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Drug resistance in breast cancer : effect of Bcl-2 level on the responsiveness of two mammary carcinoma… Sartor, Jason Robert 2002

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D R U G R E S I S T A N C E IN B R E A S T C A N C E R : E F F E C T OF BCL-2 L E V E L ON T H E RESPONSIVENESS O F T W O M A M M A R Y C A R C I N O M A C E L L LINES T O C H E M O T H E R A P E U T I C A G E N T S  by  J a s o n Robert Sartor B . S c . T h e U n i v e r s i t y of British C o l u m b i a , 1 9 9 8 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F THE REQUIREMENTS F O R T H E DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES Experimental Medicine Program F a c u l t y of M e d i c i n e W e a c c e p t this t h e s i s a s c o n f o r m i n g to the required s t a n d a r d  T H E UNIVERSITY O F BRITISH C O L U M B I A January 2002 © J a s o n Robert Sartor, 2 0 0 2  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives.  It is understood  that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  frY^tO'VLCnfal  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ^fr*.Wvf  ^7 ^  2t V'2— /  /U/rr.JA/  II  ABSTRACT  T h e B c l - 2 p r o - s u r v i v a l protein is a n important regulator of a p o p t o s i s a n d h a s b e e n implicated in m e c h a n i s m s of d r u g r e s i s t a n c e b y virtue of t h e e x p r e s s i o n l e v e l s of this protein in t h e c e l l .  In t h e s e  s t u d i e s w e s o u g h t to d e t e r m i n e t h e effect of t h e inherent B c l - 2 level in b r e a s t c a n c e r cell lines o n d r u g sensitivity a n d s u s c e p t i b i l i t y to a p o p t o s i s , a s w e l l a s attempt to s e n s i t i z e t h e s e c e l l l i n e s to c h e m o t h e r a p y by d e c r e a s i n g t h e a m o u n t of B c l - 2 in t h e c e l l .  Initial s t u d i e s i n v o l v e d t h e d o w n - r e g u l a t i o n of B c l - 2 level in  the h i g h - B c l - 2 - e x p r e s s i n g M C F - 7 a n d l o w - B c l - 2 - e x p r e s s i n g M D A - 4 3 5 / L C C 6 cell lines in vitro t h r o u g h t h e u s e of the B c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e G e n a s e n s e ™ ( G 3 1 3 9 ) . O u r f i n d i n g s i n d i c a t e d that B c l - 2 d o w n regulation v i a B c l - 2 a n t i s e n s e w a s sufficient to i n d u c e a p o p t o s i s in vitro a s m e a s u r e d b y a s e r i e s of a p o p t o t i c e n d p o i n t s , but that c h a n g e s in B c l - 2 e x p r e s s i o n l e v e l s w e r e not sufficient to s e n s i t i z e t h e cell lines to c h e m o t h e r a p y d r u g s .  A n a l y s i s of t h e effect of B c l - 2 d o w n r e g u l a t i o n v i a e s t r o g e n inhibition in  high a n d l o w B c l - 2 - e x p r e s s i n g c l o n a l p o p u l a t i o n s p r o d u c e d f r o m B c l - 2 t r a n s f e c t e d  MDA-MB-435/LCC6  a n d M C F - 7 cell lines i n d i c a t e d that this w a s not sufficient to s e n s i t i z e t h e s e lines to c h e m o t h e r a p y d r u g s . X e n o t r a n s p l a n t a t i o n of t h e s e s a m e cell lines into i m m u n e - c o m p r o m i s e d m i c e i n d i c a t e d that B c l - 2 d o w n regulation u s i n g G e n a s e n s e w a s sufficient to impair t u m o r growth a l o n e , a n d to a g r e a t e r d e g r e e w h e n c o m b i n e d with c h e m o t h e r a p y ; h o w e v e r , t h e results of t h e s e s t u d i e s e s t a b l i s h e d that t h e cytotoxicity of c o m b i n e d a g e n t s w a s additive a n d not s y n e r g i s t i c .  Interestingly, a n d in c o n t r a s t to d a t a p r e s e n t e d in  vitro, B c l - 2 l e v e l s d i d a p p e a r to c o n f e r s o m e d e g r e e o f r e s i s t a n c e in vivo to B c l - 2 a n t i s e n s e a s a s i n g l e a g e n t o r w h e n c o m b i n e d with c h e m o t h e r a p y d r u g s . T a k e n together, t h e s e results e s t a b l i s h e d that in t h e cell lines u s e d in this s t u d y , r e d u c t i o n of t h e level of B c l - 2 r e s u l t e d in i n c r e a s e d a p o p t o s i s in vitro a n d i m p a i r e d t u m o r g r o w t h in vivo, a l t h o u g h it did not s e n s i t i z e c e l l s to c h e m o t h e r a p y a g e n t s in either e n v i r o n m e n t . T h e s e r e s u l t s s u g g e s t that w h i l e B c l - 2 c l e a r l y c o n s t i t u t e s a n attractive target d u e to its role in r e g u l a t i n g a p o p t o s i s in b r e a s t c a n c e r c e l l s , additional  mechanisms  chemotherapy drugs.  a r e likely  important  in t h e control  of a p o p t o s i s  arising  from  e x p o s u r e to  iii  TABLE OF CONTENTS Abstract Table of Contents List of Figures List of Tables Abbreviations Acknowledgments Dedication  ii iii iv vii viii ix xi  C H A P T E R 1: I N T R O D U C T I O N  1  1.1 D R U G RESISTANCE  2  1.2 APOPTOSIS  8  1.3 B C L - 2 AND D R U G RESISTANCE  31  1.4 ANTISENSE OLIGODEOXYNUCLEOTIDES  44  1.5 M E T H O D S AND MATERIALS  63  1.6 SUMMARY OF HYPOTHESES  79  1.7 SUMMARY OF RESEARCH OBJECTIVES  80  C H A P T E R 2: I N V I T R O B C L - 2 A N T I S E N S E E X P E R I M E N T S  81  2.1 INTRODUCTION  82  2.2 MATERIALS AND METHODS  83  2.3 RESULTS  86  2.4 DISCUSSION 2.5 REFERENCES  C H A P T E R 3: C R E A T I O N A N D C H A R A C T E R I Z A T I O N O F C L O N E S 3.1 INTRODUCTION  105 .  