genes renders the cancerous cell resistant to an immune-mediated response as well as to many forms of chemotherapy -certain tumours may also increase the expression of FasL thereby inducing apoptosis of infiltrating anti-tumour Fas-positive immune cells such as activated T cells and macrophages (aka "Fas counter-attack") Progression Figure 6. Critical areas in which defective apoptosis may contribute to the onset and progression of cancer. D N A damage or alteration occurs within a cell in the primary organ (eg. colon) that the cell fails to repair. A t this stage the cell, v ia regulation by p53, normally stops dividing and commits to programmed cell death. However, the apoptotic machinery is not mobilized, thereby allowing cell survival and permitting cell division resulting in primary tumor formation. During metastasis, cells detach from the matrix, an event which triggers apoptosis in most adherent cells. However, cells that overexpress anti-apoptotic genes (Bcl-2, B c l - X L N F - K B , etc.) or contain abnormalities in pro-apoptotic gene expression (Bax, p53, FasL, IAP, etc.) survive and continue to divide. These cells migrate and reattach within secondary sites (eg. liver). Due to the overexpression of anti-apoptotic genes such as Bcl-2 , B c l - X L , F L I P or defective p53, these tumor cells are resistant to most forms of chemotherapy which act through the induction o f apoptosis. 38 Since the discovery of T R A I L , research has been devoted to determining what makes tumour cells more susceptible than healthy cells to TRAIL-induced apoptosis 2 0 4 2 0 5 . T R A I L is a membrane bound or soluble protein that induces apoptosis in many transformed cell lines but does not affect normal cells, even though its death domain-containing receptors ( D R 4 / T R A I L R l or D R 5 / T R A I L R2), may be expressed on both cell types 2 0 6 2 0 9 . It has been suggested that normal cells, but not transformed cells, contain at least one 'decoy' receptor ( T R I D / D c R l , 208 210 212 T R U N D D / D c R 2 ) which binds to T R A I L and attenuates the apoptotic effect of T R A I L ' Furthermore, T R A I L has been shown to mediate cell death in a variety of human haematological 213 malignancies . Unfortunately, a recent study has demonstrated that T R A I L can also induce apoptosis in normal human hepatocytes 2 ' 4 . Thus, although T R A I L did not produce any signs of 38 215 cytotoxicity in animals (mouse, monkey) or normal human cells studied to date ' , these studies indicated that the use of T R A I L may be detrimental when administered to humans 2 1 4 , 2 1 6 . Therefore, since TNF-oc, FasL and T R A I L have differing levels of cytotoxicity on normal cell types 2 1 4 , 2 1 7 , 2 1 8 5 m e feasibility of using these ligands in systemic therapeutic applications may be 215 limited. Nonetheless, activated immune cells do express these ligands and may participate in anti-tumour defense. However, the majority of studies have previously shown that T R A I L is 25 effective at inducing apoptosis in a majority of transformed cell lines but not normal cell lines . The resistance of normal cells to TRAIL-induced apoptosis was initially believed to be due to the presence of decoy receptors, T R A I L - R 3 ( D c R l ) and T R A I L - R 4 (DcR2), that lack functional 210 cytoplasmic death domains . However, differential cell susceptibility to T R A I L is not completely defined as the decoy receptor paradigm is not always true. In some cases, cells that express T R A I L - R 1 and T R A I L - R 2 but not decoy receptors are resistant to TRAIL-induced death 219 . Conversely, there are cell lines that express decoy receptors that are susceptible to T R A I L -39 mediated apoptosis . With at least five T R A I L receptors, intracellular molecules such as FLIPs, Bcl-2 family members, and anti-apoptotic signaling factors such as N F - K B or PI3-K / P K B ( A k t ) are likely to influence cell susceptibility to T R A I L . In summary, it is becoming increasingly evident that the same genetic defects that enhance the growth and survival of tumour cells may also be responsible for their resistance to various anti-cancer treatments. Increased knowledge of the molecular processes that are involved in apoptotic cell death should reveal potential chemotherapeutic targets that can selectively restore the apoptotic potential to tumour cells. 1.2.3 Development M u c h of our understanding of the role of apoptosis in development can be attributed to the studies of the nematode C. elegans in which it was first determined that cell death was 220 strictly determined in cell lineage during development . Since the initial findings that programmed cell death in C. elegans is genetically controlled, intensive studies have led to the identification of several key molecules that are involved in the regulation of apoptosis 2 2' 2 2 4 . Furthermore, the genes controlling cell death appear to be highly conserved throughout evolution from nematodes to mammals, which has led to the discovery and understanding of several 49 51 225-228 human genes that are associated with the regulation of apoptosis It is now recognized that programmed cell death is as much a part o f embryonal 229 development as cell proliferation and differentiation . Programmed cell death has evolved as the biological method of deleting cells that are harmful, unneeded or in excess during embryonic 229 development . Programmed cell death plays a crucial role in sculpting parts of the body. One example is the formation of digits in which apoptosis eliminates the cells between the developing 40 digits . M o r i et al. have found that not only does apoptosis play a role in the separation of 230 digits but also in the formation of joint cavities . Programmed cell death is also involved in the 231 formation of lumina or the removal of vestigial structures during development . It has been suggested that programmed cell death is responsible for the elimination of the Mullerian duct (forms the uterus and oviducts) in males and the Wolffian duct (forms the vas deferens, 231 epididymis and seminal vessicles) in females . In addition to its role in developmental morphogenesis, apoptosis continues to influence tissue homeostasis in the adult. Through the menstrual cycle, the process of cell death and 232 renewal in the female reproductive tract is tightly regulated . Follicular atresia and the cyclic 232 shedding of the endometrium involve the process of apoptosis . Similarly, placental apoptosis increases significantly as pregnancy progresses, suggesting a role in the normal maturation of the 232 233 placenta ' . Ce l l death is pivotal in the development of the nervous system in which virtually 234 all cell populations undergo apoptosis to some extent during early development . During 231 neuronal development, it is estimated that over 50 percent of neurons undergo apoptosis . The importance of apoptosis during neuronal development can be exemplified using caspase-3 235 knockout mice in which neuronal apoptosis does not occur . These mice are born with duplicate organs in the nervous system and the brain virtually protrudes into the back of the 235 retina and out through the skull . In contrast, the loss of cells in the human nervous system during adulthood has long been known as a hallmark of incurable degenerative disease. It has become apparent that apoptosis is frequently involved in this reduction of cell number Apoptosis also plays a critical role during the development of the heart. The level of B c l -2 expression has been reported to be lower in ventricular myocytes isolated from the right side of the heart as compared to those from the left side and the extent of myocyte cell death appears 41 237 inversely related to Bcl-2 expression . Since apoptosis affects cells of the right ventricle more 237 than the left ventricle during postnatal development , regulation of apoptosis may be responsible for the differential thickness observed in the right and left ventricles. A striking example of developmental apoptosis occurs in the inner ear of vertebrates, which acquires a precise three-dimensional arrangement of their constituent epithelial cells to form three semicircular canals, a central vestibule and a coiled cochlea. For the generation of each semicircular canal, it has been suggested that epithelial cells may 'disappear' from the 238 center of each canal by an apoptotic mechanism . In summary, apoptosis is a tightly regulated process influencing virtually all aspects of 229 embryonic and fetal development . Thus, it is understandable how exposure to certain drugs or viral infections during pregnancy which affect apoptosis may result in developmental abnormalities such as cleft palate, neural tube defects, hypospadia, syndactylism or other , , . 229,234,236,239 embryopathies 1.2.4 A g i n g The removal of damaged or senescent cells is paramount to the maintenance of multicellular organisms. There are in general, two ways in which apoptosis can play a role in the aging process: (1) by the elimination of damaged and presumably dysfunctional cells (e.g., fibroblasts, hepatocytes) which can then be replaced by new cells, thereby maintaining tissue homeostasis and (2) through the elimination of irreplaceable postmitotic cells (e.g., neurons, 240 cardiac myocytes) thereby predisposing the subject to disease . Many cell types may lose their 241 ability to undergo apoptosis with age . Thus, a general decline in organ function and/or many hyperproliferative disorders are commonly associated with aging and may, to some extent, be the 242 result of deregulated apoptosis . Although little direct evidence has been forwarded to support 42 this hypothesis, fibroblasts have been shown to lose their ability to downregulate Bcl-2 , thereby 241 rendering senescent fibroblasts resistant to apoptosis . Similarly, there is an increase in the incidence of chromosomal translocation resulting in Bcl-2 upregulation in B-cells with 241 increasing age . Other cell types, such as thymocytes and lymphocytes, appear to lose the 241 ability to initiate the death pathway due to down-regulation of the cell surface receptor Fas Increased susceptibility to infectious and autoimmune diseases is believed to be correspond with 243 advancing age in otherwise healthy individuals . Recent studies in animal models of aging have indicated that age-associated immune dysfunction may be associated with defects in T cell apoptosis 2 4 3 . Furthermore, derangement of the immune system with aging may correspond to 242 dysregulation of cytokine production. Many age-related neurodegenerative diseases are characterized by the loss of specific neurons: Huntington disease (striatum), Parkinson disease (substantia nigra), Alzheimer disease 241 (hippocampus and cortex) and amyotropic lateral sclerosis (motor neurons) . Thus, accumulating evidence implicates apoptosis as the mechanism responsible for neuronal loss in 241 many age-related neurological diseases . In other areas, recent data have suggested that hearing impairment and dysequilibrium in the elderly may be due to apoptosis occurring in the inner ear 244 . c . Others have shown that decreased numbers of retinal ganglion cells in glaucomatous eyes of 245 elderly patients is caused by apoptosis . However, many of the changes that occur during age-associated organ deterioration may be precipitated by other disease processes which damage 242 cells thereby inducing the cellular apoptotic response . 246 Estrogen deprivation in post-menopausal women causes bone loss . Recently, estrogen has been shown to inhibit bone resorption by directly inducing apoptosis of bone-resorbing 43 osteoclasts, providing evidence that the protective effects of estrogen against post-menopausal 247 osteoporosis is mediated in part by an estrogen receptor-mediated mechanism . Although alterations in apoptosis regulation has been implicated in many age-related diseases, there is still much to learn concerning the molecular mechanisms involved. Further research in this area w i l l provide pathogenic clues on a pathway to new therapeutic modalities. 1.2.5 Injury and Repair Apoptosis represents a mechanism to remove damaged, infected or unwanted cells. Human cells are continually under assault from diverse sources such as mutagens, reactive oxygen intermediates and other environmental stimuli. Many of these agents damage D N A which may be repaired by D N A repair mechanisms. If the damage is irreparable, then the cell must be eliminated via apoptosis. Occasionally, genetically-damaged cells are not destroyed and may contribute to the onset of cancer, senescence or other disease states 2 4 8 . Following tissue damage, repair involves inflammation, granulation tissue formation and scarring. Granulation tissue is derived from the connective tissue elements surrounding the injured or missing area and is comprised of small vessels, inflammatory cells, fibroblasts and myofibroblasts. A s the wound closes and progresses towards scar formation, there is a marked 249 decrease in cellularity, including the disappearance of myofibroblasts . Evidence suggests that 249 apoptosis may be responsible for the transition of granulation tissue into scar tissue . Apoptosis serves a key role in the hematopoietic system. Erythropoietin (EP) regulates red cell production by maintaining the viability of erythroid progenitors, and by stimulating their 250 proliferation and differentiation into normoblasts . Researchers have demonstrated a role for BC1-XL in late erythroid differentiation events and for caspase-3 in the apoptotic demise of 251 erythroblasts caused by E P deprivation . In certain pathologic states, E P deficiency is believed 44 to be associated with increased apoptosis of late-stage erythroblasts thereby resulting in anaemia 250,252 Investigators have shown that following experimental endothelial injury in an artery, 249 smooth muscle cells participate in the formation of intimal thickening . Apoptotic events are 249 observed in cells of thickened intima and also within human atherosclerotic plaques . In addition to atherosclerosis, apoptosis has been implicated in cardiomyopathies, paroxysmal arrhythmias, conduction disturbances and sudden death, fibromuscular dysplasia and hereditary medial degeneration of coronary arteries, and arrhythmogenic right ventricular cardiomyopathy 174,253 ^ perspective is emerging on the short- and long-term effects of exercise-induced damage of skeletal and cardiac muscle, in particular the pathogenic role of inappropriate apoptosis 2 5 4 . Apoptosis may play a role in regulating myoblast proliferation during muscle regeneration, and in the progression of dystrophinopathies. A major role of apoptosis is 254 suspected in cardiac muscle and reperfusion injury after ischemia . 1.2.6 Viral Pathogenesis Many viruses express proteins that converge with the host cells' apoptotic regulatory proteins to either permit the maintenance of latent viral infection or to preserve the viability of 255 the host cell in order to enhance the efficiency of viral replication . In addition, many viruses are known to induce apoptosis during later stages of infection 2 5 5 , 2 5 6 . 255 Viruses interact with the apoptotic pathway at many points . Several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as the tumourigenic human molluscipoxvirus, contain proteins (FLIPs) that interact with the adapter protein F A D D , thereby blocking Fas- and TNF-associated DISC formation and subsequent 45 caspase-8 activation and downstream events ' ' . Expression of the cowpox serpin C r m A prevents the caspase-8-mediated processing of caspases such as caspase-1 and caspase-3 6 3 . The IAPs (inhibitor of apoptosis proteins) constitute a family of proteins found in baculoviruses that block apoptosis induced by viral infection or by caspase-1 3 5 . Evidence has shown that IAPs may interact with T R A F 2 3 5 , however, recent experiments have demonstrated that c-IAPs may act as 259 direct inhibitors of specific caspases . Furthermore, several viral homologues of the Bcl-2 family of proteins have now been discovered 2 6 0 2 6 3 . There is also evidence for viral inhibition of N F - K B 2 6 4 . Certain viruses possess proteins, which interfere with the normal functioning of tumour suppressor genes such as p53 and the retinoblastoma (Rb) protein 2 5 6 . For D N A viruses, this inhibition may be necessary in order to control host D N A synthetic machinery and cell cycle progression 2 5 6 2 6 5 . Well-characterized examples include the human simian virus 40 2 6 6 , . . . . 267 , . 268 , , . . _ . 269 papillomaviruses , adenoviruses and hepatitis B viruses . Certain viral gene products interact with Bcl-2 family members. The Epstein Barr virus-270 271 B H R F 1 and adenovirus E1B-19 kDa proteins are Bcl-2 homologues ' . B H R F 1 and E1B-19 272 kDa proteins suppress the death-promoting activity of of the Bcl -2 homologue, B i k . Sequence analysis has identified a novel viral anti-apoptotic Bcl-2 homologue (KSbcl-2) from human herpesvirus 8 ( H H V 8 ) or Kaposi sarcoma-associated herpesvirus that does not homo- or heterodimerize with other Bcl-2 proteins 2 6 ° . It has been suggested that KSbcl -2 may have evolved to evade the negative regulatory effects of host Bax and Bak proteins 2 6 ° . Adenoviruses provide an interesting example of how a virus may utilize apoptosis and necrosis to maximize viral replication in the host (Figure 7). Adenoviruses usually infect non-273 dividing cells which are not conducive to viral, replication . To address this dilemma, adenoviruses possess a protein ( E I A ) that drives the infected cells into the S-phase, promoting 46 viral D N A replication, but at the same time, E I A also induces apoptosis, which is not conducive 274 to maximizing viral replication . However, the adenovirus E 1 B 19K protein, which is homologous to Bcl -2 , functions to prevent premature E lA- induced apoptosis while permitting 275 the progression of viral replication . Premature cell death is also averted by other adenovirus proteins such as E 1 B - 5 5 K (binds to p53 targeting it for degradation) 2 ? 6 , E3-14.7K (inhibits TNFoc-induced cytolysis) 2 7 7 and E3-10.4K/14.5K (downregulation of Fas on cell surface) 2 7 8 . The adenovirus death protein (ADP) or E3 11.6K induces cell lysis late in the virus replication cycle, allowing the release of viral progeny which accumulated during the period when apoptosis was 279 280 blocked by E 1 B 19K ' . Ce l l lysis during the late phase of adenovirus infection is accompanied by expression of high levels of A D P in contrast to its low level of expression early 279 280 279 280 in infection ' . Cells lysed by adenovirus do not exhibit an apoptotic morphology ' . Thus, adenoviruses display an ordered expression of proteins that block apoptosis and trigger necrosis in order to maximize viral replication. 47 Early Adenovirus cell entry J E1A drives cell into S-phase _ ^ Apoptosis I 0) | \ E1B-19k, E1B-55k, E3-14.7k, E3-10.4k/14.5k \ viral replication ADP expression ADP induces cell lysis Viral release Figure 7. Adenoviruses utilize apoptosis and necrosis to maximize viral replication in the host. Adenoviruses usually infect quiescent cells which do not support viral replication. The adenovirus E l A protein drives cells into S-phase promoting viral D N A replication. During this process E I A also inadvertently provokes apoptosis. However, the viral E 1B- 19K protein prevents E l A-induced apoptosis, permitting viral replication. Other adenovirus proteins (E1B-55k, E3-14.7k and E3-10.4k/14.5k) are also involved in resistance to apoptosis (refer to text). The adenovirus death protein (ADP) is expressed late in the virus replication cycle. A D P induces cell lysis (necrosis) allowing the release of accumulated viral progeny. 48 1.3 Photodynamic Therapy 1.3.1 Overview Photodynamic therapy (PDT) is a clinically approved treatment for the ocular condition age-related macular degeneration, and certain types of cancer. P D T is also under investigation for other ocular, as well as, immune-mediated and cardiovascular indications. P D T is a two step procedure. In the first step, the photosensitizer, usually a porphyrin derivative, is administered and taken up by cells. The second step involves activation of the photosensitizer with a specific wavelength of visible light. Exposure to light of an activating wavelength generates reactive oxygen species within cells containing photosensitizer. P D T with porphyrin photosensitizers induces rapid apoptotic cell death, an event that may be attributed to the close association of these compounds with mitochondria. Thus, P D T is an attractive method to treat ailments such as cancer, viral infections, autoimmune disorders and certain cardiovascular diseases in which the apoptotic program may be compromised. The present chapter examines the cellular events triggered at lethal and sublethal P D T doses and their relationship to the subsequent effects exerted upon cells. Since very little was known concerning the biochemical mechanisms of PDT-induced apoptosis at the commencement of my thesis (Figure 8 versus Figure 9), I have incorporated many of the findings of our group, including results obtained during my dissertation over the past few years into this chapter. 49 Figure 8. Level of understanding of the biochemical mechanisms of PDT-induced apoptosis as of May, 1997. There was no published literature concerning the biochemical mechanisms of verteporfm-based PDT-induced apoptosis. With respect to other photosensitizers, DNA fragmentation and membrane damage were shown to occur. Biochemical mechanisms that had been studied are shown in white boxes. Bcl-2 was shown to offer partial protection against PDT-induced apoptosis and increased ceramide levels were observed during PDT-induced apoptosis. 