109  111 112  3.2 MATERIALS AND METHODS  113  3.3 RESULTS  117  3.4 DISCUSSION  134  3.5 REFERENCES  136  C H A P T E R 4: D O W N R E G U L A T I O N O F B C L - 2 I N C L O N E S V I A E S T R O G E N A B L A T I O N  137  4.1 INTRODUCTION  138  4.2 MATERIALS AND METHODS  139  4.3 RESULTS  141  4.4 DISCUSSION  148  4.5 REFERENCES  151  C H A P T E R 5: D O W N R E G U L A T I O N O F B C L - 2 I N C L O N E S V I A B C L - 2 A N T I S E N S E IN VIVO 5.1 INTRODUCTION  153 154  5.2 MATERIALS AND METHODS  156  5.3 RESULTS  157  5.4 DISCUSSION  168  5.5 REFERENCES  172  CONCLUSIONS  174  iv  LIST OF FIGURES  CHAPTER 1 FIGURE 1.1  Pathways of Apoptosis  11  FIGURE 1.2  Bcl-2 Rheostat Theory  17  FIGURE 1.3  Mechanism of Action of Oligonucleotides  45  FIGURE 1.4  Structure of Cisplatin, Doxorubicin, Paclitaxel, and C6-Ceramide  64  FIGURE 1.5  Structure of 17(3 Estradiol and Antiestrogen Compound Faslodex  67  FIGURE 1.6  Structure of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).  71  FIGURE 1.7  Methyl Thiazolyl Tetrazolium (MTT) assay  72  FIGURE 1.8  Clonogenicity Assay  74  FIGURE 1.9  Flow Cytometry  76  FIGURE 2.1  Inherent Bcl-2 Level in MDA-435/LCC6 and MCF-7 Cells and Expression of Bcl-2 After Treatment with Bcl-2 Antisense.  87  FIGURE 2.2  Relative Bcl-2 Protein Levels in MDA-435/LCC6 and MCF-7 Cell Lines Over 96 Hours After Treatment with Bcl-2 Antisense  89  FIGURE 2.3  Relative mRNA and Protein Levels in MDA-435/LCC6 Cell Lines After Treatment with Bcl-2 Antisense  90  FIGURE 2.4  Viability of MDA-435/LCC6 Cells After Bcl-2 Antisense Treatment as Assessed by Trypan Blue Exclusion  91  FIGURE 2.5  Percentage of Non-Viable MDA-435/LCC6 and MCF-7 Cells After Bcl-2 Antisense Treatment as Assessed by Inclusion of Trypan Blue  93  FIGURE 2.6  Long Term Viability of MCF-7 Cells After Bcl-2 Antisense Treatment as Assessed by MTT  94  FIGURE 2.7  DAPI Staining of MDA-MB-435/LCC6 Cells Treated with Bcl-2 Antisense or Mismatch Control.  95  FIGURE 2.8  Mitochondrial Membrane Potential of MDA-435/LCC6 Cells Treated with Bcl-2 Antisense  97  FIGURE 2.9  Apoptotic Profile Assessed using Hoechst and PI Stains of MDA-435/LCC6 Cells Treated with Bcl-2 Antisense  98  FIGURE 2.10  Quantitative Cell Cycle Analysis of GenasenseTM and Doxorubicin Treated Cells Using Propidium Iodide Staining and Flow Cytometry  99  CHAPTER 2  V  LIST OF FIGURES CONT'D FIGURE 2.11  Cytotoxic Effect of Doxorubicin Against MCF-7 and MDA-435/LCC6 Cells After Treatment with Bcl-2 Antisense or Mismatch Control  102  FIGURE 2.12  Cytotoxic Effect of Various Chemotherapy Drugs Against MCF-7 Cells After Treatment with Bcl-2 Antisense or Mismatch Control.  103  FIGURE 2.13  Cytotoxic Effect of Various Chemotherapy Drugs Against MDA-435/LCC6 Cells After Treatment with Bcl-2 Antisense or Mismatch Control  104  FIGURE 3.1  Diagram of Plasmid Construct Bearing a Bcl-2 cDNA Transfected into MDA-435/LCC6 and MCF-7 Cells.  118,  FIGURE 3.2  Fluorescence Microscopy Images of Cells Stained with and a-Bcl-2-FITC and MTR Mitochondrial Stain.  120  FIGURE 3.3  Profile of Bcl-2 Content in MDA-MB-435/LCC6 and MCF-7 Transfected Parental Cell Lines.  121  FIGURE 3.4  Flow Cytometry Histograms of Clonal Populations Isolated from Bcl-2 Transfected MDA-435/LCC6 Cells.  123  FIGURE 3.5  Flow Cytometry Histograms of Clonal Populations Isolated from Bcl-2 Transfected MCF-7 Cells.  124  FIGURE 3.6  Calculation of P r indicating the Antibody Binding Capacity of the Cell  126  FIGURE 3.7  Flow Cytometry and Western Analysis of Selected MDA-MB-435/LCC6 Clonal Populations.  129  FIGURE 3.8  Flow Cytometry and Western Analysis of Selected MCF-7 Clonal Populations.  130  FIGURE 3.9  Western Analysis of Bax and Bak Protein Levels in MDA-MB-435/LCC6 and MCF-7 Clones.  132  FIGURE 3.10  Relative Bcl-2, Bax and Bak Levels of MD A-MB -435/LCC6 and MCF-7 Clonal Populations as Determined by Western Analysis.  133  FIGURE 4.1  Western Analysis of Bcl-2 Levels in MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell lines Exposed to Faslodex over 96 Hours.  142  FIGURE 4.2  Western Analysis of Bax and Bak Levels in Bcl-2 Transfected MDA-MB-43 5/LCC6 and MCF-7 Cells Exposed to Faslodex over 4 Days  143  FIGURE 4.3  Growth Rates of MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell lines Exposed to Faslodex over 120 Hours.  145  FIGURE 4.4  Cytotoxicity of MD A-MB-43 5/LCC6 (ER-) and MCF-7 (ER+) Cell Lines Exposed to Doxorobucin over 48 Hours.  146  CHAPTER 3  ef  CHAPTER 4  vi  LIST OF FIGURES C O N T ' D  Cytotoxicity of MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell Lines Exposed to Cisplatin over 48 Hours.  147  FIGURE 5.1  Western Analysis of Bcl-2 Levels in MDA-MB-435/LCC6 Clone 7 And Vector Tumors in Various Treatment Groups.  158  FIGURE 5.2  Western Analysis of Bcl-2 and BaxLevels in MDA-MB-435/LCC6 Clone 7 And Vector Tumors in Various Treatment Groups.  159  FIGURE 5.3  Growth of MDA-435/LCC6 Cells Implanted in Female RAG-2M Mice and Treated with Doxorubicin: Comparison of Bcl-2 Transfected and Vector Controls.  162  FIGURE 5.4  Growth of MDA-435/LCC6 Clone 7 (High Bcl-2) and Vector (Low Bcl-2) Cells Implanted in RAG-2M Mice and Treated with Bcl-2 Antisense or Reverse Polarity With or Without Doxorubicin: Comparison of Bcl-2 Transfected and Vector Controls  165  FIGURE 5.5  Growth of MDA-435/LCC6 Vector Cells (Low Bcl-2) Implanted in RAG-2M Mice and Treated with Bcl-2 Antisense or Reverse Polarity With or Without Doxorubicin  166  FIGURE 5.6  Growth of MDA-435/LCC6 Clone 7 (High Bcl-2) Cells Implanted in RAG-2M Mice and Treated with Bcl-2 Antisense or Reverse Polarity With or Without Doxorubicin  167  FIGURE 4.5  CHAPTER 5  vii  LIST OF TABLES  TABLE 1.1  Outcomes of Therapy in Advanced Disease  4  TABLE 1.2  Mechanisms of Drug Resistance  6  TABLE 1.3  Features of Apoptosis and Necrosis  9  TABLE 1.4  Pro-and Anti-apoptotic Bcl-2 Family Members  16  TABLE 1.5  Cellular Targets of Caspases  24  TABLE 1.6  Flow Cytometric Quantitation of Bcl-2 Level in MCF-7 and MDA-435/LCC6 Clonal Populations  127  TABLE 1.7  Ratio of Bcl-2 to Bax and Bak Individually or Combined in Clonal and Wild-type Cells  131  ABBREVIATIONS Av|/ A1F ALL AML ANOVA APAF AS BSA CLL Cn DAPI DiOC D M SO DNA DODAC DOPE DTIC ER FBS 6  Ig MM MP MTR MTT NAO NK ODN PBS PI PKC PNA PTP RNA RP SC1D TCR  Mitochondrial Membrane Potential Apoptosis Inducing Factor Acute Lymphocytic Leukemia Acute Myelogenous Leukemia Analysis of Variance Apoptotic Protease Activating Factor Antisense Bovine Serum Albumin Chronic Lymphocytic Leukemia Control 4',6-Diamidino-2-Phenylindole Stain Dimethyloxacarbocyanine 6 Dimethyl Sulfoxide Deoxyribonucleic Acid Dioleoyldimethylammonium Chloride Dioleoylphosphatidyl Ethanolamine Dacarbazine Estrogen Receptor Fetal Bovine Serum Immunoglobulin Mismatch Control Methyl Phosphonate Oligonucleotides MitoTracker Red Methyl Thiazolyl Tetrazolium Nonylacridine Orange Natural Killer Cells Oligodeoxynucleotide Phosphate Buffered Saline Propidium Iodide Protein Kinase C Protein Nucleic Acid Oligonucleotides Permeability Transition Pore Ribonucleic Acid Reverse Polarity Severe Combined Immunodeficient T-Cell Receptor  ix  ACKNOWLEDGMENTS First and foremost I would like to thank my supervisor, Dr. Lawrence Mayer, for all of his guidance, assistance, expertise and wisdom over the last several years. Most of all, his patience and understanding with my work and sometimes somewhat confusing and frustrating results is very much appreciated. It is to Lawrence that I owe the greatest amount of gratitude for my experiences in graduate school and for his help I am very thankful. I would also like to thank my committee members, Dr. Vince Duronio and Dr. Martin Gleave, for taking the time out of their busy schedules to agree to be on my committee. Their insights into the material I have presented is valuable and I very much appreciate the efforts Dr. Duronio and Dr. Gleave have made towards my research project. Similarly, I would like to thank Dr. Norman Wong, Dean of Experimental Medicine, for his help in administrating my graduate degree. He has always been a friendly face at my department whom I could easily approach with any questions or concerns. The research comprising this thesis would not have been possible without the help and expertise of a number of co-workers over the years. To my friend Dr. Kim Chi I owe my thanks for showing me the ropes when I first arrived and putting up with my endless questions and inquiries about the various facets of our research project. His knowledge and experience was very valuable and much of the credit for my later successes in this project are directly due to his input. I will certainly miss our great conversations and our all too short lunches! Along with Kim Chi, Frances Wong deserves my thanks for helping me understand various aspects of my research project and her help with everything from lending me buffers to checking my calculations made my life much, much easier. For this she has my gratitude. I would also like to thank Visia Dragowska for her assistance with all of the flow cytometry experiments and microscopy experiments over the past several years. Her good sense of humor and welcoming attitude always made this part of my research a pleasure. Another person who contributed a great deal to my research was Anne Wallis. Her expertise in molecular biology all but saved my research project at times and the fact that she was always willing to help me out when I was in need is certainly appreciated. Of my research group, I would lastly like to thank Daniel Menezies. He started his post-doc the same time I started graduate school and without his help none of my research would have been possible. And besides, he's a fun guy to party with even if he does collect posters from "The Odyssey"... The other group with whom I collaborated a great deal were the animal technicians. To Dana I owe my thanks (and probably a few apologies) for her help with organizing and assisting with my animal experiments and late-breaking changes in my protocol. I would also like to thank Natashia for her expert help in teaching me tissue culture and MTTs, and her great advice on good hikes was just as valuable. Most of all, her cheery attitude made coming to work a pleasure. Kudos goes to Rebecca for her help over the years, and especially at being able to find and identify my hidden and mis-labeled cell cultures! My animal work also would not have been possible without help from Sophia and Hong, and I thank them both for their assistance in this regard. I would like to thank some of my other co-workers with whom I did not necessarily work with on research projects, but who made my graduate school experience one that I will always remember and cherish. First, of course, the residents of that sacred place: The Pit. To Jennifer, thanks for the past few years, and remember, YOU WILL GRADUATE EVENTUALLY - no worries. It's been fun and its always great to have a fellow Lawrencegraduate-student to relate to ;-) And I would also like to thank Jennifer for her help with my job at UBC. I also want to mention Ludger and thank him for his help. We had some good conversations and it was a pleasure sharing The Pit with him (and I know he's eyeing my desk as I write this). Most of all, this quite unassuming PhD student will always be remembered by me as putting on the wildest, craziest house party that I've been to in years! Good luck with your mouse organ, Ludger... And then there's Sheela. Who could forget Sheela. With that infective laugh and the best sense of humor on the Lower Main, I will never forget Sheela. Sheela was a lot of fun when we went out clubbing, and I will also remember our interesting Pit conversations — usually encompassing a good juicy story or some irrevocable tale involving that studmuffin Ed (and you can tell him I said that). Thanks