50 Figure 9. Biochemical mechanisms of PDT-induced apoptosis. Verteporfin localizes to mitochondria (MT) , but may also be closely associated with ER. The initiating step of photosensitization is the absorption of a light photon by the verteporfin thereby elevating the molecule from its energy ground state to a highly unstable excited singlet state. Verteporfin returns to its ground state by transfering its energy to oxygen resulting in the production of reactive oxygen intermediates. P D T induces a rapid emptying o f E R C a 2 + stores into the cytosol that is associated with downregulation of S E R C A . P D T also instigates rapid cyt c release from mitochondria into the cytosol. Although the mechanism o f cyt c release is unclear, it the adenine nucleotide transporter ( A N T ) may be a target o f verteporfin and be responsible for a change in mitochondrial transmembrane potential (Av | /J . A I F is also released from mitochondria and may contribute to D N A fragmentation. Cyt c, ATP, Apaf-1 and pro-caspase-9 form a complex known as the apoptosome which catalyzes caspase-9 activation leading to the activation of downstream executioner caspases (Caspase-3, -6, -7).. Caspase-3 induces D N A fragmentation by cleaving I C A D / D F F which allows the translocation o f C A D into the nucleus and D N A fragmentation. Caspase-3 also cleaves structural proteins and repair enzymes as well as other caspases such as caspase-6 which cleaves nuclear lamins, or caspase-8 which cleaves B i d into a truncated form capable of inducing cyt c release thereby amplifying cyt c release. Caspase-3-mediated cleavage of cell cycle inhibitory proteins p21 and p27 may initiate premature entry into the cell cycle via C D K 2 activation. A cellular redistribution of Bax has also been observed in H U V E C and H A S M C but not in HeLa cells. Sublethal P D T may elicit an anti-apoptotic response via NF-KB-mediated transcription of anti-apoptotic genes. Increased cytosolic C a 2 + may stimulate Av|/m as well as upregulating scramblase activity and downregulating phospholipid translocase activity resulting in externalization of PS. 51 1.3.2 Introduction The original use of photosensitizers for the treatment of disease dates back to ancient 281 282 times when certain plant extracts were used to treat psoriasis and vitiligo ' . Photodynamic therapy (PDT) is now an approved treatment for several types o f cancer and recently gained approval for the treatment of age-related macular degeneration ( A M D ) , the leading cause of blindness in the elderly. PHOTOFPJN® (Axcan Pharma Inc., Quebec, Canada) was the first approved photosensitizer and is currently used clinically for the treatment of different forms of cancer (lung, esophageal, cervical, bladder, gastric) in North America, Europe and Japan. More recently, a major milestone in the field of ophthalmology was achieved when P D T gained regulatory approval in multiple jurisdictions for the use of Visudyne (benzoporphyrin derivative monoacid ring A , verteporfin, Q L T Inc., Vancouver, B C ) for the treatment of "wet" A M D , the leading cause of blindness among people over the age of 50. In the treatment of A M D , red laser 283 —— light is directed into the eye shortly after the systemic administration of Visudyne . The abnormal blood vessels responsible for the loss of visual acuity in this condition rapidly take up Visudyne and are destroyed upon light irradiation, most likely due to a combination of PDT-induced endothelial cell apoptosis, vaso-occlusion and thrombosis. In summary, Visudyne therapy of subfoveal choroidal neovascularization ( C N V ) from A M D was shown to safely reduce vision 283 loss in patients with predominantly classic C N V from A M D . In addition, Visudyne is being assessed for its ability to alleviate C N V not related to A M D , including pathologic myopia, the 284 I ocular histoplasmosis syndrome, angioid streaks, and idiopathic causes . P D T is a two-step procedure requiring 3 elements: photosensitizer, light and oxygen 2 8 5 . In the first step, a photosensitising agent (usually a porphyrin derivative) is administered intravenously (Figure 10). The photosensitizer then circulates and accumulates in most cells, 52 including the cells of interest. In the next step, the photosensitizer is exposed to non-thermal light 286 288 ' of a specific wavelength which 'activates' the drug . According to the application, a specific light delivery source may be required. Typical light sources include light emitting diodes (LED) , fluorescent tubes, or lasers for greater specificity. The initiating step of photosensitization is the absorption of a light photon by the photosensitizer elevating the molecule from its energy ground state to a highly unstable excited singlet state. The excited singlet phtotosensitizer either returns to ground state, resulting in the emission of fluorescence, or undergoes an intersystem crossover to a longer lived triplet excited state 2 8 2 , 2 8 5 , 2 8 9 ' 2 9 0 The interaction of this triplet photosensitizer with surrounding molecules results in two possible types of photo-oxidative events denoted Type I and Type II reactions. The products arising from the energised photosensitizer reacting with oxygen are peroxides, superoxide ions, and hydroxyl ions for Type I reactions and singlet oxygen CO2) for Type II reactions 2 8 2 - 2 8 5 , 2 8 9 ' 2 9 0 Although both reactions may proceed simultaneously, the generation of '02 via the Type II pathway is believed to be primarily responsible for PDT-induced cytotoxicity 282 289 291 because of its dynamic interaction with various biomolecules The anti-cancer action of P D T is likely two-fold: (1) as a consequence of direct photodynamic killing of tumour cells and (2) vascular impairment that restricts blood supply to the 285 292 region . In the absence of light, photosensitizers have little discernible biological activity. Thus, P D T is advantageous in that light may be directed specifically at the target area, limiting the degree of unwanted side effects in non-target tissues. 53 Administration of photosensitizer Figure 10. PDT is a two-step procedure. In the first step, a photosensitizing agent is administered intravenously. This is followed by an interval in which the drug circulates within the blood and accumulates within the target cells. In the second step, the photosensitizer is exposed to non-thermal light at a specific wavelength that 'activates' the drug resulting in the formation of reactive oxygen species. Photosensitizer-containing cells may undergo rapid apoptotic cell death following exposure to an activating wavelength of light. 54 PDT is a potent inducer of apoptosis in numerous experimental settings. Apoptosis has been 293 294 . . r-detected in mouse tumours as well as inflammatory cells associated with the synovial tissue of 295 arthritic rabbits treated with PDT . In PDT-treated human sarcoma xenografts transplanted into nude mice, the mechanism of tumor destruction in this model appears to be vascular damage with 292 initial apoptosis in tumor endothelial cells and delayed tumor cell apoptosis . In addition to ocular disorders and cancer, PDT is under investigation for the treatment of atherosclerosis, restenosis, allograft rejection, and immune disorders 2 9 6 3 0 1 . Photosensitizers are taken up and/or retained at higher levels in active, rapidly dividing cells. Thus, utilization of PDT for the treatment of atheromas, which are characterized by increased smooth muscle cell 159 proliferation, lipophilicity, immune cell infiltration and proliferation , is appealing. The selectivity of PDT due to preferential drug uptake, liposomal formulations of photosensitizers, increased endothelial permeability, and specific light-directed activation of the photosensitizer renders PDT an attractive therapy compared to other catheter-based approaches that are relatively non-selective and carry a substantial risk of damaging the normal arterial wall 3° 2. Extensive cellular damage has been described for atheromatous areas following light irradiation 3 ° 3 ' 3 0 4 . However, the integrity of the internal elastic lamina does not appear to be affected 3 ° 5 ' 3 0 6 . Furthermore, in studies in which the effects of PDT on isolated extracellular matrix (ECM) were assessed on EC and SMC, PDT-treated E C M enhanced EC proliferation and migration while inhibiting SMC growth, attachment and migration 3 0 ? . These in vitro studies would support a role for PDT in preferential vascular remodelling and would favour PDT for the treatment of restensosis and other vascular ailments in which reendotheliazation and reduction of SMC are desired. Furthermore, in addition to a reduction in SMC, a reduction in inflammatory infiltrates 296 was also observed following PDT . Transformed T cells in addition to normal activated T cells 55 and macrophages are highly sensitive to PDT-induced ki l l ing (apoptosis) . Thus, it is likely that within a PDT-treated atheromatous plaque that these cells would be directly eliminated via an apoptotic mechanism. The lack of inflammation in spite of extensive cell death supports the likelihood that cells within the atheroma may be eliminated by apoptosis. Furthermore, P D T may inhibit intimal hyperplasia via the local inhibition of cytokine release or activation 3 1 \ Exposure to P D T was shown to reduce levels of fibroblast growth factor (bFGF or FGF-2) , to reduce S M C mitogenesis in response to F G F - 2 , and to reduce levels of transforming growth factor-p (TGF-P) 3 U . Recently, P D T , using motexafin lutetium (Antrin) as the photosensitizer, has entered phase I 312 trials in humans . These studies have demonstrated that P D T is well tolerated in patients and 312 that P D T has the capacity to reduce intimal hyperplasia in patients with atherosclerosis . In these studies, Antrin was administered intravenously and endovascular light was delivered 312 through a cylindrical diffuser fiber 24 h after drug administration .The fiber was positioned 312 adjacent to the target area by percutaneous delivery through a guiding catheter . Thus, the future for P D T as an entity by which atheromatous plaques are eliminated is optimistic. For the treatment of immune disorders such as psoriasis, the systemic delivery of photosensitizer is followed by broad exposure of a large surface area of the patient to activating light. Low, sublethal levels of P D T can modify immune responses while limiting skin 313-315 inflammation or erythema . Large populations of infiltrating T cells are present within the skin plaques of psoriatic patients 3 ' 6 . Although the mechanisms by which P D T acts to alleviate psoriasis are not known, P D T may either have a direct impact on psoriatic keratinocytes, or may act indirectly by inducing apoptosis of plaque-associated T cells that release cytokines that alter keratinocyte growth and differentiation in this disease. To this extent, activated T cells are more 56 susceptible to PDT-induced apoptosis than resting T cells . Likely, the effects of PDT on psoriasis include both keratinocytes and T cells. Understanding of the biochemical mechanisms of PDT-induced apoptosis has advanced significantly over the past decade. PDT-induced apoptosis has been demonstrated using both in vitro and in vivo models. Further, PDT has the capacity to induce apoptosis in most normal and tumour cell types. The mitochondrial localization of certain second generation porphyrin derivatives permits rapid activation of the apoptotic program following photosensitization. The present chapter highlights recent advances in delineating the biochemical mechanisms of porphyrin-based PDT-induced apoptosis. In addition, we will discuss the cellular signalling events associated with the much less understood sublethal effects of PDT. 1.3.3 Mi tochondr ia l Regulation of PDT-Induced Apoptosis As described earlier, the search for chemical agents that target mitochondria to induce apoptosis has gained much attention in recent years. Porphyrin-derived photosensitizers may 318 319 localise to mitochondria ' . In addition, a number of groups have now shown that cyt c is released into the cytosol immediately following photosensitisation using different photosensitsers '93'32° 3 2 5 . Further evidence for a direct effect of certain photosensitizers, such as verteporfin, on mitochondria stems from the observation that unmasking of the apoptosis-associated mitochondrial 7A6 antigen occurs immediately after photosensitization and in parallel 323 with the release of cyt c in PDT-treated HeLa cells . Although it has been demonstrated that the 7A6 antigen is detected in apoptotic but not in healthy cells 3 2 6 , the role of this 38 kD mitochondrial antigen is not understood. A detailed description of intracellular apoptotic events associated with PDT is illustrated in Figure 9. 57 Porphyrin-derived photosensitizers can bind mitochondrial peripheral-type 327-329 benzodiazepine receptors (PBR) . P B R are located at the junction of the inner and outer mitochondrial membranes 3 3 0 , 3 3 ' s a pivotal site for the integration of apoptosis-related signals " 4 . Interestingly, P B R expression levels correlated with cell sensitivity to photodynamic ki l l ing with 327 porphyrins . Thus, the localization of porphyrin-derived photosensitizers to mitochondria indicates how P D T with porphyrin photosensitizers is capable of inducing rapid cyt c release and initiation of the apoptotic cascade. Identification of other mitochondrial structures that bind porphyrin photosensitizers w i l l be a future area of keen interest. 1.3.4 Role of Caspases in PDT-induced Apoptosis Caspases recognize a specific tetrapeptide sequence within their target substrates. These 53 amino acid motifs form the basis for inhibitor and synthetic substrate design . Inhibition of caspase activity prevents many of the biochemical and morphological events that occur during 53 332 apoptosis . One of the hallmarks of apoptotic death is genomic disassembly and D N A fragmentation. Caspases disrupt normal D N A repair processes by proteolytically cleaving and inactivating at least two key proteins involved in the maintenance of genomic integrity, P A R P and D N A - P K c s - Simultaneously, caspases provoke the onset of D N A fragmentation by indirectly activating an apoptosis-specific endonuclease ( C A D ) by cleavage of its cognate inhibitory peptide ( I C A D / D F F ) 3 3 . In PDT-induced apoptosis, caspase-dependent cleavage of 79 201 P A R P , D N A - P K and D F F have been observed ' . A summary of caspase substrates that have been demonstrated to be cleaved during PDT-induced apoptosis is shown in Table 2. 58 Table 2. Proteins shown to be subject to caspase-mediated cleavage during verteporfin mediated PDT-induced apoptosis. Protein Function Reference Bap31 Shuttle protein between E R and the 324 intermediate compartment and/or Golgi complex B i d Pro-apoptotic Bcl-2-related protein 193 D N A - P K c s Regulation of cell cycle in response to 201 D N A damage D F F Prevents D N A fragmentation via binding 79 to caspase-activated deoxyribonuclease focal adhesion kinase Kinase involved in regulation of cell 324 ( F A K ) adhesion Lamins Structural components of the nuclear 323 envelope P21 Cel l cycle inhibitory protein (unpublished) P27 Ce l l cycle inhibitory protein (unpublished) P A R P D N A repair enzyme 201 Ras GTPase-activating Negative regulator of the Ras signalling 324 protein (Ras-GAP) pathway 59 Although multiple caspases are active during apoptosis and possibly redundant in different cell types, lessons obtained from caspase knockout mice indicate that certain caspases 332 are essential for the normal functioning and development of specific tissues . For instance, caspases -3 and -6 are critical for proper neuronal development, while caspase-8 is necessary in 332 cardiac development and erythropoiesis . Caspase-2, on the other hand, has two isoforms, 332 caspase-2L and caspase-2s, which promote or inhibit apoptosis, respectively . Thus, deletion of 332 this gene had differential positive and negative effects in a tissue-specific pattern . Zheng et al.332 recently published an extensive review outlining the diverse phenotypes of different caspase knockout mice. Following the release of mitochondrial cyt c into the cytosol, activation of caspases-2, -3, -6, -7, -8, and -9 has been described for several cell types treated with PDT 1 9 3 3 2 3 ' 3 2 4 ' 3 3 3 During receptor-mediated apoptosis, caspase-8 is mobilized prior to cyt c release. However, in PDT-induced apoptosis caspase-8 is activated after cyt c release in both normal and transformed cell types during PDT-induced apoptosis ' 9 3 ' 3 2 4 , an event likely triggered by caspase-3 3 2 \ The later activation of caspase-8 during PDT-induced apoptosis may amplify cyt c release through caspase-8 mediated cleavage of Bid into a truncated form (tBid) that can cause cyt c release'93'324'334'335. In addition, recent studies suggested that caspase-3 catalyzes Bid cleavage and 334 triggers an amplification loop by which tBid induces further cyt c release . 1.3.5 Role of Bcl-2-Related Proteins in the Regulation of PDT-Induced Cell Death Conflicting reports have been forwarded concerning the role of Bcl-2 and related proteins in PDT-induced apoptosis. Bcl-2 expression in Chinese hamster ovary cells inhibited apoptosis and partially protected against a loss of clonogenicity in Pc4-based PDT " 6 , while in breast 60 epithelial cells Bcl-2 promotes PDT-induced apoptosis using the photosensitizer P c A l . The increased susceptibility of Bcl-2 over-expressing cells to P D T was believed to be due to a 337 photodynamic destruction of Bcl-2 that allowed Bax to exert its pro-apoptotic activity Conversely, Bcl -2 or B C 1 - X L over-expression in HL-60 cells suppressed verteporfin-based P D T -57 79 induced caspase activation and D N A fragmentation ' . B C 1 - X L overexpression inhibited D N A fragmentation indirectly by suppressing caspase-3 activity and subsequent D F F cleavage after 79 P D T . However, B C 1 - 2 / X L does not appear to prevent PDT-mediated mitochondrial alterations. In fact, over-expression of Bcl-2 or B C 1 - X L in H e L a cells did not prevent the release of mitochondrial cyt c, the unmasking of the mitochondrial 7A6 antigen or cell death induced by P D T with verteporfin 3 2 3 . Furthermore, studies of hypericin-based P D T indicated that human 338 glioma cell viability was unrelated to expression levels of Bcl-2 or Bax . In summary, the role of Bcl-2 and related anti-apoptotic proteins in the regulation of PDT-induced apoptosis is unclear but these proteins may act in a cell type- and photosensitizer- specific manner. The status of pro-apoptotic Bcl-2 family members has been assessed for normal and transformed cell types treated with PDT. Treatment of human umbilical venous endothelial cells ( H U V E C ) with verteporfin-based P D T induced cleavage of the Bcl -2 homologue B i d into its pro-apoptotic tBid form However, in contrast to receptor-mediated forms of apoptosis, B i d 193 cleavage occurred downstream of mitochondrial cyt c release . Since B i d is a caspase-8 and caspase-3 substrate 3 3 4 , 3 3 9 , 3 4 0 a n c j both are activated downstream of cyt c release following P D T 1 9 3 , it is possible that caspase-mediated B i d cleavage may amplify cyt c release in certain P D T -treated cell types. Cellular redistribution of Bax from the cytosol to mitochondria may also enhance cyt c release after P D T in normal, but not transformed cell types. In primary H U V E C , cytosolic Bax 61 levels gradually decreased following P D T but were unchanged in H e L a cells undergoing 323 PDT-induced apoptosis . The explanation for this difference is unclear. Further, cytosolic cyt c 193 323 levels increase over 2 h in PDT-treated H U V E C , but not in PDT-treated H e L a cells ' . Many tumour cell types are known to contain abnormal expression or mutations in one or more Bcl-2 -related genes l 5 " ' 9 6 , 3 4 '_ Therefore, it is possible that pro-apoptotic Bax-mediated signalling pathways may have been impaired in the transformed H e L a cell line that was used for these experiments. Thus, it is possible that in PDT-treated non-transformed cells that immediate cyt c release is attributed to direct mitochondrial effects while further cyt c release is a result of Bax and/or Bid-mediated activity. 1.3.6 Effects of P D T on Intracellular Calcium Regulation The impact of P D T on E R integrity and intracellular calcium regulation are poorly understood. Evidence suggesting a role for E R in PDT-induced apoptosis stems from studies demonstrating caspase-8 mediated cleavage of the integral E R Bap31 protein in H e L a cells 324 treated with verteporfin and light . Furthermore, certain porphyrin photosensitizers (haematoporphyrin, H P and protoporphin IX , PPIX) associate with E R to a greater degree than 342 7+ mitochondria in rat liver cells . With these photosensitizers, altered intracellular C a levels were mediated as a consequence of PDT-mediated E R damage as opposed to mitochondrial 342 9+ damage . In support of a role of Ca in PDT-induced apoptosis, for pheophorbide-treated Chinese hamster V79 cells, the intracellular calcium chelator B A P T A - A M inhibited cyt c 320 release, caspase-3 activation and apoptosis following light activation . Thus, in this system, the 9-4- • PDT-induced increase in intracellular Ca levels may instigate mitochondrial cyt c release. It 62 has not been determined whether B A P T A - A M would prevent cyt c release induced by P D T using other photosensitizers. 1.3.7 PDT, Cell Signalling and Apoptosis Although the role of protein kinases in the regulation of cell death and survival has been described in recent years the importance of these proteins in PDT-mediated cell death is unclear. Although protein phosphorylation and kinase activation have been described for PDT-treated cells 3 4 3 " 3 4 9 s their influence on cell death and/or cell survival signalling pathways is unresolved. The non-receptor tyrosine kinase Etk provided a degree of protection against PDT-induced 344 apoptosis with Pc4 in prostate cancer cells . Increased activity of stress-activated protein kinase (SAPK)/c-jun NH 2 - terminal kinase ( INK) and p38 high osmolarity glycerol protein kinase 1 (p38), in mouse 3 4 9 and human keratinocytes 3 4 6 has been demonstrated. The significance of these events in relationship to cell death or cell survival in PDT-treated cells is yet unclear. However, a recent study by Davis and colleagues suggests a requirement for S A P K 7 J N K in triggering cyt c 350 release in UV-induced apoptosis in murine embryonic fibroblasts . A role for S A P K / J N K in Fas-induced apoptosis has also been described 3 5 ' . Hypericin-based P D T activated S A P K / J N K while irreversibly inhibiting extracellularly-regulated kinase-1 ( E R K 1 ) activity in tumour cells 3 4 8 . This latter report also indicated that PDT-induced activation of S A P K / J N K was caspase independent 3 4 8 . Inhibition of p38 kinase activity impaired Pc4 mediated PDT-induced apoptosis 345 in L Y - R cells but did not affect PDT-induced apoptosis for Chinese hamster ovary cells . B y contrast, inhibition of p38 promoted apoptosis for H e L a cells treated with hypericin-based P D T 3 4 8 . PDT-induced apoptosis was enhanced by expression of the dual specificity M K P - 1 phosphatase 3 4 8 . The apparent differential activities of M A P kinases during PDT-induced 63 apoptosis may be attributable to the cell type, the photosensitizer used and/or the intensity of light applied. Clearly, the role of kinases in PDT-induced apoptosis requires further resolution. 1.3.8 Transcriptional Regulators N F - K B D N A binding activity occurs in response to various stimuli including cytokines, 352 viral infection, phorbol esters, oxidants or radiation . N F - K B is not a single protein, but is comprised of dimers of Rel family D N A binding proteins that bind closely related N F - K B 352 recognition motifs . N F - K B resides in the cytoplasm in an inactive state in association with IKB 352 proteins . Upon activation, via degradation of IKB, N F - K B translocates to the nucleus where it activates the transcription of a variety of genes involved in immune and inflammatory responses 352 . Photofrin® based P D T induced N F - K B nuclear translocation in murine L1210 lymphoma cells 3 5 3 as well as increased c-fos and c-jun gene expression 3 5 4 . The c-fos heterodimerises with c-jun to form the AP-1 transcription factor. In H e L a cells treated with Photofrin®-based P D T , activation of AP-1 but not N F - K B was observed 3 5 5 . Wi th verteporfin, as well as pyropheophorbide-a methyl ester (PPME) based P D T , decreased IKBCC levels and N F - K B activation were observed 3 3 3 , 3 5 6 . Furthermore, increased sensitivity to apoptosis was described for P D T (PPME)-treated HCT-116 carcinoma cells in which the N F - K B pathway was impaired due to an overexpression of an IKB super repressor protein suggesting that N F - K B influences cell sensitivity to P D T 3 5 7 . In addition to its role in immune responses, N F - K B stimulates the transcription of pro-survival genes such as A l , A20 , BC1-XL, TNFR-associated factors 1 and 2 ( T R A F 1 and 2) and inhibitor of apoptosis proteins (IAPs) 1 2 4 m . Thus, it is possible that low-level P D T may activate 64 the N F - K B pathway and subsequent transcription of anti-apoptotic genes. Conversely, N F - K B could activate genes such as FasL, thereby stimulating a 'counterattack' mechanism against cells 358 expressing Fas . Such an effect on keratinocytes may be beneficial for the treatment of psoriasis with P D T in which high levels of pathogenic, infiltrating, Fas-positive T cells are present in the skin. The tumour suppressor gene p53 is involved in the regulation of cell division and cell death. Altered p53 function or expression is associated with many forms of cancer and this may impact cell resistance to chemotherapy and y-irradiation. HL-60 p53 deficient cells were less 359 sensitive to Photofrin® based P D T than those transfected with wi ld type p53 . This phenomenon may be explained by the observation that p53 gene expression can, in certain cell types, upregulate Bax expression while downregulating Bcl-2 expression 3 6 0 3 6 3 which may account for the increased sensitivity of these cells to P D T . In further support of this concept, human colon cancer cell lines with a mutated p53 gene exhibited a greater sensitivity to P D T 364 following transfection with wi ld type p53 . Conversely, when p53 was specifically targeted for ubiquitin degradation in cells transfected with the E6 viral oncogene, no change in tumour cell sensitivity to P D T with Photofrin® was evident 3 6 5 . For a number of human glioma cell lines, 338 susceptibility to P D T with hypericin was unrelated to p53 expression level . Although in certain cases p53 transfection may heighten cell sensitivity to P D T , the effect is most likely due to the restoration of p53-regulated apoptosis pathways since p53 null cells are still susceptible to PDT-induced apoptosis. 