for the laughs Sheela! And good luck with your comp, PhD and making it out of AT, radiation free! (hey that even rhymes) While I spent more time in The Pit than my own apartment and got to know Ludger, Jennifer and Sheela very well, there's many more people I would also like to thank. Of course there's sweet Nancy, who plays a mean game of volleyball in spite of that nice demeanor and cute little Dos Santos laugh (yeah, you know the one). Apart from volleyball, I will always remember our fun, fun nights out with Daniel et al. at the clubs and meeting all of her friends (being the social butterfly that she is). Thanks for the memories, Nanc, I will remember those times always. Of course, I also can't forget Lincoln, my partner-in-angst when it comes to running gels or running cars. Lincoln's probably the only person in the lab who I know I could leave beer with and still get it back weeks later! Lincoln, you're a great guy and I'm glad I got to know you over that past few years. All the best. And then there's Ms. Tucker, the only other grad student crazy enough (strange enough?) to be found in the lab at 3:00 in the morning besides me. I must say that Catherine keeps a schedule that I can respect. I enjoyed helping Catherine with her project since I was there myself not that long ago, and her smile always brightens my day (and convinces me to  X  answer her questions, check her calculations, or get her sushi!). Things might seem frustrating at times, Catherine, but stay the course and you'll do fine! (I'm proof that even the most incompetent people can still succeed in science). Looking back on the last few years of graduate school, I just know that it would not been nearly as interesting had I not gotten to know our secretary Corrina. Now how could I describe Corrina... Well, if Corrina was a drink she would be an double expresso, perhaps with a shot of Baileys in there somewhere. Always chalk full of energy, her enthusiasm and glowing smile always picked up my day and it was great just to take a break and chat with her. Her easy charm and kind heart will take her a long way I know she will make an excellent teacher one day. Bella Corrina, di ringraziamento bei per i tempi di divertimento ed e la buoa fortuna! Lastly, there's Susan, who I had the privilege of greeting every day as I bounded down the stairs at some ungodly late hour in the morning. We had some good talks and some good times playing volleyball (even if she completely put me to shame) and I hope she has a great year (no injuries!). Still more people I would like to thank... My gratitude goes out to Yanping for her help and advice when I needed it. It was great to have someone like her working in my research group and the quality of her work was an example of something to strive for. I'm also a better person for having known Corinna over the past few years. Apart from my strange requests for translations into German which she was always eager to do, we had some great conversations. I'm always up to tea anytime, Corinna. I want to thank Maggie for the times that she has helped me with questions or getting papers and the great food and snacks that she always seems to bring into the lunchroom. Same goes to Gigi who was also helpful if I had questions and whose equipment I borrowed far too often. A big thanks to Ghania for her help over the years and especially with my trip to Paris where her help and insight made my trip that much more memorable. Merci encore pour la vin et la bonne chance! Kudos to Dawn on wrapping up her PhD - I can definitely relate to spending far too much time in front of the computer. I'm glad I we worked together and don't forget that that bike of yours is only a defense and a job interview away. My whole time at graduate school would never have been the same if not for Hafiza. A GREAT BIG THANKS to her, both for her wonderful help with all my requests, invoices, silly questions, etc., and for her happy hellos and warm smiles in the afternoon. Hafiza never hesitated to help me out when needed it, and she always did so with a lot of enthusiasm. I am glad she stopped flashing me though! That was just going way too out of hand... Someone else to whom I owe a great deal of gratitude and without whose help I never would have gone anywhere is, of course, Spencer. Spencer was gold when it came to answering questions and getting help with just about anything in the lab (the man can fix anything). On the personal side, Spencer is one of the nicest, most giving people I've ever met and I'm glad I got to know him over these three years. I'm going to miss our conversations about everything from kayaking to art and I wish him the best in the future. I also want to acknowledge some great people from other labs that I got to know while at the Cancer Agency. I will always remember Kevin and especially his stag (it's too bad he doesn't remember it). A good solid guy and an excellent MC, I'm sure he will go far in research. Along with Kevin there's my bud Michelle who started in my department at the same time as I did. Our ice cream trips would always cure a frustrating or bad workday and I had a lot of fun at our departmental events over the years. Someone else who I would like to thank is Gwyn, and a more down to earth person would be hard to find. I enjoyed helping him in the lab and his easy manner made him a pleasure to work with (and to drink with). Lastly, I want to acknowledge Jenn H. and say that I'm glad that I had the chance know her and share a lot of conversations and insights with her over the past several years. A talk with Jen was always a great excuse to put off work and I'll definitely miss her sense of humor and some of her perspectives on life. Whether it was climbing up to Garibaldi or partying it up in Seattle, I will remember the times we had together always. And I would be remiss not to mention a few other great people from the lab. Thanks to Jean, Daria and Margaret for their help with the IDP stuff and with putting up with my all too frequent lapses in memory. Thanks to Marcel for taking time out of his busy schedule to answer my questions when Lawrence wasn't around. Thanks to Mac and Cliff, both great guys and a lot of fun to party with (and who could forget Mac's emails). And a special thanks to all my other lab mates and colleagues that I had the pleasure to work with from time to time - Christine, Corrie, Hayes, Vincent, Euan, Janet, Norma, Lillian and Marie-Paul. Finally I would like the thank my family and friends, and specifically Mike and Pete, who, in spite of not necessarily knowing what I was doing at work or why I needed to be at the lab at midnight on a Saturday, were nonetheless helpful and understanding throughout my grad student years. My gratitude and love goes out to Chantal for putting up with me as a I labored to write my thesis. And most of all I am grateful to Jim Goldie for his wisdom and enduring help throughout my graduate degree. Without his help I would not be where I am today.  DEDICATION  To my parents, Mike and Elaine, and to my uncle Jim  xii  "Everything is simpler than you think and at the same time more complex than you imagine." — Johann Wolfgang von Goethe  CHAPTER 1 INTRODUCTION  2 1.1  D R U G  R E S I S T A N C E  1.1.1 OVERVIEW OF DRUG RESISTANCE IN CANCER Drug resistance is descriptive of a phenomenon whereby a subset of cells within a tumour is resistant to the cytotoxic effects of one or more chemotherapeutic agents used to treat the tumour. The outcome of this condition is two-fold: Not only does the tumour initially resist eradication because of the immunity of these cells, but the drug may actually select for the resistant population, resulting in a tumour that becomes even more resistant to further treatments; the former is referred to as while the latter is referred to as  acquired  intrinstic  resistance,  resistance. Thus, it is through the pre-existing occurrence of  inherent resistance mechanisms in combination with the additive process of acquired resistance that drug resistance progresses within a tumour and renders the disease increasingly untreatable. Drug resistance is associated with the neoplastic state, as populations of normal cells do not develop resistance and may even become more sensitive upon exposure to chemotherapeutic agents . 1  Therefore, there exists in cancer cells certain properties, which occur in many cancer types, that allow the cells to evade the cytotoxic activity of cancer drugs.  O n e of the most significant of these properties is  genetic instability. Cancer cells are genetically unstable in comparison with cells which have not been transformed. The origin of this genetic instability may arise from the original clonal selection of a cell that has an inherently high mutation rate due to a loss of mechanisms ensuring replicative fidelity - this, in turn, may amplify the genetic mutability as the tumour progresses . In itself, the presence of genetic instability may 2  not at first appear very significant; however, this condition gives rise to disparate populations of cells with diverse genetic make-up and results in widespread heterogeneity within the tumour mass.  It is precisely  this heterogeneity that leads to drug resistance and makes the disease so difficult to treat.  Indeed, an  advanced tumour could be described as a mosaic of many divergent cell populations, each with separate phenotypes and, more importantly, responses to given treatments.  Consequently, while one agent may  be effective against one subset of cells within a tumour, it may be completely ineffective against another; such is the source of drug resistance. Although the genetic diversity and instability of cancer cells and the heterogeneity it produces may be one of the most salient problems in drug resistance, it is certainly not the only one.  Drug  resistance may be induced or aided by a whole host of conditions, one of the most noteworthy of which is  3 t u m o r e n v i r o n m e n t : s i z e , s t a g e a n d l o c a t i o n of t u m o u r , b l o o d s u p p l y a n d g r o w t h rate, p H a n d d e g r e e of h y p o x i a , r a t e s of n e c r o s i s a n d a p o p t o s i s - all of t h e s e f a c t o r s a r e intimately a s s o c i a t e d with t h e i n d u c t i o n of d r u g r e s i s t a n c e .  A n indication of the i m p o r t a n c e of t h e s e i n f l u e n c e s in the c r e a t i o n of this c o n d i t i o n is  e v i d e n t in c o m p a r i n g the e f f e c t i v e n e s s of t r e a t m e n t of h e m a t o l o g i c a l m a l i g n a n c i e s to that of s o l i d t u m o r s . In g e n e r a l , l e u k e m i a s r e s p o n d to t r e a t m e n t better than l y m p h o m a s a n d m u c h m o r e s o t h a n s o l i d t u m o r s . In part, this m a y b e d u e to the a b s e n c e of m a n y of the c o m p l i c a t i n g f a c t o r s outlined a b o v e .  Further  e v i d e n c e of the i m p o r t a n c e of t u m o r m i c r o e n v i r o n m e n t o n d r u g r e s i s t a n c e lies in s e v e r a l s t u d i e s w h e r e d r u g r e s i s t a n c e o c c u r r e d in c e l l s g r o w n a s t u m o u r s in a n i m a l s or f o r m e d into s p h e r o i d s , but c o u l d not b e instigated in the s a m e c e l l s g r o w n a s m o n o l a y e r s in t i s s u e c u l t u r e  3 , 4 , 5  .  