65 1.3.9 Conclusions The emergence of PDT, a treatment that mainly affects cells exposed to activating light, provides several advantages over standard systemic cytotoxic agents since its activity can be controlled by the degree and targeting of light. In addition, porphyrin-derived photosensitizers have the added benefit in that they may directly affect mitochondria to induce apoptosis. This property suggests that this form of treatment may circumvent anti-apoptotic mechanisms that are present in certain types of cancer, viral infection, or diseases in which immune-mediated apoptotic elimination of damaged, injured, or unwanted cells is hindered. Over the past few years, great advances have been made in the understanding of the biochemical pathways that are activated during PDT-induced apoptosis. However, ixirther studies are necessary to delineate and understand the significance of signalling pathways that are activated in different cell types in response to sub-lethal levels of PDT. Additionally, it wi l l be important to determine the characteristics that render certain cell types, such as activated immune cells, more susceptible to PDT-induced apoptosis. With these recent advances in our understanding of P D T mechanisms, as well as the increasing approvals for the use of P D T to treat various types of disease, the advantages of this form of treatment are finally being recognized. This bodes well for further advances in the years ahead. 66 C H A P T E R II R A T I O N A L E , HYPOTHESIS AND E X P E R I M E N T A L AIMS Defective apoptosis has been suggested to play a role in the onset and progression of intimal hyperplasia associated with vascular lesions observed in patients suffering from restenosis, atherosclerosis and transplant vascular disease. Apoptosis in EC and SMC may have opposing effects in the progression of these ailments. It is likely that degradation of the endothelial lining would be detrimental whereas apoptosis of SMC in the intima may be a beneficial means of killing and removing these unwanted cells without invoking an inflammatory response. However, it has been difficult to ascertain the roles of EC and SMC death in vascular disease since very little work has been done in vitro that describes the biochemical mechanisms of apoptosis in these cells. The reason for this is uncertain, but may be due to the difficulty of working with primary cells as opposed to tumour cells combined with the use of inefficient stimuli to induce apoptosis in these cells. PDT was an attractive technique to induce apoptosis in that it was a rapid and effective means of inducing apoptosis in these cells as well as being a clinically relevant stimulus. In vivo studies have shown that site-specific delivery of verteporfin followed by photosensitization represents a safe, effective method of inhibiting the development of intimal hyperplasia 3°S. PDT has also been shown to prevent allograft rejection 3°°. Furthermore, PDT-mediated reduction in the number of SMC occurs along with the prevention of inflammatory infiltration, and development of intimal hyperplasia associated with allograft rejection 3°°. However, very little is known as to how PDT induces SMC apoptosis. On more general terms, the basic mechanisms of SMC apoptosis are poorly understood. Our preliminary studies indicated that PDT, using verteporfin as the photosensitizer, induced apoptosis. Initial studies were performed in tumour cells 67 using standard cell lines that have been used extensively to assess the effects of other drugs on apoptosis. Furthermore, these studies are relevant because P D T eradicates tumours via direct tumour cell killing as well as destroying the neovasculature leading to further tumour death due to ischemia. Thus, by studying the effects of P D T on tumour cells as well as vascular cells, our findings are relevant to the understanding of how P D T may be beneficial for the treatment of hyperproliferative disorders such as cancer as well as for the reduction of intimal hyperplasia. Furthermore, since P D T is an effective means of inducing apoptosis, it allowed us to define the biochemical mechanisms of apoptosis in E C and S M C . Since the majority of these biochemical events have never been defined for E C or S M C in response to any stimulus, these studies are directly applicable to the general understanding of vascular injury. Although presently the application of these findings are limited for the study of apoptosis in vivo, we attempted to apply some of our findings from our in vitro work to established in vivo models of transplant vascular disease. Our hypothesis was that PDT acts directly upon mitochondria to elicit rapid apoptotic cell death and involves the release of mitochondrial proteins and activation of caspases, and represents a balance of effects conveyed by members of the Bcl-2 family ofproteins. Our experimental aims were: 1. To optimize techniques used to assay apoptosis and assess the biochemical mechanisms of PDT-induced apoptosis in tumour cells. 2. To define the biochemical events related to E C apoptosis in response to PDT. 3. ' To define the biochemical events related to S M C apoptosis in response to PDT. A i m 1 w i l l not only serve to optimize experimental procedures but w i l l be critical to the understanding of the mechanisms by which P D T kills tumour cells. The vasculature is a major 68 target for PDT in the treatment of disease such as cancer, A M D , restenosis, and atherosclerosis. Results from aim 2 investigating the biochemical mechanisms of EC apoptosis in response to PDT will be especially informative as to the understanding of how PDT affects the endothelium. Furthermore, the mechanisms of EC apoptosis in general are poorly understood. Similarly, the biochemical mechanisms of SMC apoptosis in response to any stimulus, are poorly understood. Results from aim 3 will be particularly applicable to the understanding as to the effects of PDT on SMC in the treatment of ailments such as restenosis, atherosclerosis and transplant vascular disease. Increased SMC numbers within the intima are present in the formation of vascular lesions in diseases such as restenosis, atherosclerosis and transplant vascular disease. Increased SMC may be due to increased SMC proliferation or increased SMC migration. Conversely, the rate of S M C proliferation may remain constant while the rate of apoptosis is decreased. Our laboratory has an established rat heart allograft model of transplant vascular disease. Additionally, through external collaborations, we also had access to a murine heterotopic cardiac transplant model of rejection. These models of vascular injury will eventually allow us to further explore the role of apoptosis in an in vivo setting related to disease. Future studies will strive to provide evidence as to the role of apoptosis in a disease setting and will be the groundwork for future enquiry into the role of apoptosis in the pathogenesis of TVD. 69 C H A P T E R III M A T E R I A L S AND M E T H O D S 3.1 Cell Culture In vitro experiments were performed with transformed as wel l as normal cell types. Human promyelocyte leukemia HL-60 cells and human epitheloid cervical carcinoma HeLa cells have been used extensively as models systems to study biochemical pathways associated with apoptosis in response to various cytotoxic agents. In addition, large cell numbers can be attained in a short period of time. Thus, these cell lines were utilized to optimize techniques and procedures prior to using more expensive and slower growing primary cells in order to minimize the cost of optimization. Furthermore, since the apoptotic pathways in H L - 6 0 and H e L a cells in response to numerous stimuli have been well-characterized, it allowed us to compare the mechanisms of PDT-induced apoptosis to other pro-apoptotic stimuli. The use of tumour cells also allowed for more detailed analysis of the mechanisms involved in PDT-induced apoptosis that required large cell numbers. Since the apoptotic pathways for primary E C and S M C in response to any stimuli were not well-described, a similar comparison was not possible for these cell types. 3.1.1 Transformed Cell Lines 3.1.1.1 HL-60 Cells HL-60 (American Type Culture Collection ( A T C C ) , Manassas, Virginia (TCC C C L 240)) is a promyelocyte cell line derived from peripheral blood leukocytes obtained by leukopheresis from a 36-year-old Caucasian female with acute promyelocyte leukemia 3 6 6 . Cells were maintained in R P M I 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 m M L -70 glutamine, 1 m M sodium pyruvate, 1 m M H E P E S , penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco B R L , Burlington, Ontario). Transfected HL-60 cells: HL-60/neo, HL-60/Bcl-2 and HL-60/BC1-XL cell lines were generously provided by Dr. Kapi l Bhalla (Emory University School of Medicine, Atlanta, Georgia). Methods used to generate these clones have been previously 91 described . BC1-XL, Bcl-2 and Bax levels were comparable in the control (HL-60/neo) transfectants whereas Bcl-2 and Bax levels, and BC1-XL and Bax levels, were comparable in HL-60/Bcl=X L and HL-60/Bcl-2 cells respectively 9 1 . Cells were maintained at 37°C in R P M I 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 m M L-glutamine, 1 m M sodium pyruvate, 1 m M H E P E S , penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco B R L ) and G418 (1 mg/ml)(Geneticin; Life Technologies Inc., Grand Island, N Y ) . 3.1.1.2 HeLa Cells HeLa ( A T C C C C L 2 ) is a human epithelial cervical carcinoma cell line derived from a carcinoma o f the cervix of a 31-year-old African American female (Cancer Res., 12:264, 1952). Since its origin, it has been one of the most widely described cell lines. H e L a cells were maintained at 37° C in Dulbecco's Modified Eagle Medium ( D M E M ) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 m M L-glutamine, 1 m M sodium pyruvate, 1 m M H E P E S , penicillin (100 U/ml) and streptomycin (100 ug/ml). HeLa cells overexpressing Bcl-2 were generated by and obtained from Dr. C B . Thompson (University of Chicago, Chicago, IL). Briefly, Bcl-2 inserts were cloned into pSFFV-neo vectors. Vectors containing the insert (Bcl-2) or no insert (neo) were transfected into HeLa cells using electroporation. Cells containing insert were selected by their ability to survive in the presence of the G418 antibiotic. Transfected HeLa cells were maintained in D M E M supplemented with 10% 71 heat-inactivated fetal bovine serum (FBS), 2 m M L-glutamine, 1 m M sodium pyruvate, 1 m M H E P E S , penicillin (100 U/ml) and streptomycin (100 ng/ml) and G418 (10 ng/ml). 3.1.2. Primary Cells 3.1.2.1 Endothelial Cells Human Umbil ical Venous Endothelial Cells ( H U V E C ) (Clonetics, San Diego, C A ) were obtained at first passage and maintained in endothelial basal medium ( E B M ) supplemented with 10% heat-inactivated F B S , hydrocortisone (1 ug/ml), bovine brain extract (12 ug/ml), gentamicin (50 ug/ml), amphotericin B (50 ng/ml) and epidermal growth factor (10 ng/ml) (Clonetics). Cells were used for experimentation purposes from passages 2 to 7 and were then discarded. H U V E C used in our studies were obtained cryogenically frozen directly from a C a u c a s i a n male newborn. Cel l positivity was determined by morphology and for expression of von Willebrand Factor VIII and acetylated L D L . A s a negative control, cells were stained for alpha' smooth muscle actin. H U V E C also tested negative for mycoplasma, human immunodeficiency virus (HIV), hepatitis B virus ( H B V ) and hepatitis C virus ( H C V ) . 3.1.2.2 Smooth Muscle Cells Human aortic smooth muscle cells ( H A S M C ) were obtained from Clonetics. H A S M C were obtained in cryogenically-frozen vials at passage 3 isolated from a black male 7 week-old fetus. H A S M C (Clonetics, San Diego, C A ) were maintained at 37°C in smooth muscle basal medium (SmBM) (Clonetics) supplemented with 10% heat-inactivated fetal bovine serum (FBS), insulin (5 ug/ml), h F G F - B (2 ng/ml), gentamicin (50 ug/ml), amphotericin B (50 ng/ml) and epidermal growth factor (0.5 ng/ml) (Clonetics). Cel l positivity was determined by morphology and 72 for alpha smooth muscle actin positivity. A s a negative control, cells were stained for von Willebrand Factor VIII. H U V E C also tested negative for human immunodeficiency virus (HIV), hepatitis B virus ( H B V ) and hepatitis C virus ( H C V ) , mycoplasma, bacteria, yeast and fungi. 3.2 Photodynamic Therapy In vitro 3.2.1 Photosensitizer and Light Source The photosensitizer used was lipid-formulated verteporfin (benzoporphyrin monoacid ring A ) provided by Q L T Inc. (Vancouver, B C ) . Verteporfin is a chlorin-like molecule with a benzylic ring located on the ' A ' ring of the porphyrin derivative (Figure 11). Verteporfin is composed of two regioisomers with the carboxylic acid situated on the C or D ring. Verteporfin absorbs light strongly at 690 nm, a wavelength that is minimally absorbed by hemoglobin and penetrates solid tissue relatively deep 3 6 7 . Verteporfin may also be activated at wavelengths of light within the ultraviolet ( U V ) A , blue and green portions of the spectrum 3 6 7 . The red light source was manufactured by Q L T Inc. Figure 12 shows the absorption spectrum of verteporfin and the emission spectrum of the red tubes (General Electric, Model N o . F15T8-R). Light intensity was measured with a power meter supplied by Internation Light (Newburyport, M A ) . 73 o F i g u r e 11. Chemical structure of verterwrfin. 74 300 350 400 450 500 550 600 650 700 750 800 Wavelength (nm) Figure 12. Relative absorption of verteporfin and emission intensity o f the red fluorescent tubes used to activate verteporfin. The absorption spectrum for verteporfin is represented by the green line. The emission intensity spectrum for the red fluorescent light tubes is represented by the red line. 75 3.2.2 Induction of P D T 3.2.2.1 HL-60 Cells For PDT, HL-60 cells were incubated for 60 min at 37°C with verteporfin (0-200 ng/ml) in R P M I containing 2% F B S . Following incubation with verteporfin, cells were exposed to fluorescent red light (620-700 nm) delivered at 5.6 mW/cm 2 to give a total dose of 2 J/cm 2 . For inhibitor studies, cells were exposed to the following compounds: Z - A s p - D C B (110 uM), Z - A A D - C M K (100 uM), dichloroisocoumarin (DCI, 230 uM)(Sigma, Oakville, ON) , T L C K (135 uM)(Sigma), phenylmethylsulfonyl fluoride (PMSF, 100 uM)(Sigma), aprotinin (100 uM)(Sigma), or appropriately-matched vehicle concentration of dH20, methanol or dimethyl sulfoxide (DMSO). For caspase-3 inhibition studies, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-fink) (Enzyme Systems Products, Dublin, C A ) was added to the cells to give final concentrations of 10, 20 or 25 u M for the final 30 min prior to photoactivation. 3.2.2.2 HeLa Cells For PDT, HeLa cells were incubated for 60 min at 37°C with or without verteporfin (0 or 200 ng/ml) in D M E M supplemented with 2% F B S . For caspase inhibition studies, Z -DEVD-fmk (50 uM) (Enzyme Systems Prod.) or Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) (50, 100 or 200 uM) Bachem (Torrance, C A ) was added to cells for the final 30 min of the B P D - M A incubation period prior to light activation. Following drug incubation, cells were exposed to 2 2 fluorescent red light (620-700 nm) delivered at a rate of 5.6 mW/cm to give a total dose of 2 J/cm . Cells were maintained in petri dishes at 37°C until further analysis. 76 3.2.2.3 H U V E C H U V E C s (~5 x 106) were incubated for 60 min in the dark at 37°C with or without verteporfin (100 ng/ml) in E B M supplemented with 2% F B S . For caspase inhibition studies, 50 u M Z V A D - f m k (Bachem) was added to cells for the final 30 min of the verteporfin incubation period prior to light activation. Following drug incubation, cells were exposed to fluorescent red light (620-700 nm) delivered at a rate of 5.6 mW/cm 2 to give a total dose of 2 J/cm 2 . Cells were then maintained in petri dishes at 37°C until further analysis. 3.2.2.4 H A S M C H A S M C were grown on 10 cm petri dishes until they were 80-90% confluent. Cells were incubated for 60 min in the dark at 37°C with or without verteporfin (0-100 ng/ml) in S m B M supplemented with 2% F B S . Following drug incubation, H A S M C were exposed to fluorescent red light (620-700 nm) delivered 9 9 at a rate of 5.6 mW/cm to give a total dose of 2 J/cm . 3.3 Cell Viability Assays 3.3.1 M T T Assay The M T T cell viability assay is a sensitive assay for the measurement of cell proliferation based upon the reduction of the tetrazolium salt 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) . Changes in cellular metabolic activity caused by trophic factors, growth inhibitors, or inducers and inhibitors of apoptosis, may be quantified using the M T T cell viability assay. M T T is reduced to an insoluble formazan dye by mitochondrial enzymes associated with metabolic activity. The reduction of M T T is primarily due to, glycolytic activity within the cell and is dependent upon the presence of N A D H and N A D P H . 77 Other methods for determining cell viability depend upon membrane integrity (e.g. trypan blue exclusion, PI uptake), or incorporation of nucleotides during cell proliferation (e.g. B r d U or 3H-thymidine). These methods can be limited due to the impracticality of processing large numbers of samples, or by the requirement for handling hazardous materials. The M T T assay, in contrast, is a rapid and versatile method for assessing cell viability. This technique can be used to measure changes in cell proliferation or cell death. In actively proliferating cells, an increase in M T T conversion is colorimetrically quantified. Comparison of this value to an untreated control provides a relative increase in cellular proliferative activity. Conversely, in cells that are undergoing apoptosis, M T T reduction decreases, reflecting the loss of cell viability. To assess cell viability, ~5 x 10 4 cells were loaded into eight replicate wells (0.2 ml/well) of 96-well microtiter plates. A t the indicated times after P D T , 10 u L o f M T T solution (5 mg/mL) was added to each well 3 6 8 . Cells were incubated for a further 1 h at 37°C. The reaction was stopped by the addition of 150 u L of acidified isopropanol. The degree of color development was analyzed with an automated densitometer microtiter plate reader (Dynatech, Hamilton, V A ) using a 590 nm filter. 3.3.2 Propidium Iodide (PI) Uptake The PI uptake assay can be used to detect late-stage apoptotic cells and/or necrotic cells. This assay does not differentiate between apoptotic and necrotic cells 3 6 9 . To assess cell membrane integrity, 5 ul PI (2 mg/ml) was added 3, 24, 48, and 72 h after P D T , to 500 ul cells to give a final concentration of 20 ug/ml. The degree of cell permeability to PI was assessed immediately afterward with an Epics X L flow cytometer (Coulter Electronics Inc., Hialeah, F A ) . During incubation, cells were maintained in media supplemented with 10% F B S in petri dishes. 78 3.3.3 Trypan Blue Exclusion Assay To determine the number of live cells in a population, exclusion of the dye, trypan blue was used. Normal, healthy cells are able to exclude trypan blue whereas the dye readily diffuses into cells in which plasma membrane integrity has been lost. The trypan blue exclusion method provides a rough estimate of cell viability and often does not distinguish within a 10-20% difference in cell viability. Furthermore, cells that exclude trypan blue are not necessarily 369 capable of adherence and prolonged survival or proliferation . In early experiments, HL-60 cells were assessed for trypan blue exclusion. HL-60 cells were counted for trypan blue exclusion 3, 24, 48, and 72 h after P D T . Briefly, 10 pi of sample containing cells was mixed with an equal volume of trypan blue (4 mg/ml). Viable cells were counted using a hemacytometer to obtain the number of live cells per ml . This assay was discontinued for latter studies when more suitable, apoptosis-specific assays were used. 3.3.4 Cell Morphology Apoptotic morphology is still believed to be one of the most definitive criteria in determining the type of cell death. In addition to being definitive, analysis of cell morphology by light microscopy is simple, rapid and inexpensive. Further assessment of apoptotic morphology following P D T treatment was carried out by fluorescence microscopy as described in section 4.6. 3.4 Protein Analysis 3.4.1 Preparation of Cell Extracts 3.4.1.1 Whole Cell Extracts Non-adherent whole cell extracts were prepared as previously described 5 7 , 7 9 ' 2 0 1- 3 3 3- 3 4 3 Briefly, cells were washed twice with ice-cold PBS . Cell pellets were treated with in 1 ml of lysis buffer 79 (1% I G E P A L (a.k.a. NonidetP-40 (NP-40)), 20 m M Tris, p H 8, 137 m M NaCI, 10% glycerol) (Sigma) supplemented with 1 m M P M S F , aprotinin (0.15 U/ml) (Calbiochem), and 1 m M sodium orthovanadate (Sigma) for 20 min on ice. Lysates were centrifuged for 10 min at 15,800 x g at 4°C. Supernatant was then aliquoted into Eppendorf tubes and stored at -80°C until further analysis. For 1 9 3 3 2 3 3 2 4 adherent cells, a similar procedure was applied ' ' with the exception that cell lysis buffer was added directly to the plate followed by scraping of the cells off the plates and transfer of the lysate to an Eppendorf tube. Apoptotic cells that may have detached into the media were collected by centrifugation and resuspended in the cell lysate obtained from the plate for the particular treatment. Cells were then lysed on ice for 20 min and treated in a similar manner to non-adherent cells described above. For tissue segments, cell lysates were prepared by homogenization of 100-500 mg frozen tissue samples in 1 ml lysis buffer as previously described '" .Following homogenization in lysis buffer, the homogenate was left on ice for 20 min. Insoluble materials were then removed by centrifugation followed by transferring the supernatant to a new tube. Lysates were then aliquoted and stored at -80°C as described above. 3.4.1.2 Cytosolic Extracts To detect mitochondrial AIF and cytochrome c release, cytosolic protein extracts were obtained as previously described 1 9 3 ' 3 2 3 , 3 2 4 - Cells were washed twice with P B S and cell pellets were resuspended in 500 ul ice-cold buffer containing 20 m M Hepes p H 7.5, 10 m M KC1, 1.5 m M M g C l 2 , 1 m M E D T A , 1 m M E G T A , 1 m M D T T and 1 m M P M S F . Cells were disrupted by 20 strokes with the B pestle of a Kontes dounce homogenizer. Lysate was centrifuged at 10000 x g for 10 min and the resultant supernatant was further centrifuged at 100000 x g for 1 h in a Beckman Optima™ T L X ultracentrifuge (Beckman, Palo Alto, C A ) using a TL-100 rotor. 80 3.4.2 Protein Quantification Protein concentrations of cell extracts were determined with the Bicinchoninic ( B C A ) Protein assay (Pierce, Rockford, IL). The B C A reagent reacts with copper (I) complexes formed by the interaction of protein and copper (II). Reaction of B C A with copper (I) results in the formation of an intense purple colour. Protein standards ranging from 0.1 mg/ml to 2 mg/ml were prepared. To prepare the B C A reagent, 50 parts Pierce Reagent A (contains sodium carbonate, sodium bicarbonate, B C A detection reagent and sodium tartate in 0.1 M sodium hydroxide) were mixed with 1 part Pierce Reagent B (contains 4% CUSO45H2O). 100 ul of each sample or standard were pipetted into separate tubes. To each tube, 2 ml of reagent was added, mixed, and incubated at 37°C for 30 min. Samples were cooled to room temperature and absorbance was read at 562 nm. The protein concentrations were then interpolated from the standard curve obtained using the measurements for protein of known quantity. 3.4.3 Protein Analysis 3.4.3.1 Antibodies Antibodies were obtained from the following sources: mouse monoclonal anti-caspases-1, -6 and -8 (Upstate Biotechnology Inc., Lake Placid, N Y ) ; rabbit polyclonal anti-Bid, rabbit polyclonal anti-Bcl-X, rabbit polyclonal Bcl-2, goat polyclonal anti-PARP (p25), goat polyclonal anti-CPP32, mouse monoclonal anti-caspase-3 (Santa Cruz Biotechnology Inc., Santa Cruz, C A ) ; mouse monoclonal anti-caspase-7 (Transduction Laboratories, Mississauga, ON); mouse monoclonal anti-cyt c, mouse monoclonal anti-Bax, rabbit polyclonal anti-caspases-2, -4, -9, and -10 (Pharmingen, Mississauga, ON); 81 mouse monoclonal anti-PARP(C-2-10) (Biomol Research Laboratories, Plymouth Meeting, PA) . The rabbit polyclonal anti-AIF antibody 1 0 4 was a gift from Dr. Guido Kroemer (Institute Gustave Roussy, no Villejuif, France). The rabbit polyclonal D F F antibody was a gift from Dr. Xiaodong Wang (University 108 of Texas Southwestern medical school, Dallas, Texas). The rabbit polyclonal anti-Bap31 antibody was a gift from Dr. Gordon Shore (Department of Biochemistry, M c G i l l University, Montreal, Quebec). 