T h e r e f o r e , t h e s e r e s u l t s a r e not  o n l y indicative of the i m p o r t a n c e of e x t r a c e l l u l a r f a c t o r s , i n d r u g r e s i s t a n c e , but t h e y e m p h a s i z e the i m p o r t a n c e of i n c l u d i n g a d v a n c e d  in vivo  m o d e l s in s t u d y i n g d r u g r e s i s t a n c e a s t h e s e s y s t e m s r e p r e s e n t  a m o r e a c c u r a t e e n v i r o n m e n t u n d e r w h i c h this c o n d i t i o n m a y d e v e l o p . W h i l e t u m o u r m i c r o e n v i r o n m e n t is significant in the c o n t e x t of d r u g r e s i s t a n c e , there a r e yet m o r e f a c t o r s w h i c h m u s t b e t a k e n into a c c o u n t . C e r t a i n l y the i m m u n e s y s t e m p l a y s a role vis a vis the i m m u n e s t a t u s of the host. L i k e w i s e , the l o c a t i o n of the t u m o u r is a l s o important if it is p r e s e n t in i m m u n o l o g i c a l l y p r i v i l e g e d s i t e s s u c h a s the c e n t r a l n e r v o u s s y s t e m , the e y e s , or the t e s t e s . A d d i t i o n a l l y , p h a r m a c o l o g i c a l aspects must also be considered.  R e s i s t a n c e to s p e c i f i c d r u g s m a y b e r e l a t e d to t h e  particular  p h a r m a c o l o g y of a d r u g ( u p t a k e , m e t a b o l i s m , etc.) a s w e l l a s the t o l e r a n c e of n o r m a l c e l l s to the cytotoxicity of this d r u g . F u r t h e r m o r e , this r e s i s t a n c e m a y b e i n c u r r e d t o w a r d s a particular d r u g or to the w h o l e c l a s s to w h i c h the d r u g b e l o n g s . E s s e n t i a l l y , d r u g r e s i s t a n c e in c a n c e r o c c u r s a s the result of the c o m b i n a t i o n of a v a r y i n g set of f a c t o r s . A t the level of the c e l l , g e n e t i c instability is a k e y f e a t u r e in the d e v e l o p m e n t of d r u g r e s i s t a n c e ; at the level of the n e o p l a s m , t u m o u r m i c r o e n v i r o n m e n t h a s a role in p r o d u c i n g this c o n d i t i o n ; at the level of the w h o l e o r g a n i s m , i m m u n o l o g i c a l a n d p h a r m a c o l o g i c a l f a c t o r s a r e important.  A l t h o u g h all of t h e s e  e l e m e n t s a r e significant, the i n c i d e n c e of d r u g r e s i s t a n c e in c a n c e r c a n n o t s i m p l y b e r e d u c e d to this d i s c r e e t s e t of c o m p o n e n t s ; invariably, the d r u g resistant state is m u c h m o r e c o m p l e x t h a n this.  1.1.2  CLINICAL IMPLICATIONS OF DRUG RESISTANCE  T h e i m p o r t a n c e of d r u g r e s i s t a n c e in the t r e a t m e n t of c a n c e r c a n n o t b e u n d e r s t a t e d . many  malignancies are  inherently  resistant  r e s i s t a n c e d u r i n g the c o u r s e of t r e a t m e n t . 6  to  chemotherapeutic  agents  and  others  will  Indeed, develop  F u r t h e r m o r e , the c l i n i c a l r e l e v a n c e of d r u g r e s i s t a n c e is e v e n  m o r e a p p a r e n t w h e n o n e c o n s i d e r s that, other t h a n in s o m e p e d i a t r i c t u m o r s a n d l y m p h o m a s , c a n c e r is often i n c u r a b l e o n c e it h a s p r o g r e s s e d b e y o n d the point of l o c a l treatment.  T a b l e 1 illustrates the fact that  in m a n y f o r m s o f c a n c e r , c h e m o t h e r a p y p r o v i d e s little o r n o s u r v i v a l b e n e f i t in m e t a s t a t i c d i s e a s e .  Much  of the i n e f f e c t i v e n e s s of c h e m o t h e r a p y in this s t a g e of d i s e a s e m a y b e r e l a t e d to d r u g r e s i s t a n c e .  Table 1.1  Outcomes of Therapy in Advanced Disease . 7  Not Curable - Improved Survival  Curable  > > > > >-  > > > > > >  Choriocarcinoma Acute lymphocytic leukemia in children Hodgkin's Disease Diffuse histiocytic lymphoma Nodular mixed lymphoma Testicular cancer Ovarian cancer Acute myelogenous leukemia Wilms' Tumor Embryonal Rhabdomyosarcoma Burkitt's lymphoma  > > > > > > > > > > > > > > > > >  Breast Carcinoma Prostate Cancer Chronic Myelogenous Leukemia Chronic Lymphocytic Leukemia Small Cell Carcinoma of the Lung Soft Tissues sarcomas Gastric Carcinoma Malignant Insulinoma Endometrial Carcinoma Adrenal Cortical Carcinoma Medulloblastoma Neuroblastoma Polycythemia Vera Glioblastoma Bladder Carcinoma Osteosarcoma Ewing Sarcoma  T h e information p r e s e n t e d in T a b l e 1.1 early s t a g e s and localized.  Not Curable - Minimal or No Improved Survival  > > > > > > > > > >  Non Small Cell Lung Cancer Carcinoma of the Cervix Colon Carcinoma Malignant Melanoma Head and Neck Carcinoma Renal Cell Carcinoma Malignant Carcinoid tumors Thyroid Carcinoma Rectal Carcinoma Hepatocellular Carcinoma  s t r e s s e s the i m p o r t a n c e of treating c a n c e r w h i l e it is in  W h i l e this c o n c e p t is a p p a r e n t f r o m e m p i r i c a l c l i n i c a l o b s e r v a t i o n s , it is  s u p p o r t e d b y theoretical m o d e l s a s w e l l . W h e n o n e c o n s i d e r s that s p o n t a n e o u s m u t a t i o n is r e s p o n s i b l e for the initial f o r m a t i o n of d r u g r e s i s t a n c e within t u m o u r s , t h e n the n u m b e r of d r u g r e s i s t a n t c e l l s will d e p e n d o n both this rate of mutation a s w e l l a s the o v e r a l l t u m o u r s i z e (in t e r m s of cell n u m b e r ) . A s s u m i n g the mutation rate r e m a i n s c o n s t a n t within a n u n t r e a t e d t u m o u r , o n e might t h e n s u r m i s e that the n u m b e r of resistant c a n c e r c e l l s w o u l d i n c r e a s e in a linear relation to the t u m o u r s i z e . not the c a s e .  