3.4.3.2 SDS-Polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis ( S D S - P A G E ) under reducing conditions was utilized to separate proteins for subsequent analysis. Electrophoresis of proteins was carried out under denaturing conditions that ensure dissociation of proteins into their polypeptide subunits and minimize protein aggregation. Ce l l lysates were mixed 50:50 with sample buffer and boiled for 5 min prior to loading into the gel. Approximately 20-30 ug/ml protein was loaded per well . SDS detergent was used in the sample and running buffers. SDS disrupts non-covalent interactions of proteins. 2-mercaptoethanol (2-Me) is also added to sample buffer to reduce disulphide bonds. SDS binds to proteins at a ratio of approximately one SDS for every two amino acid residues which gives a complex of SDS and denatured protein a large net negative charge that is roughly proportional to the mass of the protein. The negative charge obtained due 370 to SDS binding is much greater than the charge on the native protein . Thus, during electrophoresis, when a voltage is applied, the negatively charged SDS-protein moiety travels towards the positively charged anode. Since the amount of SDS bound is proportional to the molecular weight of the polypeptide and is independent of its sequence, the SDS-protein complexes migrate through polyacrylamide gels according to their size 3 6 9 . Small proteins move rapidly through the gel whereas larger proteins migrate slower. Conversely, in non-denaturing 82 gels, protein mobility is determined by protein size, charge and conformation . Polyacrylamide gels are composed of chains of polymerized acrylamide that are crosslinked by bisacrylamide. These gels form as a consequence of polymerization of the monomeric acrylamide to polymeric chains that are cross-linked by bisacrylamide. The polymerization reaction is initiated by adding ammonium persulfate (APS) and is further catalyzed by adding T E M E D . T E M E D catalyzes the formation of free radicals from A P S . To polymerize gels, 75 uL 10% A P S and 7.5 uL T E M E D were added and vortexed for 10 seconds.The gel was pipetted into prepared gel plate apparatus, leaving enough space for inserting the comb. The top of the gel was coated with water-saturated butanol and allowed to polymerize for 1 h. The gel was then rinsed with water and the 4% stacking gel was added consisting of: 1.0 m L 30% acrylamide 5.0 m L d H 2 0 1.5 m L Laemmli stacking gel buffer 100 u L 10%> ammonium persulfate (APS) 10 u L T E M E D The comb was placed in apparatus and the gel polymerized at room temperature for -15 minutes. Each protein sample was diluted in anequal volume of SDS-sample buffer containing a final concentration of 5% 2-Me and heated at 95°C for 5 min followed by cooling to room temperature prior to loading into its respective well. Once the gels were loaded, a Mini-Protean II Cel l electrophoresis unit was used to run the gel (Bio-Rad). The middle and outside of gel apparatus were filled with Laemmli running buffer and covered with the l id. The voltage was set at constant setting of 80 V for 20 min and then increased to 125 V for the remainder of the running time. 83 3.4.3.3 Western Immunoblotting After electrophoresis, gels were equilibrated in Transfer buffer for 20-30 min at room temperature with gentle shaking. While the gels were equilibrating, nitrocellulose membranes were soaked in Transfer buffer for at least 10 min at room temperature under gentle agitation. The transfer cassette apparatus was set up as follows: First Layer: Fiber pad that has been soaked in Transfer buffer. Second Layer. 2 pieces of Whatman 3 papers that have been soaked in Transfer buffer. Third Layer. Nitrocellulose membrane that has been cut to the size of the gel. Cut one corner of the membrane to maintain sample orientation. Fourth Layer. Gel Fifth Layer. 2 pieces of Whatman 3 papers that have been soaked in Transfer buffer. Sixth Layer. Wet fiber pad. Cassettes were placed into the transfer apparatus with the gel side facing the cathode. The container was filled with ice-cold Transfer buffer and a cooling apparatus, the l id was attached and plugged into the power apparatus. The transfer was performed for 1 h at 100 V . The cooling pack was placed in the buffer tank to absorb heat generated during the transfer. After the transfer, membranes were removed and rinsed twice in P B S - T (Appendix I). Membranes were blocked by placing each membrane in Blocking buffer (5 % skim milk powder in PBS-T ween) for 30 min at room temperature. A 10 m L primary antibody solution was then prepared (usually 1 pg/ml in Blocking buffer) as recommended by the manufacturer for Western blotting. The primary antibody solution was added under agitation for 45 min at room temperature. Membranes were then rinsed twice with PBS-T, washed once for 15 min in PBS-T 84 followed by two washes for 5 min in PBS-T. Diluted HRP-conjugated secondary antibody solution (usually 1:5000 in Blocking buffer) were prepared. Membranes were incubated in diluted secondary antibody for 30 min at room temperature. To detect biotinylated molecular weight markers, membranes were incubated for 15 min in Streptavidin-HRP conjugate solution. Membranes were then rinsed twice and washed once for 15 min, and twice for 5 min in PBS-T as described above. HRP-labelled proteins were visualized using the Amersham enhanced chemiluminescence (ECL) protocol. Briefly, equal volumes of Amersham Detection Solution 1 and Amersham Detection Solution 2 were added to the protein side of each membrane for 1 min. The final volume required is 0.125 m L / c m 2 membrane. Excess buffer was removed from the membrane and the membrane was carefully wrapped in plastic wrap while gently removing any air pockets. In a dark room, a sheet of autoradiography film was placed on top of the membrane (protein side up), and exposed for times ranging from 30 sec to 1 h. To develop the film, film was placed in Developer solution (Kodak) for 2 min, washed in water for 30 sec, placed in Fixative Solution (Kodak) for 2 min, and finally washed in water for 1 min. Films were then digitally scanned for presentation and computer storage purposes. 3.4.3.4 Fluorometric Quantification of Caspase Activity Two methods were used to study caspases in PDT-treated cells: (1) fluorometric quantification of caspase activation using specific synthetic fluorogenic peptide substrates and (2) immunoblot detection of pro- and activated caspases and the cleavage of their specific substrates. The first method is rapid and quantitative, however, although caspase cleavage activities of different specificities can be discriminated, the assay can not fully distinguish which specific caspases are activated. The latter method is less quantitative, but can define precisely whether a particular 85 caspase family member is activated in response to a particular treatment or a specific caspase substrate is processed or cleaved during apoptosis. Cell-free protease assays were performed by incubating approximately 50 ug of cell lysate protein (as assessed using the B C A protein assay described above) in 150 ul of reaction buffer (1% I G E P A L , 20 m M Tris, p H 7.5, 137 m M N a C l , 10% glycerol) containing 100 u M of caspase-1 (4-(4-dimethylaminophenylazo) benzoyl-Tyr-Val-Ala- Asp- Ala-Pro-Val-5 [(2-aminoefhyl) amino] -napthalene-1 -sulfonic a c i d ) ( D A B C Y L - Y V A D A P V - E D A N S ) , caspase-3 (Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin ( A c - D E V D - A M C ) ) (Calbiochem, Cambridge, M A ) , caspase-6 (Ac-Val-Glu-I le-Asp-AMC) or caspase-8 (Z-Ile-Glu-Trir-Asp-7-Arnino-4-trifluoromethylcoumarin (Z-57 79 193 20 1 324 IETD-AFC)) substrate in 96-well microtiter plates as previously described . Lysates were incubated at 37°C for 4 h and cleaved substrate fluorescence levels were determined using a CytoFluor™2350 (PerSeptive Biosystems, ON) set at excitation and emission wavelengths of 360 nm and 460 nm. Fluorogenic (AMC/AFC-label led) substrates were used instead of colorimetric substrates (p-Nitroanilide) substrates because they provide a 10- to 100-fold increase in . . . 369 sensitivity . 3.5 Flow Cytometry 3.5.1 DNA Fragmentation (PI staining) 371 372 A PI staining procedure was used to detect changes in the status of cellular D N A ' . PI staining was chosen instead of T U N E L labeling for flow cytometry studies because this assay is more rapid, inexpensive and produced equivalent results for the quantification of D N A fragmentation. During apoptosis, in apoptotic cells D N A is fragmented. Low-molecular weight fragments of D N A diffuse out of the nuclei. These cells wi l l therefore show less staining than their 86 normal counterparts . Although, the assay by itself does not distinguish between apoptosis and necrosis, it is a useful assay in combination with other biochemical assays as an endpoint of which to measure apoptosis. D N A gel electrophoresis was discontinued as a means to assess D N A fragmentation in apoptotic cells because it is time consuming and is only qualitative whereas PI staining is rapid and quantitative 3 6 9 . A t various times following PDT, approximately 1 x 10 6 cells were washed twice with ice-cold PBS then permeabilized and fixed in 80% ethanol at 4°C for 1 h. Cells were washed twice in ice-cold PBS and treated with RNAse (5 U /ml , DNAse-free) (Sigma) and stained with PI (50 u.g/ml) in PBS. Samples were analyzed by flow cytometry. The percentage of cells containing hypodiploid levels of D N A was calculated from single parameter flow cytometry 372 for PI fluorescence using an Epics X L flow cytometer (Coulter Electronics Inc., Hialeah, F A ) . 3.5.2 7A6 Antigen Detection Detection of the mitochondrial epitope 7A6 by the mouse monoclonal phycoerythrin (PE)-373 labelled Apo2.7 antibody has been utilized as a tool to measure apoptosis . H A S M C were prepared for flow cytometry by washing twice with P B S followed by incubation for 5 min at 37°C in P B S containing 0.05% trypsin and 0.53 m M E D T A . Cells were permeabilized with 0.1% digitonin in P B S . Mitochondrial 7A6 antigen expression by apoptotic H A S M C was assessed using - 323 flow cytometry . 3.6 Direct and Indirect Immunofluorescent Microscopy 3.6.1 Mitotracker Staining Although conventional fluorescent stains for mitochondria, such as rhodamine 123 and tetramethylrosamine, are readily sequestered by functional mitochondria, they are subsequently washed out of the cells upon the loss of mitochondrial membrane potential. Thus, their use in 87 experiments in which cells must be treated with aldehyde fixatives or other agents that affect the energetic state of the mitochondria are limited. However, MitoTracker probes are mitochondrion-selective stains that are concentrated by active mitochondria and are well retained during cell fixation. Because the MitoTracker Red probe is retained after permeabilization, the sample retains the fluorescent staining pattern characteristic of live cells during subsequent processing steps for immunocytochemistry, in situ hybridization or electron microscopy. Furthermore, MitoTracker reagents can eliminate some of the difficulties of working with pathogenic cells because, once the mitochondria are stained, cells can be treated with fixatives prior to analysis. MitoTracker probes are cell-permeant mitochondrion-selective dyes that contain a mildly thiol-reactive chloromethyl moiety. The chloromethyl group may be responsible for retaining dye association with mitochondria following fixation. To label mitochondria, media was removed from cells. Cells were incubated in a media containing Mitotracker Red (250 ng/ml) for 30 min at 37°C. Cells were then washed twice with prewarmed (37°C) P B S . Cells were subsequently fixed with 2% paraformaldehyde for 15 min at room temperature. A t this point, the mitochondria-labelled cells were ready for subsequent immunocytochemical labelling (described below). 3.6.2 Immunocytochemistry H U V E C were grown on type I collagen-coated 8-well chamber slides ( V W R Canlab, Mississauga, ON) . Following treatment with either media alone, or 2 h post-PDT (100 ng/ml verteporfin; 2 J/cm red light, 620-700 nm), cells were coated with acetone for 20 min at -20°C. Cells were then washed with P B S and incubated with 5% skim milk powder in P B S for 30 min at 88 37°C. Cells were washed with P B S and incubated with or without rabbit anti-active caspase-3 antibody (Pharmingen) (1:20) for 30 min at 37°C. Cells were washed and incubated with 1% normal goat serum for 30 min at 37°C. Goat serum (Biosource, Camarillo, C A ) was removed and goat anti-rabbit IgG FITC-conjugated antibody (Biosource, Camarillo, C A ) (1:100) in 0.1% normal goat serum was added for 30 min at 37°C. Wells were coated with 100 ul Antifade reagent (Molecular Probes; Oregon). Fluorescent images were acquired through a standard FITC filter set (Omega, Vermont) using a 16-bit cooled C C D camera (1024X1024 pixels; Photometries, Arizona) mounted on 2.5X adaptor at the bottom-port of an Axiovert SI00 T V microscope (Zeiss, Canada) and coupled to the signal acquisition and processing software SlideBook (Intelligent Imaging Innovations). A 6 3 X Zeiss objective was used for obtaining detailed pictures. H A S M C were grown on type I collagen-coated 8-well chamber slides. Following treatment with media alone or P D T , cells were coated with 2% paraformaldehyde for 15 min at RT . Cells were washed with P B S and incubated with 5% skim milk powder in P B S for 30 min at 37°C. Cells were washed with P B S and incubated with or without mouse anti-cyt c Lamin A / C or Bax monoclonal antibodies (1:20) or rabbit polyclonal antibody (1:20) for 30 min at 37°C. Cells were then washed and incubated with 1% normal goat serum for 30 min at 37°C. Goat serum (Biosource, Camarillo, C A ) was removed and goat anti-mouse or rabbit IgG Alexa 488-conjugated or goat anti-mouse IgG Alexa 590 antibody (Molecular Probes, Oregon) (1:200) in 0.1% normal goat serum was added for 30 min at 37°C. Wells were coated with 100 ul Antifade reagent (Molecular Probes). Mitotracker was utilized to detect mitochondria as per the manufacturer's instructions. Fluorescence images were acquired using a laser scanning confocal system (Noran OZ) on an inverted microscope equipped with a lOOx oil-immersion lens. The 89 cells were illuminated using the 488 nm and the 568.2 nm line of an Argon-Krypton laser. Fluorescence emission was collected in a photomultiplier tube after it passed through a 525/52 B P or a 590LP filter. Image analysis was performed in ImagePro Plus and all data was stored on C D - R O M . 3.6.3 Calcium Imaging Calcium studies were performed in collaboration with Dr. D . Ruehlmann, a post-doctoral fellow in Dr. C. van Breemen's laboratory. For calcium studies, media was removed and cells were rinsed once with Hank's calcium and magnesium free balanced salt solution (Hank's B S S ; BioWhittaker, Walkersville, M D ) . Cells were incubated at 37°C in media containing various amounts of verteporfin (0-100 ng/ml) for 1 h. Cells were then incubated at room temperature for 10 min at 37°C in Hank's B S S . Cells were incubated in Hank's B S S supplemented with 3 u M Fluo-3 in media containing verteporfin for an additional hour. Media was supplemented with histamine (1 uM) , cyclopiazonic acid (CPA) (1 uM) or ionomycin (1 uM) as indicated. Cells were exposed to red light while on the microscope stage. Observations of C a 2 + changes were made using a Noran Oz laser scanning confocal microscope through an air 60x lens on a N i k o n Eclipse TE200 inverted microscope (Figure 13). The cells were illuminated using the 488 nm line of an Argon-Krypton laser and emission and collected by a high-gain photomultiplier tube after it had passed through a 525/52 B P filter. Samples of approximately 20 cells were assessed for each experiment. A t least three experiments were done per treatment. Fluorescence was digitized and quantified using ImagePro Plus software. The average fluorescence and range for each experiment was recorded and plotted. 90 Figure 13. Intracellular C a 2 + imaging using confocal microscopy. Ce l l permeable F l u o - 3 - A M is taken up by cells. Upon entry, esterases degrade the A M from Fluo-3, at which time, Fluo-3 can not be readily released from the cell. Free cytosolic C a 2 + binds to Fluo-3. Upon excitation with 488 nm laser light, Ca 2 +-bound Fluo-3 emits a fluorescence of526 nm light which is allowed to pass through the dichroic mirror and through the imaging aperture. The signal is amplified through the photomultiplier, digitized and stored. 91 C H A P T E R IV R E S U L T S 4.1 Aim I Effects of PDT on Transformed Cells 4.1.1 HL-60 Cells 4.1.1.1 Protease Activity and DNA Fragmentation A high proportion of HL-60 cells treated with P D T (verteporfin and light) exhibited D N A fragmentation (91.9 ± 5.3%; n = l l ) within 3 h of photosensitization compared to only 4.3 ± 2.9% (n=l l ) of untreated cells (Figure 14). N o increase in the number of cells exhibiting D N A fragmentation was evident for cells exposed only to the light source alone or in cells incubated with verteporfin and protected from light. In preliminary work to determine whether non-specific proteolytic activity might be involved with PDT-induced apoptosis, various broad range biochemical inhibitors were added to the cells after verteporfin incubation but prior to their exposure to light (Figure 14). Preliminary experiments indicated that D N A fragmentation in HL-60 cells was not evident for at least 40 min following light treatment. The extent of D N A fragmentation was determined 3 h post-irradiation since PDT-treated HL-60 cells exhibited high levels of D N A fragmentation by this time. The general caspase family inhibitor, Z - A s p - D C B as well as the serine protease inhibitors D C I and T L C K completely blocked PDT-induced D N A fragmentation. The tripeptide granzyme B inhibitor ( Z - A A D - C M K ) did not block D N A fragmentation. Other inhibitors (PMSF and aprotinin) and vehicle solutions had no effect on D N A fragmentation. 92 Figure 14. Effects of various protease inhibitors on PDT-induced D N A fragmentation in human promyelocytic leukemia cells. HL-60 cells were incubated with verteporfin (total of 60 min) and the indicated inhibitor and/or vehicle solution for the final 30 min prior to light irradiation. Cells were fixed at 3 h after P D T and assessed for D N A status using PI staining and flow cytometry. Results are sumarized as the average and SD from a series of experiments (verteporfin: n=l 1; light: n=3; light: n=3; PDT: n=l 1; each protease inhibitor: n=6; each vehicle (dH 2 0 , D M S O , Methanol): n=2) 93 To further examine the nature of the proteolytic activity induced by P D T , protease assays were performed to measure caspase-1 (YVAD-ase) and caspase-3 (DEVD-ase) activity (Figure 15). There was no evidence of caspase-1-like (YVAD-ase) activity. These findings were supported by the absence of caspase-1 processing using Western blot analysis. However, DEVD-ase activity, indicative of caspase-3/7-like activity, was present in lysates from cells treated with verteporfin and light but not in control cell lysates. To examine whether P D T induced caspase-3 processing and P A R P cleavage, HL-60 cells were treated with P D T and lysed 0, 15, 30, 60, or 120 min after light activation (Figure 16). Cell lysates were separated by S D S - P A G E , transferred to nitrocellulose and probed with anti-caspase-1, anti-caspase-3 or anti-PARP antibodies. Pro-caspase-1 processing was not evident following PDT. However, caspase-3 processing, as evidenced by decreased levels of the proform as well as appearance of the 12 k D subunit, was complete within 15 min after light irradiation. P A R P was fully cleaved by 60 min following light treatment. A small amount of the p i 2 subunit was detected in the untreated cells. This was likely due to background levels of apoptosis in the culture. However minimal P A R P cleavage was detected. The caspase-3 antibody did not detect the 17 k D fragment of caspase-3 since it was specific for the amino terminus of the p 12 subunit of the 32 k D pro-caspase-3 protein. The peptide inhibitor Z - A s p - D C B did not prevent caspase-3 processing at the concentrations employed (Figure 17). However, the monopeptide did block the cleavage of P A R P . Processing of caspase-3 and P A R P cleavage were both inhibited by T L C K or D C I treatment prior to light treatment (Figure 18). 94 st ^ o ?3 d) Q ISO a (0 ra O 500 450 400 350 300 250 200 50 0 50 100 150 200 250 2500 100 150 Time (min) 250 Figure 15. Increased caspase-3/7-like (DEVD-ase) but not caspase-1-like (YVAD-ase) protease activity in PDT-treated human promyelocytic HL-60 cells. Cells were incubated with verteporfin for 1 h, follwed by exposure to red light. Whole cell extracts were prepared 1 h after P D T and assessed for DEVD-ase or YVAD-ase cleavage activity over a 4 h period. Ce l l extracts were prepared from untreated (hollow circle), verteporfin-treated (filled square), or PDT-treated (filled circle) cells .Error bars represent the SD of the average of 6 replicates. 95 -25 kD fragment Figure 16. P D T induces rapid activation of caspase-3, but not caspase-1 in human promyelocytic leukemia cells. HL-60 cells were incubated with verteporfin (100 ng/ml) for 1 h followed by exposure to red light (2 J/cm 2). Whole cell extracts were prepared at the indicated times after light exposure and assessed for the status of caspase-1 and -3 and P A R P cleavage using Western blotting. 96 X J o o o a. o tr > PDT + Z-Asp-DCB (nM) -pro-caspase -3 caspase-3 (p12) -PARP -25 kD fragment Figure 17. Z - A s p - D C B prevents the proteolytic cleavage of P A R P but does not inhibit caspase-3 processing in PDT-treated HL-60 cells. HL-60 cells were lysed 60 min after P D T (100 ng/ml verteporfin; 2 J/cm 2 light). Cel l lysates were subjected to S D S - P A G E , transferred to nitrocellulose and probed with either polyclonal (A) anti-caspase-3 or (B) anti-PARP antibodies. 97 •o 1= CD £ £ PDT + DCI (p.M) 240 1 -pro-caspase-3 § > 0 60 120 0 -caspase-3 (p12) - P A R P -25 kD fragment B c m I— 4-1 o (0 Q. > 1 0 135 270 540 1 ® PDT + T L C K (p.M) 54  H-pro-caspase-3 -caspase-3 (p12) - P A R P -25 kD fragment Figure 18. Caspase-3 processing and cleavage activity can be blocked by broad range serine protease inhibitors. H L - 6 0 cells were incubated with verteporfin (100 ng/ml) for 1 h followed by exposure to red light (2 J/cm 2). Cells were incubated with increasing concentrations of D C I (A) or T L C K (B) for 30 min prior to light exposure as indicated. Whole cell extracts were prepared at the indicated times after light exposure and assessed for the status of caspase-3 processing and P A R P cleavage using Western blotting. 98 To determine whether caspase-6 was activated and whether its processing was caspase-3-dependent, HL-60 cells were treated with different amounts of the caspase-3 inhibitor Z -DEVD-fmk prior to photosensitization of verteporfin. The presence of Z - D E V D - f m k did not affect PDT-induced caspase-3 processing and the appearance of the 12 k D active subunit at the concentrations tested (Figure 19). However, when the membrane was probed with either anti-caspase-6 or anti-P A R P antibodies, it was evident that PDT-induced cleavage of these proteins was greatly reduced at a concentration of 25 u M of Z-DEVD-fmk. The PDT-induced increase in hypodiploid D N A ( D N A fragmentation) was also blocked at this concentration of the tetrapeptide inhibitor. A lower concentration of Z-DEVD-fh ik (10 uM) did not block the processing of pro-caspase-6 or P A R P nor affect the appearance of hypodiploid levels of D N A (Figure 19). These results demonstrate that caspase-6 is activated by P D T and, furthermore indicate that caspase-6 activation occurs downstream of caspase-3 processing. 99 100 c -PARP (p116) "§ | I 0 10 20 25 | 1 o P D T + DEVD-fmk (nM) 3 0) > Figure 19. Caspase-6 activation may occur downstream of caspase-3 activation. HL-60 cells were treated with increasing concentrations of the caspase-3/7 inhibitor D E V D - f m k prior to P D T (100 ng/ml verteporfin; 2 J/cm light). With increasing concentrations of the inhibitor, D N A fragmentation (n= 1), as well as processing of caspase-6 and P A R P were decreased. Processing of the caspase-3 p 12 subunit was unaffected by the presence of D E V D - f m k . 100 4.1.1.2 Overexpression of Bcl-2 Blocks Caspase-3 and Caspase-6 Processing To assess the influence of Bcl-2 overexpression on cell susceptibility to photodynamic killing, HL-60/neo or HL-60/Bcl-2 cells were incubated with or without verteporfin and then exposed to red light. Cells were lysed 1 h after light activation of verteporfin. The status of Bcl-2, caspase-3 and caspase-6 in whole cell extracts was determined by Western immunoblotting. The Bcl-2-transfected cells exhibited a greater band intensity of Bcl-2 than the the control (HL-60/neo) cells (Figure 20). The electrophoretic status of Bcl-2 was unaffected by the P D T treatment of either cell type and no Bcl-2 cleavage products were detected by the antibody that was used (HL-60/neo or HL-60/Bcl-2). For cells treated with verteporfin and light, caspase-3 and caspase-6 cleavage was evident for HL-60/neo cell lysates but not in the HL-60/Bcl-2 cell extracts (Figure 20). These results suggested that Bcl-2 influences caspase-3 and caspase-6 activation produced by PDT. 4.1.1.3 Overexpression of Bcl-2 or BC1-XL Inhibits Caspase-3-Iike Protease Activity Cel l lysates from control (neo)-transfected and Bcl-2-transfected H L - 6 0 cells were assayed for their capacity to cleave a fluorescently-labelled caspase-3 substrate ( A c - D E V D -A M C ) (Figure 21). Lysates prepared from HL-60/neo cells treated with verteporfin and light exhibited high A c - D E V D - A M C cleavage activity, whereas lysates from HL-60/Bcl -2 cells treated with the same levels of verteporfin and light exhibited baseline levels o f substrate cleavage. Whole cell extracts prepared from HL-60 cells with B c l - X L overexpression exhibited minimal caspase-3-like proteolytic activity in contrast to the HL-60/neo cells treated with the same level of verteporfin and light (Figure 22). These results further support the observation that overexpression of Bcl -2 blocks activation of caspase-3 instigated by verteporfin photoactivation. 101 •caspase-2 (p12) •pro-caspase-6 -caspase-6 (p20) Figure 20. Overexpression of Bcl-2 prevents the activation of caspase-3 and caspase-6 in PDT-treated human promyelocytic leukemia cells. Control-transfected HL-60 (Neo) or B c l -2-transfected HL-60 (Bcl-2) cells were incubated with 100 ng/ml verteporfin (PDT-100) or 200 ng/ml verteporfin (PDT-200) followed by exposure to red light (2 J/cm 2). Whole cell extracts were prepared 1 h after P D T and probed for the presence of Bcl -2 , caspase-3 or caspase-6 by Western blotting. 102 1 0 0 0 0 F • • • c o Q. Q> "C O > o o Q D_ o o CM a a. HL-60/neo HL-60/Bcl-2 Figure 21. Bcl -2 prevents caspase-3/7-like activity in PDT-treated human promyelocytic leukemia cells. Control-transfected HL-60/Neo or Bcl-2-transfected HL-60 /Bc l -2 cells were treated with 100 ng/ml verteporfin (PDT-100) or 200 ng/ml verteporfin (PDT-200) followed by exposure to red light (2 J/cm 2). Whole cell extracts were prepared 1 h after P D T and assessed for caspase-3/7-like (DEVD-ase) protease activity as described. Error bars represent SD of the mean values obtained from 3 experiments using replicates of 5 for each treatment sample. 103 1000 F £ 9 0 0 £ 800 | | 700 j^g 600 e: •«* *? Q 500 { § 2 . 4 0 0 Q . {g 300 o 200 100 0 B c < o z *S Q 2 it- -* 70 60 : so : 40 30 20 10 0 untreated verteporfin PDT untreated verteporfin PDT Figure 22. Overexpression of B c l - X L blocks PDT-induced caspase-3/7-like (DEVD-ase) activity and D N A fragmentation. (A) A t 1 h after treatment, lysates of HL-60/neo (empty bars) or H L -60 /Bc l -X L (solid bars) cells were prepared and assayed for their capacity to cleave the fluorescently-labelled caspase-3 substrate ( A c - D E V D - A M C ) . Cytosolic extracts were prepared from cells treated with light (untreated), verteporfin (100 ng/ml) or PDT. (B) HL-60/neo (empty bars) and H L -60 /Bc l -X L (solid bars) cells were assessed for D N A fragmentation by PI staining 3 h after the indicated treatments. 104 4.1.1.4 Overexpression of Bcl-2 or BC1-XL Inhibitss PDT-induced DNA Fragmentation HL-60/neo and HL-60/Bcl -2 cells were analyzed for their D N A status 3 or 24 h following verteporfin photoactivation (Figure 23). The majority of HL-60/neo cells treated with verteporfin and light contained hypodiploid levels of D N A as compared to untreated cells and cells treated with light or verteporfin alone. However, there was no evidence of PDT-induced D N A fragmentation (cells<2N D N A ) for HL-60/Bcl -2 cells treated with verteporfin and light at 3 h after photoactivation and only a slight increase in hypodiploid D N A by 24 h. HL-60/neo and H L - 6 0 / B c l - X L cells were also assessed for the presence of D N A fragmentation 3 h after photoactivation of verteporfin (Figure 23). The majority of HL-60/neo cells treated with verteporfin and light exhibited D N A fragmentation as compared to untreated cells or cells exposed to verteporfin alone. However, an increase in D N A fragmentation was not evident in H L - 6 0 / B C 1 - X L cells treated with verteporfin and light at 3 h after photoactivation (Figure 22B). These results indicate that overexpression of Bcl-2 or B c l - X L in H L - 6 0 cells inhibits D N A fragmentation induced by verteporfin and light. 105 i f S z | C M SS i2 < o Z S Z 2 — < o Z Q b 100F 90 \ 80; 70 \ 601 501 401 30^  20^  10§ 0^ 100p 90^  80 \ 70; 60 \ 50; 40; 30 \ 20; 101 0^  3 h post-PDT J L J L • 24 h post-PDT untreated verteporfin PDT-100 PDT-200 Figure 23. Overexpression of Bcl-2 inhibits D N A fragmentation in PDT-treated human promyelocytic leukemia cells. Control-transfected HL-60 (empty bars) or Bcl -2-transfected H L - 6 0 (solid bars) cells were treated with 100 ng/ml verteporfin (PDT-100) or 200 ng/ml verteporfin (PDT-200) followed by exposure to red light (2 J/cm 2). A t 3 h and 24 h after PDT, D N A fragmentation was assessed using flow cytometry as described. Mean values with SD for 3 independent experiments are shown. 106 4.1.1.5 Overexpression of Bcl-2 in HL-60 Cells Provides Partial Protection Against Cell Death at Low Levels of PDT Our final step was to examine the effect of Bcl-2 overexpression on the viability of cells treated with verteporfin and light. HL-60/neo and HL-60/Bcl-2 cells were incubated with titrated amounts of verteporfin and exposed to light 1 h later. Bioreduction of M T T (Figure 24), PI uptake (Figure 25), and trypan blue exclusion (Figure 26) were used to compare the viability of each cell type at 3, 24, 48 and 72 h after PDT. Overexpression of Bcl-2 provided no observable protection at concentrations of verteporfin at 50 or 100 ng/ml as determined by all three cell viability assays over a 72 h period. However, with lower concentrations of verteporfin (10 or 25 ng/ml), Bcl-2 overexpression did provide partial protection against P D T for a small proportion of cells which exhibited recovery over the 72 h test period. Thus, Bcl-2 provides partial protection against apoptosis induced by low levels, but not high levels of P D T in HL-60 cells. 107 100 10 25 50 100 10 25 50 100 100 100 10 25 50 100 10 25 50 100 verteporfin (ng/ml) Figure 24. Overexpression of Bcl-2 inhibits the loss of cell viability in human promyelocytic leukemia cells treated with low but not high doses o f PDT. Control-transfected H L - 6 0 (empty) or Bcl-2-transfected HL-60 (solid) cells were treated with increasing levels of verteporfin followed by exposure to red light (2 J/cm 2). A t 3, 24, 48 or 72 h after PDT, cell viability was assessed using the M T T cell viability assay as described. Error bars represent the SD of mean values from 16 replicates for each treatment. 108 Figure 25. Overexpression of Bcl -2 in PDT-treated human promyelocytic leukemia cells partially inhibits loss of cell viability as determined by PI uptake studies. HL-60/neo ( • ) and HL-60 /Bc l -2 ( • ) cells were incubated with titrated amounts of verteporfin (0-100 ng/ml) and then exposed to red light 1 h later. Cel l viability was assayed 3,24,48 and 72 h after P D T using PI uptake to distinguish between live and dead cells. Each measurement is expressed as a percentage of the total cells gated that are permeable to PI. 109 100 90 80 70 60 50 40 30 20 10 0 3h 10 25 50 100 100 90 80 70 60 50 40 30 20 10 0 24 h r* r 10 25 50 100 100 90 80 70 60 50 40 30 20 10 0 48 h J 10 25 50 100 100 90 80 70 60 50 40 30 20 10 0 72 h 10 25 50 100 verteporfin (ng/ml) Figure 26. Bcl -2 does not provide protection against PDT-induced cell death at verteporfin concentrations of 50 ng/ml or greater as determined by trypan blue dye exclusion. H L -60/neo (empty bars) and HL-60/Bcl -2 (solid bars) cells were incubated with titrated amounts o f verteporfin (0-100 ng/ml) for 1 h and then exposed to red light (2 J/cm 2). Ce l l viability was assessed 3, 24, 48, and 72 h later by counting viable cells using trypan blue exclusion. Data is expressed as a percentage of the result obtained with untreated (control) cells counted at each timepoint.(n= 1) 110 4.1.1.6 DFF: A Link Between BC1-XL, Caspase-3 Activation and DNA Fragmentation Since levels of caspase-3-like activity as determined by the protease assay directly corresponded to subsequent levels of D N A fragmentation, our next aim was to examine possible 78 links between these two events. L i u et al. identified a novel protein (DFF) in staurosporine-treated HeLa cells that was proteolytically cleaved during apoptosis. It was demonstrated that caspase-3 could cleave this protein into active subunits that were required to induce D N A fragmentation in isolated cell nuclei 7 8 . W i l d type HL-60 cell lysates were prepared at 5, 15, 30 or 60 min following PDT. Immunoblot analysis showed that pro-caspase-3 cleavage was evident within 5 min after P D T (Figure 27). Furthermore, virtually all D F F was processed by 15 min post-PDT, whereas P A R P was not completely cleaved until 30 min post-PDT. The earlier cleavage of D F F by caspase-3 may be attributable to the cytosolic localization of D F F and to the nuclear localization of P A R P . Our observation that D F F was cleaved in PDT-treated cells undergoing apoptosis is similar to that of L i u 78 et al. in which staurosporine was used as the pro-apoptotic stimulant in HeLa cells. Treatment of the HL-60 cells with the caspase-3 inhibitor, Z -DEVD-fmk , prior to light activation of verteporfin, prevented D F F and P A R P cleavage (Figure 28). The observation that Z -DEVD-fmk blocks P A R P cleavage is consistent with results using other stimuli obtained by other 69 375 376 investigators ' ' . Our D F F results for HL-60 cells treated with P D T support evidence by L i u et 78 al. suggesting that D F F is a substrate of caspase-3 . To further examine upstream events regulating PDT-induced D F F activation, the role of the anti-apoptotic B C 1 - X L proto-oncogene was assessed. The status of B C 1 - X L , caspase-3, P A R P and D F F in cytosolic extracts from PDT-treated cells was determined by Western immunoblotting (Figure 29). A low level of B C 1 - X L expression was detected in the HL-60/neo cells as compared to 111 the H L - 6 0 / B C 1 - X L transfectants (Figure 29). N o apparent cleavage or size alteration of BC1-XL was observed in either the PDT-treated HL-60/neo or H L - 6 0 / B C 1 - X L cells. Following light activation of verteporfin, caspase-3, P A R P and D F F were cleaved in the HL-60/neo cell lysates but not in the H L - 6 0 / B C 1 - X L cell extracts (Figure 29). These results indicate that caspase-3-dependent D F F cleavage and subsequent D N A fragmentation by P D T is regulated indirectly by B C 1 - X L . 112 Time after PDT (min) 0 5 15 30 60 1" -m*-imm* nj00Kmm* ^MMMHW -25 kD fragment Figure 27. P D T induces rapid caspase-3 activation followed by D F F and P A R P cleavage. H L -60 cells were incubated with verteporfin (100 ng/ml) for 1 h followed by exposure to red light (2 J/cm 2). Cells were lysed at the indicated times post-PDT and assessed via Western blotting for caspase-3, DFF, and PARP. 113 I I Q ,S > TJ «F Hi 5 o Q S 8- + 3 > D_ D. •DFF -PARP -25 kD fragment Figure 28. D F F is cleaved by a caspase-3-like protease. A t 1 h after PDT, cell lysates were prepared. Proteins were separated by S D S - P A G E followed by Western immunoblotting using anti-DFF, or anti-PARP antibodies. HL-60 cells were treated with light (control), verteporfin (100 ng/ml), verteporfin (100 ng/ml) and light (2 J/cm2) (PDT) or verteporfin (100 ng/ml) and Z - D E V D - f m k (20 u M ) and light (2 J/cm2) (PDT + Z - D E V D - f m k ) . 114 % "'""! •Bcl-XL -pro-caspase-3 -caspase-3 (p12) PARP -25 kD fragment -DFF -30 kD fragment Figure 29. Overexpression of B c l - X L in HL-60 cells blocks the cleavage of pro-caspase-3, PARP, and D F F induced by PDT. Cells were lysed 1 h after light irradiation. Cel l lysates were separated by S D S - P A G E followed by Western immunoblotting. Cells were treated with red light (untreated), verteporfin (100 ng/ml) or P D T (verteporfin +red light). 115 4.1.2 HeLa Cells 4.1.2.1 Cell Shrinkage and Membrane Blebbing in PDT-Treated HeLa Cells HeLa cells were examined by phase-contrast microscopy to assess their morphology following PDT. Cel l shrinkage and membrane blebbing, characteristic of apoptotic cells, were observed for a small portion of cells within 1 h following light irradiation of cells treated with verteporfin (Figure 30). These morphologic changes were evident for the majority of cells by 2-3 h post-PDT. Red light alone or verteporfin in the absence of light did not induce any morphological alterations indicative of apoptosis. 4.1.2.2 Caspases Play a Critical Role in PDT-Induced HeLa Cell Apoptosis The morphological changes associated with PDT-induced HeLa cell apoptosis corresponded to the degree of caspase-3/7-like activity (Figure 31). DEVD-ase activity was detected in cell lysates by 1 h post-PDT. Treatment of cells with verteporfin in the absence of light had no effect on caspase activity or cell viability. We previously demonstrated that pretreatment with caspase inhibitors or overexpression of Bcl-2 or BC1-XL blocked PDT-induced caspase-3 activation and D N A 57 79 201 fragmentation in HL-60 cells ' ' . T o confirm that caspases were involved in PDT-induced cell killing of HeLa cells, cells were pretreated with increasing concentrations of the broad range caspase family inhibitor Z V A D - f m k prior to PDT. With increasing concentrations of Z V A D - f m k , morphologic apoptotic changes such as membrane blebbing and cell shrinkage became less evident for PDT-treated cells (Figure 32). These results demonstrate that caspases are required for the formation of the apoptotic phenotype observed for HeLa cells following PDT. 116 untreated PDT (0 h) PDT (1 h) PDT (2 h) ~ y ' ';;! HHHBflHHHHHH ,1 ] - -' rs r ' 7 " V \ r-• PDT (3 h) PDT (4 h) PDT (5 h) verteporfin 5 h Figure 30. P D T induces cell shrinkage and membrane blebbing in H e L a cells. Cells were incubated with or without verteporfin (200 ng/ml) for 1 h and exposed to red light (2 J/cm 2) or protected from light. Cells were visualized using phase-contrast microscopy. 117 800 r Figure 31. P D T induces increased caspase-3/7-like activity in H e L a cells. Cells were incubated with or without verteporfin (200 ng/ml) for 1 h and exposed to red light (2 J/cm 2) or with verteporfin in the absence of light. Whole cell extracts were prepared and assessed for caspase-3/7-like proteolytic activity. 118 PDT + increasing concentrations of ZVAD-fmk Figure 32. Morphological changes associated with PDT-induced H e L a cell apoptosis are caspase-dependent. H e L a cells were incubated with or without (untreated) verteporfin (200 ng/ml) for 1 h and exposed to red light (2 J/cm 2). PDT-treated cells were incubated with increasing concentrations of the general caspase family inhibitor Z V A D - f m k prior to photosensitization. Cells were visualized at 2 h post-PDT by phase-contrast microscopy. 119 4.1.2.3 PDT Induces the Activation of Caspases 3,6,7,8 and 9 in HeLa Cells Our next step was to assess the status of several members of the caspase family during PDT-induced apoptosis of HeLa cells. Caspases 3, 6, 7, and 8, but not caspases 1, 2, 4 and 10, were processed following P D T (Figure 33). Our group demonstrated at a later date that caspase-9 323 activation was also involved PDT-induced apoptosis . Interestingly, caspase-8 processing was not evident prior to cleavage of caspases-3, -6 and -7. To further support this notion, the caspase-3/7 inhibitor D E V D - f m k was capable of inhibiting caspase-8 processing (Figure 34). The involvement of caspase-8 as an early upstream effector caspase in receptor-mediated apoptosis instigated by 35 377 378 FasL and T N F has been well documented ' ' . However, with the exception of U V irradiation, 379 which may directly trigger the Fas pathway , at the time, we were unaware of a chemotherapeutic agent that had been shown to activate caspase-8. To further discern the role for caspases in PDT-induced apoptosis, we examined cleavage of the established caspase substrates P A R P , D F F and Ras-GAP. A l l three proteins were cleaved following P D T (Figure 35). Light alone did not activate any of the above caspases that were examined. 120 TJ 0) +J n 0) u C 2 Time after PDT (h) c t 0 a -pro-caspase-1 -pro-caspase-2 -pro-caspase-3 -caspase-3 (p12) pro-caspase-4 -pro-caspase-6 -pro-caspase-7 -pro-caspase-8 pro-caspase-10 Figure 33. P D T induces the activation of caspases-3, -6, -7, and -8 in cervical carcinoma cells. HeLa ceils were incubated with or without verteporfin (200 ng/ml) for 1 h and exposed to red light (2 J /cm) or with verteporfin in the absence of light. Whole cell extracts were prepared and assessed for the status of various caspase family members using Western blotting. 121 1200 > 1000 E 800 '•= o £ ; ^ 600 *? Q tn id 400 ro w ro O -Bap31 -p20 - P A R P -p85 -DFF -p30 -p11 -pro-caspase-8 -p43 o •*-> ro o c O Q. 0) t > Q a. o • N + I-Q D_ o o a + i -Q Q. O o Q N + I-Q Q. o Q > 111 Q + I-Q a. Figure 34. PDT-induced caspase-8 activation occurs downstream of caspase-3 and-7 activation. HeLa cells were pretreated with Z V A D - f m k or D E V D (as indicated) for 30 min during verteporfin (200 ng/ml) incubation prior to photosensitization. Whole cell extracts were prepared and assessed for caspase-3/7-like activity (top) and immunoblotted for Bap31, PARP, D F F and caspase-8 (bottom). 122 V 0 re o Time after PDT (h) c t 0 o. o t > -DFF -p30 -p11 -PARP -p85 -Ras-GAP Figure 35. P D T induces caspase-mediated cleavage of DFF, P A R P and Ras-GAP. HeLa cells were lysed at the indicated times following P D T (200 ng/ml verteporfin; 2 J/cm 2 light). Cel l extracts were assessed for the status of DFF, P A R P and Ras -GAP using Western blotting. 123 4.1.2.4 Mitochondrial Cyt c Release Occurs Prior to Caspase Activation in HeLa Cells Since it has been well documented that caspase-8 is the initial caspase activated in FasL, 30 35 380 T R A I L and TNF-induced apoptosis , we sought to determine whether caspase-8 activation occurs prior to, as in Fas and TNF-induced apoptosis, or after cyt c release. Whole cell extracts and cytosolic (S-100) protein extracts were analyzed for cyt c content by Western blotting (Figure 36). Detection of cyt c in the cytosolic fraction corresponds to the release of cyt c from the mitochondria 23 . Cyt c was readily detectable within the cytosolic fraction prepared immediately following light activation indicating that cyt c release is an early event in PDT-induced apoptosis. The levels of cyt c in the cytosol did not change over 5 h following PDT. Cyt c was undetectable in the cytosolic fractions of untreated cells or cells treated with verteporfin in the absence of light indicating that the cytosolic preparation was effective. Caspase-8 activation was assessed by Western blotting and by a cell-free protease assay that measures the cleavage activity of the caspase-8-specific fluorogenically-labeled IETD tetrapeptide (Figure 36) 5 6. IETD-ase activity was present at increasing levels up to 2 h post-PDT at which time levels decreased. Cleavage of the caspase-1 and -8 substrate Bap31 was evaluated in parallel over the 5 h period following light activation. Caspase-8 cleavage and activation, as well as Bap31 cleavage occurred at least 1-2 h after the appearance of cyt c in the cytosol indicating that caspase-8 activation is not responsible for the induction of cyt c release during PDT-induced apoptosis. Since caspase-1 is not activated and caspase-8 is the only other caspase known to bind to and cleave 108 Bap31 , the occurrence of Bap31 cleavage during PDT-induced apoptosis provides further support of the existence of caspase-8 activity in these cells. 124 -cyt c (whole) -cyt c (cytosolic) pro-caspase-8 -Bap31 -p20 ® • C 3 1 3 Time after PDT (h) t 0 Q. 0) t Figure 36. Caspase-8 activation and cleavage of the ER-localized Bap31 substrate occur after mitochondrial release of cyt c during PDT-induced apoptosis. HeLa cells were lysed at the indicated times following P D T (200 ng/ml verteporfin; 2 J/cm light). Cel l lysates were analyzed for caspase-8-like (IETD-ase) cleavage activity using a cell-free protease assay. Whole cell lysates were separated by S D S - P A G E followed by Western immunoblotting and probed for cyt c, caspase-8 or Bap31. Cytosolic extracts were also prepared to assess the presence of cyt c in the cytosol. 125 In summary, these results demonstrated, for the first time, the rapid release of cyt c from mitochondria during PDT-induced apoptosis. Furthermore, they were the first of a series of studies to demonstrate that caspase-8 activation occurs downstream of cyt c release in non-receptor mediated apoptosis and to suggest that caspase-8 activation may act in an amplification loop to further enhance cyt c release or caspase activation. However, increased cyt c release over time after P D T was not observed in HeLa cells. Further studies demonstrated, using the caspase-3-specific D E V D - f m k inhibitor, that caspase-8 processing and Bap31 cleavage occur downstream of caspase-3 (Figure 36). Thus, caspase-8 is a downstream caspase during PDT-induced apoptosis. Light alone did not induce caspase activation. 4.1.2.5 Role of Bcl-2 and Bcl-XL in HeLa cell apoptosis Further studies were done in conjunction with others in the laboratory to determine the 323 effects of Bcl-2 and BC1-XL on HeLa cell apoptosis . These studies demonstrated that Bcl-2 and BC1-XL do not block early mitochondrial events such as cyt c release and 7A6 expression but do inhibit caspase activation. However, in contrast to HL-60 cells, Bcl -2 overexpression in HeLa cells did not provide protection against cell death at any dose of P D T . Our studies found that cyt c release occurs in a P D T dose-dependent manner (Figure 37) and we also reconfirmed our previous findings that Bcl-2 does not block cyt c release (Figure 37). Furthermore, in H e L a cells, an intracellular redistribution of the pro-apoptotic Bcl-2 family member Bax was not evident (Figure 37). Light alone did not induce cyt c release in either cell type. 126 C CN c XJ if-Ci) Jr cu verteporfin (ng/ml) © i 25 50 100 200 1 > -Bax (HeLa/neo cells) -Cyt c (HeLa/neo cells) -Cyt c (HeLa/Bcl-2 cells) Figure 37. Mitochondrial release of cyt c in PDT-treated HeLa/neo and HeLa/Bcl-2 cells. HeLa/neo or HeLa/Bcl-2 cells were incubated with increasing levels of verteporfin for 1 h followed by exposure to red light (2 J/cm 2). A t 1 h after PDT, cytosolic extracts were prepared and then assessed for the status of Bax or cyt c via Western blotting. 