T h i s , h o w e v e r , is  5 T h e G o l d i e - C o l d m a n h y p o t h e s i s d e f i n e s the probability of the p r e s e n c e of d r u g r e s i s t a n c e c e l l s b a s e d u p o n mutation rate a n d t u m o u r s i z e . T h e following f o r m u l a d e s c r i b e s this r e l a t i o n s h i p : 8  Po = e" a(N  1)  Where: > P is the probability of finding no drug resistance cells > a is the mutation rate > N is the cell number 0  A s a n e x a m p l e , a c c o r d i n g to this f o r m u l a a t u m o u r c o n t a i n i n g a h u n d r e d t h o u s a n d c e l l s a n d a mutation rate of o n e in a million w o u l d h a v e a 1 0 % c h a n c e of c o n t a i n i n g r e s i s t a n t c e l l s .  Surprisingly  h o w e v e r , a n i n c r e a s e in t u m o u r s i z e by only 1.5 l o g s (5 X 1 0 c e l l s ) w o u l d result in a 9 9 % c h a n c e of the 7  e x i s t e n c e of d r u g resistant c e l l s . T h u s , there a p p e a r s to b e a n a r r o w , critical s t a g e in the growth of the t u m o u r in w h i c h d r u g r e s i s t a n c e m a y e x p a n d a n d amplify.  A l t h o u g h t h e a b o v e is r e p r e s e n t a t i v e of s t e m  c e l l s in a t u m o r , this result is n o n e t h e l e s s significant s i n c e m o s t c l i n i c a l l y d e t e c t a b l e t u m o u r s u s u a l l y c o n t a i n a r o u n d 1 0 c e l l s , a n d therefore w o u l d a l r e a d y b e r e p l e t e with d r u g r e s i s t a n t c e l l s . 9  A n a d d i t i o n a l c o m p l i c a t i o n c o m p o u n d i n g t h e p r o b l e m of d r u g r e s i s t a n c e is that c h e m o t h e r a p y d r u g s m a y a c t u a l l y g e n e r a t e d r u g r e s i s t a n c e o n their o w n ; that is, t h r o u g h their u s e , c h e m o t h e r a p y a g e n t s produce r e s i s t a n c e to t h e m s e l v e s . T h i s o c c u r s b e c a u s e m a n y c h e m o t h e r a p e u t i c d r u g s d a m a g e D N A ; i n d e e d , this is p r e c i s e l y w h y t h e y a r e u s e d a n d is the m a n n e r in w h i c h t h e y kill c e l l s .  However,  d a m a g e d D N A , in a d d i t i o n to killing s o m e c e l l s , m a y a l s o g e n e r a t e m u t a t i o n s in o t h e r s - m u t a t i o n s that m a y result in the cell e v a d i n g the c y t o t o x i c a c t i o n of the d r u g , or in other w o r d s , d e v e l o p i n g r e s i s t a n c e to it.  C o n t i n u e d u s e of the d r u g then g e n e r a t e s s e l e c t i v e p r e s s u r e for this c l o n e a n d s p a w n s w i d e s p r e a d  r e s i s t a n c e within the t u m o u r m a s s .  1.1.3  MECHANISMS OF DRUG RESISTANCE  In c o n s i d e r i n g d r u g r e s i s t a n c e thus far, the c a u s e s a n d contributing f a c t o r s of its d e v e l o p m e n t h a v e b e e n e v a l u a t e d a s w e l l a s the c l i n i c a l i m p l i c a t i o n s of this p h e n o m e n o n .  In this next s e c t i o n , t h e  6 m a n y f o r m s of d r u g r e s i s t a n c e a n d t h e m e c h a n i s m s b y w h i c h it t a k e s p l a c e a r e d i s c u s s e d .  T a b l e 1.2  outlines nine b a s i c m e c h a n i s m s of drug r e s i s t a n c e .  Table 1.2 Mechanisms of Drug Resistance Resistance Mechanism  9  Decreased uptake Increased efflux Decrease in drug activation Increase in drug catabolism Increase in the level of target enzyme Alterations in target enzyme Inactivation by binding of sulfahydryls (e.g. glutathione) Increased D N A repair Decreased ability to undergo apoptosis  Drug Methotrexate, nitrogen mustard, melphalan, cisplatin Anthracyclines, vinca alkaloids, etoposide, taxanes Antimetabolites Antimetabolites Methotrexate, topoisomerase inhibitors Methotrexate, other antimetabolites, topoisomerase inhibitors Alkylating agents, cisplatin, anthracyclines Alkylating agents, cisplatin, anthracyclines, etoposide Alkylating agents, cisplatin, anthracyclines, etoposide  O n e m e c h a n i s m of drug r e s i s t a n c e that is e x t r e m e l y important, is d r u g r e s i s t a n c e that o c c u r s w h e n c e l l s a r e i m p e r v i o u s to t h e p r o c e s s o f a p o p t o s i s . A p o p t o s i s is a p r o g r a m m e d a n d r e g u l a t e d s e r i e s of cellular e v e n t s that results in t h e d e s t r u c t i o n of t h e c e l l , a n d it is t h e p r i m a r y m e c h a n i s m that t h e b o d y u s e s to d e s t r o y d a m a g e d or old c e l l s . T h e role of a p o p t o s i s is significant in c a n c e r , not o n l y b e c a u s e m i s regulation of this p r o c e s s m a y p l a y a role in t h e d e v e l o p m e n t of u n c o n t r o l l e d cell g r o w t h , but b e c a u s e t h e c h e m o t h e r a p e u t i c a g e n t s u s e d to treat c a n c e r a r e thought to i n d u c e a p o p t o s i s in t u m o u r  cells  1 0 , 1 1 , 1 2  .  H e n c e , a n inability to i n d u c e a p o p t o s i s w o u l d result in t h e d e c r e a s e d e f f i c a c y of t h e s e d r u g s a n d t h e d e v e l o p m e n t of r e s i s t a n c e . A s a regulator of a p o p t o s i s , Bcl-2 h a s b e e n i m p l i c a t e d in t h e d e v e l o p m e n t of d r u g r e s i s t a n c e . T h i s protein is f o u n d primarily in the m i t o c h o n d r i a a n d is a d o w n s t r e a m regulator of a p o p t o s i s a n d m a y protect the cell f r o m b e i n g triggered into a p o p t o s i s b y s i g n a l s f r o m p53. C e l l s e x p r e s s i n g high l e v e l s of B c l - 2 m a y b e inordinately resistant to induction into a p o p t o s i s w h e n t r i g g e r e d to d o s o through c e l l u l a r d a m a g e from chemotherapy agents.  T h e o u t c o m e of e n h a n c e d B c l - 2 l e v e l s a n d t h e s u b s e q u e n t  s u p p r e s s i o n of a p o p t o s i s w o u l d b e t h e c l o n a l e x p a n s i o n of a d e f e c t i v e c l o n e into a p o p u l a t i o n of c e l l s that c o n t a i n s significantly d a m a g e d D N A a n d is a l s o r e s i s t a n t to a p o p t o s i s .  