127 4.1.2.6 Intracellular Ca l c ium Regulation To assess whether intracellular C a 2 + release corresponded to the degree of light irradiation, HeLa/Neo and HeLa/Bcl-2 cells were incubated with verteporfin (100 ng/ml), followed by incubation with the C a 2 + indicator Fluo-3 and then activated with 0.5, 1 or 2 J/cm 2 red light (Figure 38). Each experiment was repeated 3 times and fluorescence was assessed for at least 20 gated cells using immunofluorescence confocal microscopy. Following light activation, there was an immediate increase in cytosolic C a 2 + levels. The level of the C a 2 + spark was light dose-dependent. A t the highest light dose, the level of intracellular C a 2 + release in the Bcl -2-transfected H e L a cells was approximately 25% greater than the HeLa/Neo cells. Following P D T , cell C a 2 + levels returned to basal levels indicating that C a 2 + was either being sequestered back 2+ 2"P into the E R or that C a was being extruded via the plasma membrane C a ATPases. To assess whether C a 2 + was sequestered back into the E R , when C a 2 + levels had returned to normal after P D T , histamine was added to determine whether there was any C a 2 + retained in the E R . There was a spark in the cells that had been treated with 0.5 J/cm , but only a minimal response was observed for the 1 J /cm 2 and 2 J/cm 2 doses. Similar to the previous light dose studies, there was a 2+ slightly larger response in the Bcl-2-transfected cells suggesting that Bcl -2 may increase E R Ca load. Since the response to histamine decreased with increasing doses of P D T , one could assume that C a 2 + was not sequestered into the E R but was extruded out of the cell v ia plasma membrane C a 2 + ATPases ( P M C A ) or the N a + / C a 2 + exchanger. 128 0 10 20 30 40 50 60 70 80 Time frames (5 sec/frame) Light exposure Histamine Time frames (5 sec/frame) Figure 38. PDT-induced intracellular C a 2 + release occurs in a light dose-dependent manner. HeLa/neo (A) or HeLa/Bcl-2 (B) cells were incubated with 100 ng/ml verteporfin. Intracellular C a 2 + levels were detected using the C a 2 + indicator Fluo-3 as described. Cells were exposed to 0 ,1 , or 2 J/cm 2 red light. A t 200 sec (40 frames), cells were exposed to 1 u M histamine to empty E R C a 2 + stores. Measurements were recorded at 5 sec intervals (5 sec=l frame). Error bars represent the standard error of the mean taken from the average fluorescence of approximately 20 cells from each of 3 separate experiments. 129 Doses of 0, 50 or 100 ng/ml verteporfin and 2 J /cm 2 red light were assessed in HeLa/Neo and HeLa/Bcl-2 cells to determine i f the response was drug dose dependent (Figure 39). Similar to our first set of experiments, the PDT-induced spark in cytosolic C a 2 + was immediate and occurred in a drug dose-dependent manner. Following photosensitization, at both 50 ng/ml and 100 ng/ml verteporfin, the levels of intracellular C a 2 + were higher in the HeLa/Bcl-2 cells than the HeLa/Neo cells. There was no increase in cells treated with no verteporfin and light. When histamine was added after light exposure there was a marked increase in the levels of intracellular C a 2 + in the untreated (light alone) HeLa/Bcl-2 cells compared to the HeLa/Neo. Thus, Bcl-2 appears to increase E R C a 2 + load. Since both the light-dose and drug-dose studies were performed in C a 2 + containing media and E R C a 2 + loads were depleted after P D T , it is likely that C a 2 + levels returned to basal levels via PMCA-mediated extrusion. However, since the studies were done in C a 2 + containing media, we could not rule out the possibility that increased C a 2 + was due to the influx of C a 2 + into the cell. Since the previous studies had indicated that P D T induced a higher level of C a release in Bcl-2 overexpressing cells, we set out to determine whether this phenomenon was due to an increase in intracellular C a 2 + load in Bcl-2 overexpressing cells. HeLa/neo and HeLa/Bcl-2 cells were treated, in C a 2 + -free media, with ionomycin, which induces release of C a 2 + from all internal stores (Figure 40). Following the addition of ionomycin, there was an immediate rise in intracellular C a 2 + in both cell types. However, the rise in intracellular C a 2 + was significantly greater in Bcl -2 overexpressing cells indicating that Bcl-2 overexpression is associated with 9-1-increased C a load. 130 A 140 p g 120 f c Light exposure Histamine i i u Q 100 : 80 ; 60 ; I 40 20 0 0 B 140 120 & 100 tn c m -~-i i | 8 c in O Q go o 3 LL 80 60 40 20 Verteporfin: • 0 ng/ml • 50 ng/ml A 100 ng/ml 10 Light exposure 20 30 40 50 Histamine 60 70 80 10 20 30 40 50 60 Time frames (5 sec/frame) 70 80 Figure 39. Intracellular C a 2 + release occurs in a verteporfin dose-dependent manner. (A) HeLa/neo or (B) HeLa/Bcl-2 cells were incubated with 0, 50 or 100 ng/ml verteporfin. Intracellular C a 2 + levels were detected using Fluo-3 as described. Cells were exposed to 2 J/cm 2 red light. A t 200 sec (40 frames), cells were exposed to 1 p M histamine to empty E R C a 2 + stores. Measurements were recorded at 5 sec intervals (5 sec=l frame). Error bars represent the standard error of the mean taken from the average fluorescence of approximately 20 cells from 3 separate experiments. 131 Figure 40. Increased Bcl-2 expression is associated with increased intracellular C a + load. HeLa/neo or HeLa/Bcl-2 cells were incubated in media containing E G T A (ie. Ca2 +-free media) and exposed to ionomycin. Ce l l C a 2 + levels were measured using the fluorescent C a 2 + indicator as described.Results at each time point represent the standard error of the average fluorescence recorded from approximately 20 cells from each of 3 separate experiments. 132 In order to determine the source of intracellular C a after P D T , HeLa/neo and HeLa /Bc l -2 cells were depleted o f E R C a 2 + stores by treating the cells in Ca 2 +-free media with histamine (Figure 41). Following histamine-mediated depletion of E R C a 2 + stores, P D T induced a marked increase in intracellular C a 2 + in the HeLa/neo cells but only a minimal increase in the HeLa /Bc l -2 cells. These results indicate that P D T also induces the release of C a 2 + from another intracellular source, most likely the mitochondria. Furthermore, Bcl-2 may offer some degree of protection from the mitochondrial release of C a 2 + . To determine whether all intracellular C a 2 + stores are depleted during P D T , HeLa/neo and HeLa/Bcl-2 cells were treated in Ca 2 +-free media with P D T followed by ionomycin (Figure 42). A minimal response to ionomycin was observed for HeLa/neo cells whereas the HeLa/Bcl-2 cells exhibited a marked increase in C a 2 + levels after ionomycin. These results suggest that not all C a 2 + stores are depleted by P D T in Bcl-2 overexpressing cells. In the next set of experiments, HeLa/neo or HeLa/Bcl-2 cells were treated with the potent S E R C A blocker cyclopiazonic acid (CPA)(Figure 43). Similar to P D T , C P A induced a rapid increase in cytosolic C a 2 + levels in both cell types. Again, a larger response was noted in the CPA-treated HeLa/Bcl-2 cells suggesting that increased Bcl-2 expression is associated with increased E R C a 2 + load. These cells were then subjected to histamine to deplete E R C a 2 + stores. Histamine treatment induced a further increase in intracellular C a 2 + which was again higher in the Bcl-2-transfected cells. Thus, these experiments further support the notion that Bcl-2 increases E R C a 2 + load. Furthermore, it suggests the possibility that S E R C A may be a potential target of P D T . 133 80 r 0 20 40 60 80 100 120 Time (5 sec/frame) Figure 41. Bcl -2 provides protection against intracellular C a 2 + release in cells depleted of E R - C a 2 + stores. HeLa/neo or HeLa/Bcl-2 cells were treated with Ca2 +-free media containing verteporfin (100 ng/ml) and histamine (1 uM) which depletes E R - C a 2 + stores. Media was removed and replaced with another dose of Ca2 +-free media containing verteporfin and histamine to ensure E R C a 2 + depletion. Cells were then exposed to 2 J/cm 2 red light. Error bars represent the standard error of the average fluorescence of 20 cells from each of 3 separate experiments. 134 EGTA Figure 42. Increased release of C a 2 + from Bcl-2 overexpressing H e L a cells however, not all intracellular C a 2 + stores are depleted in Bcl-2 overexpressing cells. In the presence of 1 u M E G T A , cells were incubated with verteporfin and then exposed to light. Cells were then exposed to 1 u M ionomycin to determine the extent of intracellular C a 2 + depletion. Intracellular C a 2 + was assessed by measuring Fluo-3 fluorescence as described. Error bars represent the standard error of the average fluorescence of 20 cells from each of 3 separate experiments. 135 Histamine 0 111 II II II II II i II II II II 11 II II i II 11 II II 11 II i II II II H i m 111 II 11 II 1111 II i II II 11 II 11 * <£> tf> # # # » # # # # # ^ ^ # « ^ # # # # # # Time (sec) Figure 43. Treatment of HeLa/neo and HeLa/Bcl-2 cells with the potent S E R C A blocker C P A induces an immediate rise in intracellular C a 2 + levels. Levels o f Fluo-3 fluorescence, indicative of intracellular C a 2 + concentration, were measured at 5 sec intervals. Cells were first exposed to the S E R C A blocker, CPA(1 u M ) (blue arrow). C P A (1 p M ) was then removed by replacing the media.Cells were then exposed to histamine (1 pM) . The time o f histamine addition is indicated by the red arrow for HeLa/neo cells and the black arrow for HeLa/Bcl-2 cells. 136 Since the S E R C A isoform S E R C A 2 has been shown to be sensitive to oxidative stress , and the potent S E R C A blocker C P A elicited a rapid rise in cytosolic C a 2 + levels, we decided to examine the effects of P D T on S E R C A 2 protein levels using Western blot analysis. Our first set of experiments assessed the timing of S E R C A 2 modification/degradation (Figure 44A). HeLa/Neo or HeLa/Bcl-2 cells were incubated with verteporfin for 1 h and activated with 2 J/cm 2 red light. Decreased levels of S E R C A 2 protein were detected immediately after light irradiation in both cell types indicating that S E R C A 2 modification/degradation is an early event in response to P D T . Interestingly, in the Bcl-2 transfected cells, the level of S E R C A 2 seemed to slightly reappear by 1 h post-PDT. To ensure that the observations were true, experiments were repeated several times and blots were also stained with Bax, whose levels are unchanged in this cell type, to ensure equal loading was attained in gels. Additionally, gels were stained with Gelcode Blue reagent in standard procedures to ensure equal loading of protein. Thus, Bcl-2 appeared to be associated with increased S E R C A 2 levels that may aid to explain our earlier observations that Bcl -2 overexpression was associated with increased C a 2 + load. H e L a cells were incubated with verteporfin doses ranging from 0 to 200 ng/ml for 1 h followed by exposure to 2 J/cm red light (Figure 44B). Cells were lysed immediately after photosensitization. A dose-dependent decrease in S E R C A 2 protein detection by Western blotting was observed in both the HeLa/Neo and HeLa/Bcl-2 cells. Neither light alone nor drug in the absence of light exhibited any evidence of S E R C A 2 protein alteration. 137 TJ m ro CO I r HeLa/neo [« HeLa/Bcl-2 [*< Time after PDT (h) 0.5 1 4 22 24 -SERCA2 -SERCA2 B TJ CD •»-» c o 0) i_ c 3 HeLa/neo^ HeLa/Bcl-2 [ verteporfin (ng/ml) + light r 10 25 50 100 150 200 -SERCA2 -SERCA2 Figure 44. S E R C A 2 degradation in HeLa/neo and HeLa/Bcl-2 cells.(A) Cells were treated with P D T (100 ng/ml verteporfin; 2 J/cm 2 light) and whole cell extracts were prepared at the indicated times after PDT. Cel l lysates were assessed for the status of S E R C A 2 using Western blotting as described. (B) Cells were treated with increasing doses of verteporfin and light (2 J/cm 2). Immediately after PDT, cells were lysed and protein extracts were assessed using Western blotting for the status of S E R C A 2 . 138 The effect of caspases on PDT-mediated decreased S E R C A 2 levels was evaluated (Figure 45). The general caspase family inhibitor Z V A D - f m k did not protect against the P D T -induced modification of S E R C A 2 levels indicating that this event is caspase-independent. Light treatment alone did not alter S E R C A 2 protein levels. The latter observation was further substantiated by the fact that S E R C A 2 modification occurs immediately following P D T whereas caspases are activated in H e L a cells during the first hour post-PDT (Figure 33 and 36). The influence of increased intracellular C a 2 + levels on mitochondrial cyt c release was assessed using the cell permeable C a 2 + chelating agent B A P T A - A M (Figure 46). HeLa cells were incubated with B A P T A - A M prior to P D T to determine whether increased Ca is required for the induction of mitochondrial cyt c release. B A P T A - A M did not prevent mitochondrial cyt c release or caspase activation indicating that elevated C a 2 + with P D T may be a parallel event to these apoptosis-related phenomena. 139 T3 0 Q < > N S + c 3. C t : O t o !£> o a a O Q a> t : > > N > S & a & PDT + ZVAD (M ) 50 100 200 -SERCA2 Figure 45. Degradation/modification of S E R C A 2 does not require caspases. HeLa cells were pretreated with increasing amounts of the general caspase inhibitor Z V A D - f m k prior to photosensitization. Cells were lysed immediately after P D T (100 ng/ml verteporfin; 2 J/cm 2 light) and the status of S E R C A 2 was assessed using Western blotting. 140 •D 3 co <o i_ -t-> c 3 C t o Q. <D O > Q CL Q. < m < + i E CL <E < O CQ O Q > CL O CO w 0) o G) O — — -Cytc Figure 46. B A P T A or Bcl-2 overexpression individually or in combination does not influence mitochondrial release of cyt c in PDT-treated HeLa cells. HeLa/neo or HeLa/Bcl-2 cells were treated with verteporfin (100 ng/ml) for 1 h followed by light (2 J/cm 2). Cells were incubated with or without B A P T A (10 u M ) for 30 min prior to light exposure. (A) HeLa/neo or (B) HeLa/Bcl-2 cells were treated as indicated and cytosolic extracts were prepared 1 h after treatment. Cytosolic extracts were assessed for cyt c or Bax (B) using Western blotting. 141 4.2 Aim II: Effects of PDT on Primary Endothelial Cells 4.2.1 Cell Shrinkage and Membrane Blebbing in PDT-Treated H U V E C H U V E C were examined by phase-contrast microscopy to assess their morphology following PDT. Cell shrinkage and membrane blebbing, characteristic of apoptotic cells, were observed for the majority of cells within 3 h following P D T (Figure 47). H U V E C that were treated with the general caspase family inhibitor Z V A D - f m k prior to P D T did not exhibit shrinkage and membrane blebbing after treatment (Figure 47). Red light alone or verteporfin in the absence of light did not induce any morphological alterations indicative of apoptosis. Evidence of the importance of caspase-3 in H U V E C apoptosis is shown in Figure 48A whereby untreated and PDT-treated cells were probed for the presence of active caspase-3 using indirect immunofluorescence microscopy. Apoptotic cells visualized using open field microscopy corresponded to positively-labeled cells containing active caspase-3. The efficacy of Z V A D - f m k on its ability to inhibit caspase activity was assessed by obtaining whole cell extracts from PDT-treated cells treated in the presence or absence of Z V A D - f m k (Figure 48B). Cells pretreated with Z V A D - f m k did not exhibit caspase-3 processing or P A R P cleavage indicating that the tripeptide inhibitor was effectively blocking caspase activation. 142 untreated verteporfin (100 ng/ml) PDT PDT + ZVAD-fmk Figure 4 7 . PDT-induced H U V E C cell shrinkage and membrane blebbing are caspase-dependent. Prior to light exposure P D T (100 ng/ml verteprofin; 2 J/cm 2 red light)-treated cells were incubated with or without the general caspase family inhibitor Z V A D - f m k (50 uM). Cells were visualized 3 h post-PDT using phase contrast microscopy. Arrows represent examples of apoptotic cells exhibiting cell shrinkage and membrane blebbing. 143 Figure 48. Caspase-3 activation is present only in apoptotic endothelial cells. H U V E Q were incubated with or without verteporfin (100 ng/ml) for 1 h and then exposed red light (2 J/cm). (A) H U V E C were assessed via indirect fluorescence microscopy for active caspase-3. Cells were visualized using open field microscopy as well as fluorescence microscopy. (B) Whole cell extracts were prepared 2 h post-PDT. The status of P A R P and caspase-3 was assessed by Western blotting. 144 4.2.2 Altered Cytosolic Levels of Bax and Cyt c During H U V E C Apoptosis H U V E C were incubated with verteporfin for 1 h and exposed to 2 J/cm red light. Whole cell protein extracts and cytosolic protein extracts were prepared at 0, 1 and 2 h after photosensitization. Western blot analysis of whole cell protein extracts indicated that the overall expression level and molecular mass of Bax and cyt c did not change, whereas B i d was undetectable by 2 h post-irradiation (Figure 49). However, cytosolic levels of Bax decreased and were not detectable by 2 h post-treatment. In contrast, cytosolic cyt c was evident immediately and its levels increased over 2 h post-treatment (Figure 49). Morphological evidence of apoptosis was not observed prior to the initial release of cyt c and decrease in cytosolic Bax. These results differed from our findings with PDT-treated HeLa cells in two aspects. Firstly, the levels of cyt c in the cytosol following P D T increased with time in H U V E C whereas in HeLa cells the levels were unchanged up to 5 h post-PDT. Secondly, the decline in cytosolic Bax levels that was observed in H U V E C was not observed in HeLa cells in response to PDT. 4.2.3 Activation of Caspases -2, -3, -6, -7, -8 and -9 in HUVECs The involvement of caspase family members other than caspase-3 during endothelial cell death was unknown, thus, we took advantage of the apoptosis-inducing characteristics of verteporfin-based P D T to assess the status of various caspase family members in this cell type. Caspase -2, -3, -6, -7, -8, and -9 activation were assessed using Western blotting. Processing of caspases, as determined by either the disappearance of the proform of the enzyme and/or detection of one of the cleavage intermediates or active subunits (caspase-2, 3 8 and 9 only), was apparent for caspases 2, 3, 6, 7, 8, and 9 within 1-2 h post-treatment (Figure 50). To further support our findings, cell lysates were assessed for their relative levels of caspase-3/7-like, caspase-6-like or caspase-9-like activity using a cell-free protease assay as described (Figure 51). Red light alone did not induce an increase in caspase activity when compared to controls. 145 TJ CD co £ h post-treatment c T : r i = 0 1 2 Whole -Bax Cytochrome c Cytosolic [ -Bax *-Cyt c Figure 49. Intracellular distribution of Bax and cyt c and cleavage of B i d during endothelial cell apoptosis. H U V E C were treated with or without (untreated) verteporfin (100 ng/ml), followed by exposure to red light (2 J/cm ). Whole or cytosolic extracts were prepared at the indicated times post-PDT. The status of Bax, cyt c and B i d were assessed using Western blotting. 146 T J CD •*•> CO j= h post-PDT i 1 0 1 2 3 - 1 -pro-caspase-2 -pro-caspase-3 -pro-caspase-6 -pro-caspase-7 -pro-caspase-8 •pro-caspase-9 Figure 50. Activation of multiple caspases during endothelial cell apoptosis. H U V E C were treated with or without (untreated) verteporfin (100 ng/ml), followed by exposure to red light (2 J/cm 2). Cells were lysed and protein extracts were prepared at the indicated times post-PDT. The status of different caspases were assessed using Western blotting. Caspase processing was determined by either a decreased in the pro-form of the enzyme, and/or the appearance of one or more of the subunits (denoted by lines). 147 7000 c 6000 < 5000 « 4000 <j> 3000 ^ (0 (0 2000 : CL CO 1000 : ra t O o >, 1200 > 1000 800 < <*> 600 0) </) 400 ra CL (0 ra O > o < I © (0 ra CL (0 ra O 200 140 120 100 80 60 40 20 0 g Time post-PDT (h) c 3 Figure 51. Increased caspase-3/7-, -6 and -9-like proteolytic cleavage activity during endothelial cell apoptosis. H U V E C were incubated with or without verteporfin (100 ng/ml) for 1 h, and exposed to red light (2 J/cm 2). Whole cell protein extracts were prepared at the indicated times after PDT. Cel l extracts were assessed for caspase-3/7-, caspase-6 or caspase-9-like proteolytic activity. 148 Peak caspase-9 activity was observed at 1 h after P D T and declined thereafter whereas caspase-3/7 and caspase-6 protease activity plateaued at 2 h after P D T and then declined. Thus, these studies would suggest that caspase-9 activation may procede that of caspase-3, -7, or - 6 . Similar to our findings with tumour cells, cleavage of B i d , P A R P and F A K were observed following PDT. Furthermore, the status of the cell cycle inhibitory proteins p21 and p27 were assessed (Figure 52). I T U V E C were treated with verteporfin and red light and whole cell lysates were prepared. Cleavage of p21 and p27 was observed within 1-2 h after photosensitization. Although protein levels of C D K 2 were unchanged after PDT, however, the phosphorylation status or kinase activity of C D K 2 was not examined. 149 Figure 52. Decreased levels of cell cycle inhibitory proteins p21 and p27 after PDT. A t the indicated times after P D T (100 ng/ml verteporfin; 2 J/cm 2), cells were lysed and analyzed via Western blotting for the status of p21, p27 and C D K . Although C D K 2 levels were not significantly altered, p21 levels diminished and were associated with the increased expression of a smaller band indicative of a cleavage product. Decreased p27 levels were evident at a later timepoint. 150 4.3 Aim III Effects of PDT on Primary Smooth Muscle Cells 4.3.1 PDT Induces Apoptosis in H A S M C Morphological indicators of apoptosis such as cell shrinkage and membrane blebbing were assessed for untreated, verteporfin-treated or PDT-treated H A S M C at 0, 1, 2 and 3 h post-PDT (Figure 53). Evidence of cell shrinkage and membrane blebbing was observed by 1 h and approximately 50-60% of cells exhibited an apoptotic morphology by 3 h following PDT. The extent of D N A hypodiploidy indicative of D N A fragmentation was assessed using PI staining. Baseline levels of hypodiploid D N A (-9%) were unchanged immediately after P D T when compared to untreated or verteporfin-treated H A S M C (Figure 54). B y 1 h after photosensitization, levels of hypodiploid D N A were increased to approximately 24% and by 5 h after photosensitization, greater than 40% of cells contained hypodiploid D N A . Light alone did not affect H A S M C levels of hypodiploid D N A . To further characterize PDT-induced H A S M C apoptosis, mitochondrial 7A6 antigen expression was assessed using flow cytometry (Figure 55 and 56). The mitochondrial 7A6 antigen was not detectable for untreated or verteporfin-treated H A S M C . However the 7A6 epitope was readily detectable immediately after P D T and expression levels increased over the the next 2 h (Figure 55). The general caspase inhibitor Z V A D - f m k did not affect 7A6 antigen expression after P D T indicating that increased levels of the 7A6 antigen occurs in a caspase-independent manner (Figure 56). 151 untreated verteporfin 0 h 1 h 2h 3h Figure 53. Morphological alterations of H A S M C following PDT. H A S M C were incubated with verteporfin (100 ng/ml) for 1 h followed by light exposure (2 J/cm 2). Cells were visualized using phase contrast microscopy. 152 Figure 54. Induction of D N A fragmentation in H A S M C following PDT. PDT-treated H A S M C were incubated with verteporfin (100 ng/ml) for 1 h followed by exposure to red fluorescent light (2 J/cm 2). Untreated (light alone) and verteporfin (dark)-treated H A S M C were also assessed. Bars represent mean values for 3 experiments +/- SD. 153 PDT 7A6 antigen expression (Fluorescence intensity) Figure 55. The apoptosis-associated mitochondrial 7A6 antigen is detectable immediately after treatment of H A S M C with PDT. A t the indicated times after P D T (100 ng/ml verteporfin; 2 J/cm 2), cells were prepared and analyzed using flow cytometry for the 7A6 antigen using the monoclonal Apo2.7 antibody. Results for isotype controls are shown in shaded areas. 154 untreated verteporfin PDT ZVAD-fmk + PDT jtJr \ ^ ^y^1' *v... ^ \ 7A6 antigen expression (Fluorescence intensity) Figure 56. Expression of 7A6 in PDT-treated H A S M C is not caspase-dependent. 7A6 expression in untreated, verteporfin(100 ng/ml)-treated, PDT(100 ng/ml verteporfin; 2 J/cm2)-treated and PDT+ZVAD-treated H A S M C was detected by the Apo2.7 monoclonal antibody and assessed using flow cytometry. Results for isotype controls are shown in shaded histograms. Cells were assessed 1 h post-PDT. 155 H A S M C were immunostained for active caspase-3 (Figure 57A) or with the mitochondria-specific mitotracker red stain and nuclear lamins A and C (Figure 57B). Active caspase 3 was not detected in untreated H A S M C whereas in PDT-treated H A S M C active caspase 3 was readily detectable thus providing additional evidence that PDT induces H A S M C apoptosis. Additionally, membrane blebbing can be observed in cells containing active caspase-3. Lamins, major constituents of the nuclear envelope, have been shown to be processed by caspases during apoptosis 83 381 . Lamin A/C nuclear staining was markedly altered for the PDT-treated HASMC. Similar to 339 studies L i et al. , clustering of mitochondria was also observed however, we believe this may be artifactual and due to the rounding up and/or shrinkage of the cells. 156 Figure 57. P D T induces caspase-3 activation and degradation of nuclear lamins in H A S M C . (A) Untreated (light alone) or P D T (100 ng/ml verteporfin; 2 J/cm2)-treated H A S M C were fixed 1 h after photosensitization and immunocytochemically probed for the presence of active caspase-3 (green) and assessed using bright field microscopy (40x mag.). (B) Untreated or PDT-treated H A S M C were stained using mitotracker red and fixed 1 h after photosensitization and immunocytochemically probed for nuclear lamins A and C (green).(1 OOx mag). 157 4.3.2 Cellular Redistribution of Bax, Cyt c and AIF During H A S M C Apoptosis The cellular redistribution of Bax, cyt c and AIF were examined via Western blot analysis of cytosolic and whole cell lysates as well as by confocal microscopy. Analysis of whole cell extracts indicated that the overall expression level or molecular mass of Bax and cyt c were not altered over the 2 h timeperiod following PDT. (Figure 5 8A). However, decreasing cytosolic levels of Bax were observed and Bax was minimally detectable by 2 h post-treatment. Untreated and PDT-treated H A S M C were immunostained with an anti-Bax monoclonal antibody and analyzed by indirect fluorescence microscopy. Bax immunostaining exhibited a more punctate cellular distribution in the apoptotic H A S M C (Figure 58B) in agreement with Western blot data indicating a cellular redistribution of Bax during PDT-induced apoptosis. In contrast to Bax, cyt c was not present in the cytosolic fraction of untreated cell lysates, but was evident in lysates prepared immediately following P D T and increased steadily over 2 h post-treatment. B y 2 h post-PDT, cell shrinkage had occurred to such an extent that cellular localization of Bax was impractical. Levels of cyt c in whole cell extracts did not change whereas cytosolic levels of cyt c were evident immediately after P D T but were not detected in untreated cells (Figure 5 8A). Cyt c levels increased in the cytosol over a 2 h period after PDT. Confocal microscopy revealed a very specific, string-like staining pattern for cyt c in untreated cells and more dispersed staining in PDT-treated cells (Figure 58B), providing further support for cyt c release during PDT-induced H A S M C apoptosis. It should be noted that cyt c staining was not uniformly dispersed throughout the cell at 0 h or 1 h post-PDT indicating that not all cyt c is redistributed immediately from mitochondria during PDT-mediated apoptosis. Due to cell shrinkage and rounding up, it was difficult to determine whether all cyt c was released by 2 h post-PDT. To determine the occurrence and timing of mitochondrial 7A6 antigen unmasking in response to PDT, the monoclonal Apo2.7 antibody followed by flow cytometry was utilized at time points corresponding to 158 those for cyt c and Bax assays (Figure 55). Increased levels of 7A6 antigen corresponded with increased levels of cyt c present in the cytosol. Light alone did not induce cyt c release. Similar to the results obtained with cyt c, cytosolic levels of A I F increased over 2 h after photosensitization (Figure 59A). Furthermore, when assessed using indirect immunofluorescence microscopy, we observed a redistribution of AIF from mitochondria to nuclei (Figure 59B). These results indicate that the mitochondrial AIF pathway is functional during S M C apoptosis and is likely involved in chromatin condensation and stage I D N A fragmentation. 4.3.3 Caspase 3,6, 7,8 and 9 Activation During PDT-Induced H A S M C Apoptosis Processing of caspases, as determined by either the disappearance of the proform of the enzyme (caspases 6) and/or detection of one or more of the active subunits (caspase 3, 7, 8 and 9), was apparent for all caspases tested and these changes increased in a time-dependent manner after P D T (Figure 60). Although the general caspase family inhibitor Z V A D - f m k did not affect mitochondrial 7A6 expression, it did block the morphological characteristics of apoptosis (Figure 63 A ) and caspase-3 activation (Figure 6IB) induced by P D T indicating that H A S M C slirinkage and membrane blebbing are caspase-dependent. Cells treated with verteporfin in the absence of light or light alone did not induce caspase activity nor elicit a change in cellular mophology. 159 A B CB £ h post-PDT 5 Whole Cytosolic Extract L B Bax Cytc -Bax -Cytc -Bax -Cytc Figure 58. Cellular redistribution of Bax and mitochondrial release of cyt c during PDT-induced smooth muscle cell apoptosis. Cells were treated with 2 J/cm 2 light alone (untreated) or verteporfin (100 ng/ml) for 1 h followed by exposure to 2 J/cm 2 red light. (A) Whole cell protein extracts and cytosolic protein extracts were prepared and assessed using Western blotting for Bax or cyt c. (B) Indirect fluoresence microscopy depicting the intracellular localization of Bax and cyt c at 0 h, 1 h and 2 h after PDT. 160 A Figure 59. Cellular redistribution of A I F during H A S M C apoptosis. Cells were treated with 2 J/cm 2 light alone (untreated) or verteporfin (100 ng/ml) and 2 J/cm 2 light and then assessed by (A) Western blotting (cytosolic extracts) or (B) indirect immunofluorescence at the indicated times after PDT. 161 73 Qi +-> CO 2 h post-PDT § ' 0 1 2 1 pro-caspase-3 — pro-caspase-6 sas sw-pro-caspase-7 pro-caspase-8 •4 Figure 60 Activation of caspases-3, -6, -7, -8 and -9 during H A S M C apoptosis. Untreated or P D T (100 ng/ml verteporfin; 2 J/cm2)-treated H A S M C were lysed at the indicated times after treatment and assessed for the status of caspases-3, -6, -7, -8 or -9 by Western blotting. Arrows point to cleavage fragments detected with each antibody. 162 untreated PDT PDT + ZVAD B > 700 O eoo re 0 500 " 7 400 O 300 200 O CO re Q. 100 CO 8 ' -pro-caspase 3 S re c 3 Q a. Q < + i -Q 0. Figure 61. Z V A D - f m k inhibits morphological changes and caspase activity associated with PDT-induced H A S M C apoptosis. (A) A t 3 h post-PDT, untreated (2 J/cm 2 light on ly) , verteporfin (100 ng/ml)-treated or P D T (100 ng/ml verteporfin; 2 J/cm2)-treated cells were examined using phase-contrast microscopy. (B) Caspase 3/7-like activity and caspase-3 processing were assessed for untreated, PDT-treated or PDT+ZVAD-fmk-treated cells. 163 C H A P T E R V D I S C U S S I O N Over the past decade increasing evidence has implied a role for apoptosis, or lack thereof, in the pathogenesis of many common vascular diseases 2 6 . The intimal hyperplasia associated with the formation of vascular lesions may be due to increased S M C proliferation and/or decreased S M C apoptosis thereby allowing S M C to accumulate. In addition E C apoptosis has been observed in numerous vascular disorders. Thus, a greater understanding as to how E C and S M C apoptosis are regulated is necessary. The biochemical mechanisms of E C and S M C apoptosis, in response to any type of stimuli, are poorly understood. The use o f P D T as a method to induce apoptosis in these cells provided us with the opportunity to study E C and S M C apoptosis in a clinically relevant situation. Results from these studies are applicable not only to the effects of P D T on endothelium and smooth muscle per se, but also with respect to the general understanding of E C and S M C response to toxic stimuli. Additionally, mechanistic studies with E C and tumour cells are informative with respect to the understanding as to how P D T eradicates tumours and the potential contribution of a vascular effect. P D T is a clinically approved treatment for various types of cancer and A M D . In both of these cases, an abnormal neovasculature is a primary target for P D T . In A M D , the abnormal blood vessels responsible for the loss of visual acuity rapidly take up Visudyne and are destroyed upon light irradiation, most likely due to a combination of PDT-induced E C apoptosis, vaso-285 367 382 occlusion and thrombosis . In the case of PDT-treated tumours, the anti-tumour effect of P D T likely involves a combination of PDT-induced E C apoptosis, vaso-occlusion and thrombosis in addition to direct tumour cell apoptosis, necrosis and possibly an immune-164 mediated anti-tumour response . Thus, an understanding of PDT-induced E C apoptosis in addition to the mechanisms of tumour cell apoptosis is clinically relevant. P D T is currently under investigation for the treatment of other oncologic and ocular disorders as well as atherosclerosis, restenosis, allograft rejection and autoimmune disorders 382 384 . Prior to the commencement of my studies in early 1997, P D T had been shown to catalyze the formation of reactive oxygen species and rapidly induce apoptosis in a variety of cell types following light irradiation 2 9 3 , 3 3 6 ' 3 8 5 3 8 6 However, the biochemical mechanisms related to P D T -induced apoptosis were not known. In particular, virtually nothing had been published with regards to the biochemical effects of verteporfin (a.k.a. Visudyne) and light with respect to apoptosis in any cell type. 5.1 Mechanisms of PDT-Induced Tumour Cell Apoptosis Preliminary studies were performed using transformed cell lines in order to optimize experimental techniques prior to using the more expensive and time-consuming primary E C and S M C culture models. These preliminary studies, using verteporfin as the photosensitizer, revealed that P D T induced apoptosis and rapid caspase activation as early as 15 min following treatment in human promyelocyte leukemia HL-60 cells whereas in human cervical carcinoma 201 324 387 H e L a cells, caspase activation was not evident until 1 h after treatment ' ' . Comparatively, we examined caspase activation in coxsackievirus B 3-induced cytopathic effect in which 388 caspase-3 was activated by 8 h post-infection . Thus, different stimuli induce apoptosis and caspase activation at differing rates within a particular cell type. Other stimuli such as U V , staurosporine, AraC , T R A I L and FasL were tested in either H e L a cells or H L - 6 0 cells and also exhibited a much slower onset of apoptosis. However, P D T in combination with agents such as 165 FasL and/or TRAIL did appear to elevate apoptotic activities such as caspase activation and 389 D N A fragmentation . The timing of mitochondrial cyt c release was evaluated in HeLa cells as well as EC and SMC. In PDT-treated HeLa cells, release of cyt c into the cytosol was detected immediately after 323,324 photosensitization followed by the activation of caspases 2, 3, 6, 7, 8 and 9 in HeLa cells Thus, it is likely that PDT exerts direct effects on mitochondria. In further support of this concept, we also noted that in PDT-treated HeLa cells, that increased expression of the mitochondrial, 323 apoptosis-specific 7A6 antigen corresponded to the degree and timing of cyt c release . Porphyrin-derived photosensitizers such as verteporfin can bind mitochondrial peripheral-type benzodiazepine 327-329 receptors (PBR) . PBR are located at the junction of the inner and outer mitochondrial membranes 3 3 ° ' 3 3 ' , a pivotal site for the integration of apoptosis-related signals 1 M . Interestingly, other investigators have shown that PBR expression levels may correlate with cell sensitivity to 327 photodynamic killing with porphyrins . Thus, the localization of verteporfin to mitochondria via PBR binding offers an attractive hypothesis as to how verteporfin-based PDT is capable of inducing rapid cyt c release and initiation of the apoptotic cascade. However, in recent studies, we have demonstrated that this is not the case. The Jurkat lymphoma cell line expresses very low levels of 390 PBR . Surprisingly, Jurkat cells were not resistant to PDT-induced apoptosis but rather were 308 actually more sensitive to PDT-induced apoptosis that other cell types studied . In further 391 collaborative studies, the identification of other mitochondrial targets for PDT was pursued . In these studies verteporfin-based PDT was found to kill lymphoma cells by an apoptotic process involving a dissipation of the mitochondrial inner transmembrane potential (Ai|/m). PDT-induced A\|/m in isolated mitochondria was suppressed by bongrekic acid and partially by cyclosporin A 391 (CsA) . In these studies, Belzacq et al. also showed that PDT permeabilized proteoliposomes 166 containing the semipurified permeability transition pore complex (PTPC) or the purified PTPC component adenine nucleotide translocator (ANT) , yet had no effect on protein-free control liposomes. Verteporfin phototoxicity on A N T proteoliposomes was mediated by reactive oxygen 391 species and was prevented by recombinant Bcl-2 or the adenine nucleotides A T P and A D P Thus, A N T is another potential mitochondrial target for verteporfin. Identification of other mitochondrial structures that bind porphyrin photosensitizers w i l l be a future area of keen interest. A s further support of mitochondria as a primary target for verteporfin-based P D T , an immediate release of both mitochondrial and E R C a 2 + stores was observed immediately after photosensitization. Consequently, the E R may also be a direct target of verteporfin. Indeed cell localization studies indicate that verteporfin not only localizes to mitochondria, but may also localize to E R (unpublished results). Furthermore, other porphyrin-derived photosensitizers, hematoporphyrin and protoporphyrin, have been reported to accumulate to a greater degree in 342 E R versus mitochondria . Additionally, an indirect immunofluorescence study using a monoclonal Connexin antibody to label E R exhibits an altered staining pattern following P D T (unpublished results). These results suggest that the E R may also be a primary target of P D T . To further evaluate a link between P D T and the E R intracellular C a levels were measured in response to PDT. Since Bcl-2 proteins are localized to E R in addition to mitochondria, and have been shown to affect intracellular C a 2 + regulation, the responses were measured in HeLa/Neo and HeLa/Bcl-2 cells. P D T induced an immediate spark in intracellular C a 2 + in a light- and drug-dependent manner in both cell types. However, a higher degree of intracellular C a was released in the Bcl-2-transfected cells suggesting that Bcl-2 may increase either mitochondrial and/or E R Ca load. When histamine, an E R C a 2 + depleting agent, was added to either cell type, there was a larger C a 2 + release in the HeLa/Bcl-2 cells supporting the notion that Bcl-2 increases E R C a 2 + load. When 167 E R were depleted of Ca using histamine, and then treated with PDT, there was still a rise in the HeLa/neo cells and a reduced response in the Bcl-2-transfected cells indicating that Ca was also released from a source other than the E R such as mitochondria. Due to the close association of verteporfin with mitochondria, it is most likely that the other source was mitochondria. Furthermore, these studies would suggest that Bcl-2 may inhibit mitochondrial release of Ca in response to PDT. However, the E R was the predominant source of C a 2 + release after PDT. The decreased response of cells to histamine after P D T suggests that the E R are no longer capable of sequestering C a 2 + . Since we could rule out the E R playing a role in the removal of excess C a 2 + , the ability of the cell to remove excess C a 2 + must have been due to the P M C A would suggest that P D T does not damage the cell membrane. This concept is supported by observations suggesting that verteporfin localizes to the E R and mitochondria. Previous studies have indicated that depletion of E R C a levels with thapsigargin (TG) 139 2+ causes apoptosis . Our studies demonstrated an immediate increase in intracellular Ca similar to that which ocurred after P D T upon addition of C P A . Both T G and C P A are potent S E R C A blockers. Furthermore, S E R C A - 2 has been shown in other models to be highly sensitive to 143 oxidative stress generated by free radicals , therefore, in a reactive oxygen-induced injury such as PDT, we speculated that damage of S E R C A 2 and subsequent E R C a 2 + depletion may be a primary event involved in PDT-mediated cell death. S E R C A are the proteins responsible for pumping intracellular C a 2 + into the E R and thus, maintaining C a 2 + homeostasis within the cell. The effects on S E R C A 2 protein levels were examined by Western blotting. In our first set of experiments, S E R C A 2 levels were assessed at various intervals after PDT. Surprisingly, S E R C A 2 levels were diminished immediately after P D T suggesting that these proteins were highly sensitive to PDT. Whether the proteins were degraded, downregulated or underwent a conformational change 168 resulting in the loss of the epitope recognized by the aht i -SERCA2 antibodies is uncertain. However, it is unlikely that P D T affected its translation due to the rapidity of its disappearance. Furthermore, a conformational change is also unlikely since the protein samples were denatured prior to electrophoresis and Western blotting. Furthermore, it was determined that the altered S E R C A 2 levels occurred in a caspase-independent manner since pretreatment with the general caspase family inhibitor Z V A D - f m k did not prevent loss of S E R C A 2 . Furthermore, S E R C A 2 alteration ocurred in a verteporfin dose-dependent manner and appeared to correspond directly to cell viability. S E R C A are thiol containing enzymes that are inactivated in response to thiol oxidation 3 9 2 . It is possible that verteporfin-based P D T leads to thiol group oxidation. Thus, the replenishment of E R C a 2 + stores and maintainance of internal C a 2 + homeostasis is likely abolished in response to lethal levels of P D T due to the oxidative damage caused to S E R C A 2 . Altered levels of cytosolic C a 2 + may contribute to many physiological changes manifesting the apoptotic phenotype. Several studies have suggested a role for C a 2 + in the activation of 129 endonucleases associated with D N A fragmentation during apoptosis as well as in the activation of proteases involved in apoptosis ' 3°. C a 2 + is also required for the externalization of phosphatidylserine (PS) to the outer leaflet of the cell membranes of apoptotic cells 1 3 P S is one of many cell surface markers now identified that facilitate the recognition and uptake of apoptotic 132 133 bodies by phagocytes ' . Early recognition and phagocytosis of apoptotic cells by neighboring cells is essential for the rapid removal of these cells before they lose their membrane integrity and release their contents. C a 2 + has been shown to activate calcineurin, a C a 2 + -dependent phosphatase, resulting in the dephosphorylation of the pro-apoptotic Bcl-2 family member known as Bad 1 3 5 . Upon dephosphorylation, Bad has been shown to migrate to mitochondria, bind to BC1-XL and induce cytochrome c release 1 3 5 . Bad is retained in an inactive, anti-apoptotic state when it is 169 phosphorylated via its binding to 14-3-3 proteins. Thus, it is possible that a link may exist between S E R C A - 2 damage, increased intracellular C a 2 + , calcineurin activation, and apoptosis via calcineurin-mediated Bad dephosphorylation. Alternatively, increased intracellular Ca may stimulate cyt c release directly, or involve a combination of both mechanisms. Although it is well-known that Bcl-2 and BC1-XL localize to the outer membranes of mitochondria, E R and nuclei, the vast majority of work on these proteins has focussed on mitochondria and very little is understood as to how these proteins affect the E R or nuclei. Interestingly, Bcl-2 overexpression has been shown to block increased intracellular C a 2 + levels associated with apoptosis in certain cell types ' 3 7 ' ' 3 8 . However, although intracellular C a 2 + has been shown to contribute to cell death, the effects of Bcl-2 on PDT-mediated intracellular C a 2 + levels were unknown. In our studies, Bcl-2 did not block the rise in intracellular C a 2 + after PDT. Rather, Bcl-2 may actually have increased the amount of C a 2 + released in response to PDT. To investigate the role of Bcl-2 in PDT-mediated C a 2 + release further, the effects of Bcl-2 on S E R C A 2 degradation was compared between control (HeLa/neo) and Bcl-2-transfected (HeLa/Bcl-2) cells. Overexpression of Bcl-2 did not alter S E R C A 2 levels when compared to a that of HeLa/neo cells when protein levels were assessed for the status of S E R C A 2 immediately after PDT. However, when S E R C A 2 levels were assessed in a time-course experiment over 24 h, the levels of S E R C A 2 in Bcl-2-transfected cells seemed to reappear whereas in control cells S E R C A 2 levels dropped immediately and remained undetected. Thus, Bcl-2 expression may indirectly affect S E R C A 2 protein levels. In support of this notion, recent studies in breast epithelial cells have demonstrated 138 that increased Bcl-2 expression is associated with increased S E R C A 2 m R N A and protein levels . Furthermore, Bcl-2 and S E R C A 2 may interact with each other as determined by co-immunoprecipitation studies 1 3 8 . Thus, Bcl-2 may maintain E R C a 2 + stores via the upregulation of 170 S E R C A 2 . Indeed Bcl-2 can prevent thapsigarin-induced emptying of E R Ca stores. Recent evidence in tumour cells suggests a role for Bcl-2 in the regulation of calcium uptake by the E R 1 3 7 ' 1 3 8 . Previous studies have indicated that depletion of E R C a 2 + levels with thapsigargin (TG) causes apoptosis . Bcl-2 has been shown to preserve E R Ca levels by maintaining Ca uptake in the 137 E R . In support of the latter concept, Bcl-2 has been shown to form cationic-selective pores in liposomes 1 4 ° ' 1 4 ' . Interestingly, similar to Bcl-2, S E R C A - 2 may also localize to the nucleus as well as 142 9+ the E R . However, the effects of S E R C A - 2 or Bcl-2 on nuclear Ca are not known. S E R C A 2 is 143 highly sensitive to free radicals , thus, in reactive oxygen-induced injury associated with PDT, damage of S E R C A 2 and subsequent E R depletion may be a primary event involved in cell death. In rat fibroblasts transfected with Bcl-2 specifically targeted to the ER, Bcl-2 was shown to block 144 Myc-induced but not Etoposide-induced apoptosis , suggesting that Bcl-2 expression pertaining specifically to the E R provides a protective effect to some, but not all forms of apoptosis. Conversely, Bcl-2 may also maintain mitochondrial C a 2 + stores and subsequent changes in 393 74 -transmembrane potential . Indeed, we found evidence of increased Ca load in both mitochondria and E R associated with Bcl-2 overexpression in HeLa cells. Furthermore, when treated with PDT, histamine, ionomycin in C a 2 + -free media, or C P A , that Bcl-2-transfected cells exhibited a marked increase in intracellular C a 2 + release compared to controls. C a 2 + can facilitate or induce mitochondrial cytochrome c release . A link between Ca , mitochondrial cyt c release and was investigated by preincubating HeLa cells with the cell-permeable C a 2 + chelator B A P T A - A M prior to photosensitization. Cyt c release was not affected by B A P T A - A M suggesting that increased C a 2 + is not responsible for inducing cyt c release. Our results differ from recently published results demonstrating that B A P T A blocked cyt c release and caspase-394 3 activation in response to pheophorbide-based P D T . However, in collaboration with Dr. J. 171 Piette's group (University of Liege, Belgium) we determined that cyt c release in response to 395 pheophorbide-based P D T occurs much slower that with verteporfin-based P D T . Thus, different photosensitizers have different targets and may utilize different biochemical pathways to initiate apoptosis. Furthermore, it is likely that S E R C A 2 degradation and increased C a 2 + levels may be associated with a separate response and possibly the activation of other biochemical pathway(s) following PDT. Downstream of cyt c release, 7A6 antigen expression, and C a 2 + release, the activation of -193 caspases-3, -6, -7, -8 and - 9 was observed in HeLa cells . In addition to being the first studies to detail the involvement of multiple members of the caspase family in PDT-induced apoptosis, these studies demonstrated for the first time, that caspase-8 activation occurred downstream of cyt c release in mitochondria-mediated apoptotic pathways such is the case with PDT-induced apoptosis 3 2 4 . Since these initial studies, work performed by our laboratory as well as other investigators have demonstrated that the phenomenon of caspase-8 activation downstream of cyt c release may be involved in an amplification loop in which caspase-8 processes B i d into its 193 323 324 334 396 truncated form (tBid) that is capable of inducing cyt c release . In HL-60 cells we demonstrated that caspase-6 activation occurs downstream of caspase-3 activation Other researchers using different stimuli to instigate apoptosis have now confirmed these results. In HL-60 cells, we studied the effects of Bcl -2 and BC1-XL on 57 79 verteporfin-based PDT-induced apoptosis ' . Overexpression of Bcl -2 or BC1-XL in HL-60 cells completely inhibited caspase activity and D N A fragmentation. Furthermore, studies with BC1-XL overexpressing HL-60 cells were the first to suggest that BC1-XL prevented D N A fragmentation through prevention of caspase-3 activation and subsequent caspase-3-mediated cleavage of D F F 79 . Subsequent studies in PDT-treated HeLa cells revealed that although Bcl-2 or BC1-XL 172 expression blocks caspase activity, it did not block the release of cyt c from mitochondria nor 323 cell death at any dose of P D T The role of Bcl-2 and related proteins in PDT-induced apoptosis has been a subject of intense debate. Bcl -2 expression in Chinese hamster ovary cells inhibited apoptosis and partially protected against a loss of clonogenicity in Pc4-based P D T " 6 , while in breast epithelial cells Bcl-2 promoted PDT-induced apoptosis using the photosensitizer P c A l 3 " . The increased susceptibility of Bcl -2 over-expressing cells to P D T was believed to be due to a photodynamic 337 destruction of Bcl -2 that allowed Bax to exert its pro-apoptotic activity . Conversely, Bcl-2 or BC1-XL over-expression in HL-60 cells suppressed verteporfin-based PDT-induced caspase 57 79 activation and D N A fragmentation ' . BC1-XL overexpression inhibited D N A fragmentation 79 indirectly by suppressing caspase-3 activity and subsequent D F F cleavage after P D T . Bcl-2 also provided partial protection against PDT-induced apoptosis using low but not high concentrations of verteporfin. However, in H e L a cells Bcl -2 or BC1-XL did not appear to prevent PDT-mediated mitochondrial alterations. In fact, over-expression of Bcl -2 or BC1-XL in H e L a cells did not prevent the release of mitochondrial cyt c, the unmasking o f the mitochondrial 7A6 323 antigen or cell death induced by P D T with verteporfin at any concentration . Furthermore, studies of hypericin-based P D T indicated that human glioma cell viability was unrelated to 338 expression levels of Bcl -2 or Bax . In summary, the role of Bcl -2 and related anti-apoptotic proteins in the regulation of PDT-induced apoptosis is unclear but these proteins may act in a cell type- and photosensitizer- specific manner. 5.2 E C a n d S M C Apoptosis A s the mechanisms of PDT-induced apoptosis in tumour cells became clearer, the effects of P D T on ECs and S M C s were evaluated. The vasculature is a central target for P D T in the 173 majority of treatment regimes and photosensitizers are known to accumulate at sites of vascular injury. However, prior to our studies, the mechanisms of PDT-induced apoptosis of ECs or S M C s have never been described. Since it is generally understood that tumour cells have one or 397 more genetic defects that relate to altered responses to pro-apoptotic stimuli , it could not be assumed that P D T would elicit the same effect on all cell types. This rationale is further supported by the fact that different cell types differ remarkably in their relative sensitivities to PDT-induced apoptosis (eg. Jurkat or HL-60 cells versus H e L a cells, et cetera). For instance, P D T induced complete caspase-3 processing in H L - 6 0 cells within 15 min after photosensitization whereas caspase-3 activation is not observed for 1-2 h in H e L a cells, H U V E C or H A S M C . In vivo studies have shown that site-specific delivery of verteporfin followed by photosensitization represents a safe, effective method of inhibiting the development of intimal hyperplasia 3 ° \ P D T has also been shown to prevent allograft rejection 3°°. Furthermore, P D T -mediated reduction in the number of S M C occurs along with the prevention of inflammatory infiltration, and development of intimal hyperplasia associated with allograft rejection 3°°. However, very little is known as to how P D T induces S M C apoptosis. On more general terms, the basic mechanisms of S M C apoptosis are poorly understood. Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in E C s were largely undefined. Our studies defined several novel events pertaining to E C apoptosis in response to PDT-mediated oxidative stress 193 and the general understanding of E C apoptosis . Apoptosis was induced in E C using verteporfin as the photosensitizer and light. In tumour cells, alterations in mitochondrial integrity, transmembrane potential and cyt c release into the cytosol are key events that drive 174 apoptosis. In PDT-treated H U V E C , release of mitochondrial cyt c into the cytosol was detectable immediately and accumulated over two hours after photosensitization while cytosolic levels of the pro-apoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another pro-apoptotic Bcl-2 family member, B i d , was observed by 2 h after treatment. Although B i d cleavage had been previously shown to occur as an upstream event responsible for inducing cyt c release, we demonstrated that B i d cleavage could also occur after cyt c release. One problem that we, and others have found with studying the mechanisms of apoptosis (especially caspases) in primary cell types such as H U V E C is that often difficult to generate enough protein for detailed Western blot analysis. Furthermore activation of caspases is often more difficult to detect using primary versus transformed cell lines because the cell population is more diverse and each cell may respond to a given, stimulus at a different rate. This worked to our advantage when studying caspase activation in primary E C in response to PDT. P D T appeared to induce apoptosis fairly uniformly in these cells. Activation of caspases -2, -3, -6, -7, -8 and -9 was detected following the release of cyt c, and cleavage of specific downstream substrates was observed. Prior to our studies, only the status of caspase-1 and -3 had been examined in E C apoptosis. In addition to caspase-1 and -3 we demonstrated the involvement of caspase -2, -6, -7, -8 and -9 in E C apoptosis. In summary, these studies were the first to demonstrate the involvement of cellular Bax redistribution, B i d cleavage, mitochondrial release of cyt c, followed by the activation of multiple caspases during E C apoptosis in response to any , . 193 stimuli . The mechanisms of E C apoptosis in response to verteporfin and light help to explain the mechanisms by which P D T elicits its effects on abnormal, rapidly dividing blood vessels in 175 angiogenic disorders such as cancer and A M D . The ability of verteporfin to be taken up and retained at a higher rate in the rapidly-dividing versus normal blood vessels makes this photosensitizer an attractive means to treat unwanted angiogenesis. Furthermore, the increased permeability associated with the tumour neovasculature allows for verteporfin to accumulate in neoplastic tissues. Tissue eradication can be further specified by the use of laser light directed specifically on the target area. Defective S M C death in addition to increased proliferation have been postulated to play a 187 398 role in the pathogenesis of many common vascular ailments ' . However, the biochemical mechanisms of S M C apoptosis are poorly understood. In addition, P D T is under investigation for the treatment of intimal hyperplasia therefore, biochemical responses of S M C to P D T are of clinical importance. We induced apoptosis in H A S M C with verteporfin and light. Immediate release of mitochondrial cyt c into the cytosol was detectable in H A S M C by Western blotting and continued to increase over two hours following treatment while levels of the pro-apoptotic i . . 399 Bcl-2 family member Bax decreased reciprocally during this time period . Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells whereas Bax staining became more localized in these cells suggesting an intracellular redistribution of this protein possibly to mitochondria. Increased expression of the mitochondrial 7A6 antigen, a marker of apoptosis, was also confirmed by flow cytometry as well as confocal microscopy. Following cyt c release, caspase -3, -6, -7, -8 and -9 activation was observed followed by the degradation of nuclear lamins. In summary, these results demonstrate, for the first time, the phenomenon of cellular Bax redistribution and mitochondrial cyt c release during S M C apoptosis and provide further insight into the understanding as to how S M C respond to toxic stimuli. 176 Further studies are required to determine the role of Bad and possibly the role of the PI3 kinase pathway in response to P D T in these cells. A l F has been previously shown using other pro-apoptotic stimuli in tumour cells to be released from mitochondria whereupon it translocates to nuclei and stimulates chromatin condensation and incomplete 50 kb D N A fragmentation referred to as stage I D N A fragmentation 4°°. This stage of apoptosis is caspase-independent. Complete (stage II) D N A fragmentation into 200 bp fragments requires caspase-3-mediated activation of C A D as previously discussed. The status of A I F was evaluated following PDT-treatment of S M C . Cytosolic levels of A I F increased over a 2 h period following P D T . Moreover, cellular redistribution into the nucleus was evident. Thus, it is apparent that the caspase-independent phenomenon of mitochondrial A I F release and its involvement in stage I nuclear apoptosis are observed during S M C apoptosis. To our knowledge, this is the first demonstration for the involvement of A I F not only during PDT-induced apoptosis, but also in S M C apoptosis in response to any stimuli. Cleavage of the cell cycle inhibitory proteins p21 and p27 were also observed following PDT. These proteins regulate cell cycle progression by binding to, and inhibiting the activity of C D K 2 . It has been speculated that some of the morphological features of apoptosis may be attributed to premature entry into the cell cycle. It is uncertain whether this is true, but caspase-mediated cleavage of these cell cycle inhibitory proteins would help to enable C D K 2 to promote cell division. Further studies are required to explore this possibility. 177 C H A P T E R VI CONCLUSIONS AND F U T U R E PROSPECTS During the tenure of my graduate studies, I have had the opportunity to explore the role and mechanisms of apoptosis in a number of in vitro and in vivo settings ranging from PDT-induced apoptosis in transformed and normal cell lines, to the apoptotic mechanisms associated with coxsackievirusB-induced cytopathic effect, to the role and mechanisms of T V D using murine and rat models of acute and chronic cardiac allograft rejection. Prior to the commencement of my studies, very little was understood regarding the mechanisms of verteporfin-based PDT-induced apoptosis. We have made significant strides towards understanding the effects of P D T on both adherent and non-adherent tumour cells in addition to vascular cells. The information that was obtained from these studies has contributed to several facets of scientific knowledge in addition to being clinically relevant. The U S F D A , as well as numerous other jurisdictions approved verteporfin, also known as Visudyne®, for the treatment of A M D , the leading cause of blindness in the elderly. In the treatment of A M D , the abnormal blood vessels responsible for the loss of visual acuity in this condition are believed to rapidly take up Visudyne® and are destroyed upon light irradiation, most likely due to a combination of PDT-induced E C apoptosis, vaso-occlusion and thrombosis. Since minimal biochemical work had been published on the biochemical effects of P D T on E C , results obtained from our studies were highly applicable to the understanding as to how P D T alleviates A M D . Furthermore, studies on the effects of P D T on E C were applicable to the mechanisms by which P D T eliminates the abnormal neovasculature in the treatment of tumours. Our understanding of the mechanisms by which P D T eliminates tumours was enhanced by the study of the biochemical effects of P D T on tumour cells. These studies provided evidence that P D T has the ability to eradicate tumours that are otherwise resistant to other forms of chemotherapeutics. Admittedly, i f one adds enough of a particular 178 chemotherapeutic they can k i l l even the most resistant tumour cells in vitro. However, when injected into animals, the amount of drug that can be added is limited, among other factors, to its relative cytotoxic specificity towards tumour cells. In the case of P D T for the treatment of tumours, verteporfin, in the absence of light, is not cytotoxic. Furthermore, the dose of P D T can be increased by either increasing the amount of drug or the amount of light. Therefore, a much higher degree of cytotoxicity can be attained due to the ability of lasers to specifically direct light to a particular target area. However, P D T does have its limitations in that it can only be applied to areas in which light can be delivered. The studies on the effects of P D T on S M C are applicable to clinical studies pertaining to the use of P D T in the treatment of restenosis, atherosclerosis and T V D . One of the primary contributors to these ailments is the increase of S M C due to increased proliferation, migration or possibly a decrease in apoptosis. Since, previous studies have indicated that active, rapidly dividing cells take up verteporfin at a higher rate than their resting counterparts, it is likely that verteporfin may be taken up at a higher rate in actively dividing S M C . Futhermore, verteporfin is a liposome based formulation, and lipid uptake and accumulation in S M C is higher in a disease setting which may facilitate increased drug uptake. Thus, in vivo, the effects of P D T on the reduction of intimal hyperplasia are complex. It is likely that P D T affects not only S M C , but also immune cells. To this extent, activated T cells and macrophages, which are often considered to be pathogenic in the formation of intimal hyperplasia, appear to be more sensitive to P D T than other cell types. However, one also has to consider the effects of P D T on the endothelium. We have previously shown that P D T can induce endothelial apoptosis. It is possible due to increased endothelial permeability, increased lipophilicity, as well as increased uptake of verteporfin by rapidly dividing immune cells and S M C that E C may take up less verteporfin. In such a scenario, it may be possible 179 to apply enough light to induce immune cell and S M C apoptosis while sparing endothelial integrity. We have previously demonstrated that low level P D T induces N F - K B activation in HL-60 cells. It is likely that the same may be true for E C . If the latter is the case, N F - K B activation promotes the transcription of anti-apoptotic genes which may further enhance E C survival and possibly re-endothelialization. Further experimentation is required to concur whether these hypotheses are viable. The work related to the biochemical mechanisms of E C and S M C apoptosis are also of significant interest to the general scientific community, excluding photodynamic therapists. The definition of the apoptotic pathway in PDT-treated E C was among the first studies to detail the phenomena of intracellular Bax migration, B i d cleavage, cyt c release and activation of caspases. It may be argued that the majority of these events were observed in PDT-treated tumour cells and whether it was worth examining in other types of cells. However, it is quite apparent that different cell types respond differently to PDT, or any stimuli for that matter. Furthermore, events such as intracellular Bax migration and a gradual increase in cytosolic levels of cyt c were not observed for PDT-treated HeLa cells, but were observed in PDT-treated E C and S M C . One of the key foci for apoptosis in the years ahead wi l l be to decipher what characteristics renders one cell type more susceptible to a particular apoptotic stimuli than another. Defining the genetic and phenotypic characteristics that render different cell types more or less resistant to apoptosis w i l l lead to new avenues by which to manipulate these features to stimulate or inhibit the death of a particular group of cells. In diseases in which cell loss due to apoptosis is detrimental, including many myocardial or neuronal diseases, methods to enhance the survival of these cells would be extremely beneficial. On the contrary, in hypoproliferative disorders whereby the apoptotic pathway may be defective 180 resulting in the accumulation of a particular cell type, methods to detect and target the protein that is responsible preventing the apoptotic elimination of these cells would be equally valuable. Increased vascular endothelial permeability of coronary arteries may be associated with 398 transplant vascular disease . Increased endothelial permeability, possibly due to the effects of SMC-mediated release of V E G F on the endothelium, may allow lipoproteins access to the 398 subendothelial space . These lipids can bind to vessel wall proteoglycans, which causes these lipids to become more susceptible to oxidative modification. These oxidized lipoproteins are then engulfed by macrophages and S M C ultimately leading to the formation of foam cells that contribute to plaque formation and instability. Work in support of this hypothesis is in progress and the focus of a recently funded grant. However, since verteporfin accumulates at sites of increased vascular permeability, it is possible that these cells may preferentially take up more verteporfin rendering them as preferential targets of P D T . Whether verteporfin would be preferentially accumulated by macrophages and S M C is uncertain, however, this would offer a mechanisms by which P D T could alleviate some of the effects of vasculopathy and chronic cardiac allograft transplant rejection. Furthermore, activated T cells are more susceptible to P D T 309 than their resting counterparts . Similarly, activated macrophages are also more susceptible to 402 verteporfin-based photodynamic ki l l ing than their resting counterparts . Thus, P D T may be an effective means to suppress allograft rejection by (1) inducing S M C apoptosis thereby reducing intimal hyperplasia and (2) reducing the number and contribution of inflammatory cells associated with the disease process. To this extent, LaMuraglia and colleagues have demonstrated, using a model by which infrarenal aortas of A C I rats were P D T - treated and then orthotopically grafted in Lewis rats, that P D T eliminates both the inflammatory infiltration and intimal hyperplasia associated with transplantation 3°°. Furthermore, it should be noted that P D T 181 is now in clinical Phase I trials for the treatment of human peripheral atherosclerosis, which suggests a promising future for photoangioplasty in the treatment of primary atherosclerosis and 312 prevention of restenosis . To date, we have not examined the effectiveness of verteporfin in the treatment and prevention of intimal hyperplasia associated transplant vascular disease. However, the effects of verteporfin on other models of intimal hyperplasia have shown promise 3 0 6 , m _ Taking into 309 402 consideration in vitro studies on the effects of P D T on activated immune cells ' as well as S M C 399 404 300 , and in vivo studies using other photobiological techniques by our laboratory and others , the prospects for verteporfin-based P D T are promising. However, whereas the induction of E C apoptosis is beneficial for the treatment of angiogenesis-related phenomena associated with cancer and A M D , damage to the endothelium is not advantageous for the treatment of vessels associated with transplant vascular disease. Much of the endothelial lining at the sites of intimal hyperplasia is already damaged following transplantation prior to reendothelialization. Thus, endothelial damage may not be a major concern. Furthermore, through increased vascular permeability allowing increased infiltration of verteporfin into the intima, in combination with the increased uptake by rapidly dividing cells and high sensitivity of activated immune cells to verteporfin-based PDT, the endothelium is likely not the primary target. Future studies may examine the feasibility of P D T for the treatment of acute and chronic cardiac allograft rejection. In summary, although we have made several breakthroughs in the understanding of the biochemical mechanisms of apoptosis over the course of my dissertation, the journey has only just begun. In the understanding of the role that apoptosis plays in disease, future studies w i l l be aimed at defining the susceptibility, mechanisms, roles and contributions of apoptosis between different cell types in concert with one another. In the case of the study of T V D , this w i l l require 182 the use of co-culture systems or animal models. To adequately utilize a co-culture model for the study of T V D , one would imagine that both an allogeneic as well as a syngeneic co-culture system would be required. Obviously, these systems are currently not very practical. However, with recent advances in stem cell work, it may be soon possible to derive different cell types from the same stem cell, which may provide the opportunity to study events such as immune cell interactions with the endothelium in a more syngeneic environment. Wi th respect to animal studies, the tools by which to detect and study the mechanisms of apoptosis are still limited and dependent specifically on TUNEL-labe l l ing , immunohistochemistry and possibly the use of transgenic mice. With recent advancements in techniques such as laser capture microdissection and D N A microarrays, we w i l l soon be able to distinguish not only the specific genetic alterations between specific cell types from normal and diseased states, but we may be able to remove a specific subset of cells from a tissue for more specific analysis. To summarize my dissertation, I have listed below what I consider to be the major consider to be the major contributions of my work to the scientific community. Contributions to scientific knowledge 1. Provided landmark mechanistic studies as to how P D T kil ls tumour cells. 2. Detailed the effects of Bcl-2 on PDT-mediated events such as cyt c release, caspase activation, and C a 2 + release. These results also describe the differential effects of Bcl-2 on C a 2 + release from E R and mitochondria in response to P D T . 3. First studies to describe the biochemical effects of P D T on endothelial cell viability. The biochemical effects of PDT-induced E C apoptosis have never been addressed prior to our studies. Furthermore, E C are the primary target for P D T in the treatment of A M D , the 183 leading cause of blindness in the elderly and the most widely applied P D T clinical treatment in the world. 4. Many of the events described for PDT-induced E C apoptosis were the first to delineate these mechanisms for E C in response to any pro-apoptotic stimulus. 5. Although several studies have demonstrated that P D T reduces intimal hyperplasia, little had been published regarding the effects of P D T on S M C viability. We not only demonstrated that P D T induced apoptosis in S M C , but also outlined in detail the mechanisms by which apoptosis occurs in these cells. 6. 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