T h e ultimate result o f this, not  surprisingly, is a p e r s i s t e n t a n d powerful r e s i s t a n c e to c h e m o t h e r a p e u t i c a g e n t s .  7  1.1.4 1  REFERENCES  G o l d i e , J . H . , a n d C o l d m a n , A . J . D r u g R e s i s t a n c e in C a n c e r . C a m b r i d g e U n i v e r s i t y P r e s s 1 9 9 8 . p. 1 2 .  L o e b L A . M u t a t o r p h e n o t y p e m a y b e r e q u i r e d for m u l t i s t a g e c a r c i n o g e n e s i s . Cancer Res 1991 J u n 1 5 ; 51(12): 3 0 7 5 - 9 .  2  T e i c h e r B A , H e r m a n T S , H o l d e n S A , W a n g Y Y , Pfeffer M R , C r a w f o r d J W , F r e i E . T u m o r r e s i s t a n c e to alkylating a g e n t s c o n f e r r e d b y m e c h a n i s m s o p e r a t i v e only in v i v o . Science. 1 9 9 0 M a r 2 3 ; 2 4 7 ( 4 9 4 9 P t 1):1457-61  3  K o b a y a s h i H, M a n S , G r a h a m C H , Kapitain S J , Teicher B A , Kerbel R S . A c q u i r e d multicellular-mediated r e s i s t a n c e to a l k y l a t i n g a g e n t s in c a n c e r . Proc Natl Acad Sci. 1 9 9 3 A p r 1 5 ; 9 0 ( 8 ) : 3 2 9 4 - 8 .  4  G r e e n S K , F r a n k e l A , K e r b e l R S . A d h e s i o n - d e p e n d e n t multicellular d r u g r e s i s t a n c e . Anticancer Des. 1 9 9 9 A p r ; 1 4 ( 2 ) : 1 5 3 - 6 8 .  5  P i n e d o , H e r b e r t M . , a n d G i a c c o n e , G u i s e p p e . 1 9 9 8 . Drug Resistance C a m b r i d g e : C a m b r i d g e U n i v e r s i t y P r e s s , p. 1 9 9 .  6  7  Drug  in the Treatment of Cancer.  P i n e d o , H e r b e r t M . , a n d G i a c c o n e , G u i s e p p e . 1 9 9 8 . p. 2 0 0  G o l d i e J H , C o l d m a n A J . A m a t h e m a t i c m o d e l f o r relating t h e d r u g s e n s i t i v i t y o f t u m o r s to their s p o n t a n e o u s mutation rate. C a n c e r T r e a t R e p . 1 9 7 9 N o v - D e c ; 6 3 ( 1 1 - 1 2 ) : 1 7 2 7 - 3 3 .  8  T a n n o c k , l a n F., a n d Hill, R i c h a r d P . 1 9 9 8 . The Scientific Basis of Oncology. p. 3 9 7 .  9  Toronto: McGraw-Hill,  S e a r l e J , L a w s o n T A , A b b o t t P J , H a r m o n B , K e r r J F . A n e l e c t r o n - m i c r o s c o p e s t u d y of t h e m o d e of cell d e a t h i n d u c e d b y c a n c e r - c h e m o t h e r a p e u t i c a g e n t s in p o p u l a t i o n s of proliferating n o r m a l a n d n e o p l a s t i c c e l l s . J Pathol. 1 9 7 5 J u l ; 1 1 6 ( 3 ) : 1 2 9 - 3 8 . 1 0  A n i l k u m a r T V , S a r r a f C E , H u n t T , A l i s o n M R . T h e nature of c y t o t o x i c d r u g - i n d u c e d c e l l d e a t h in m u r i n e intestinal c r y p t s . BrJ Cancer. 1 9 9 2 A p r ; 6 5 ( 4 ) : 5 5 2 - 8 . 1 1  K a u f m a n n S H , E a m s h a w W C . Induction of a p o p t o s i s b y c a n c e r c h e m o t h e r a p y . Exp Cell Res. 2 0 0 0 A p r 1 0 ; 2 5 6 ( 1 ) : 4 2 - 9 1 2  8  1.2 APOPTOSIS T h e p r o c e s s of a p o p t o s i s is a n important a r e a of c a n c e r r e s e a r c h , a n d s t u d i e s c o n c e r n i n g this topic h a v e p r o g r e s s e d f r o m a f e w p u b l i c a t i o n s in the e a r l y 1 9 7 0 s to t h o u s a n d s of p a p e r s p e r y e a r t o d a y . W h i l e the g r o w t h of c e l l s h a s a l w a y s b e e n r e c o g n i z e d a s the p r i n c i p l e e l e m e n t in o n c o g e n e s i s , with e x p a n d e d i n v e s t i g a t i o n s into a p o p t o s i s , the s i g n i f i c a n c e of cell d e a t h in the d e v e l o p m e n t of c a n c e r is finally b e i n g r e a l i z e d . In addition to its s i g n i f i c a n c e in o n c o l o g y , a p o p t o s i s is p r o v i n g to b e v e r y important in a w i d e r a n g e of m e d i c a l d i s c i p l i n e s , a n d further d i s c l o s u r e of the m e c h a n i s m s that control this p r o c e s s will i n v a r i a b l y result in t h e r a p e u t i c o p p o r t u n i t i e s .  1.2.1 T W O M O D E S O F C E L L D E A T H : A P O P T O S I S A N D N E C R O S I S  A l t h o u g h the c o n c e p t of p r o g r a m m e d cell d e a t h h a s b e e n p r e s e n t s i n c e the n i n e t e e n t h century, it was  only thirty y e a r s a g o that this p h e n o m e n o n w a s a d e q u a t e l y d e s c r i b e d a n d a s s i g n e d a  apoptosis\  S i n c e that t i m e this p r o c e s s h a s g a r n e r e d m u c h attention, e s p e c i a l l y within t h e p a s t d e c a d e  w h e n m a n y of the p r o c e s s e s u n d e r l y i n g it w e r e e l u c i d a t e d . remove  name:  aged, damaged  or  infected  cells from  the  body,  A p o p t o s i s is a n innate p r o c e s s u s e d to and  it is n e c e s s a r y to  maintain  tissue  h o m e o s t a s i s . F u r t h e r m o r e , a s the natural m e t h o d of deleting s u p e r f l u o u s c e l l s , it is a n important e l e m e n t in the f o r m a t i o n of s t r u c t u r e s during the d e v e l o p m e n t a l p r o c e s s a s w e l l .  For these very s a m e reasons,  h o w e v e r , a b n o r m a l i t i e s in its regulation m a y c a u s e d e l e t e r i o u s e f f e c t s . P r i o r to the e s t a b l i s h m e n t of a p o p t o s i s a s a b o n a - f i d e m e c h a n i s m of cell d e a t h , n e c r o s i s w a s the r e c o g n i z e d a n d e s t a b l i s h e d m o d e o f c e l l u l a r d e a t h within the b o d y . A s m o r e i n f o r m a t i o n w a s u n c o v e r e d c o n c e r n i n g a p o p t o s i s a n d n e c r o s i s , a s e r i e s of h a l l m a r k e x c l u s i v e l y a s s o c i a t e d with either m e c h a n i s m .  p r o c e s s e s e m e r g e d w h i c h s e e m e d to  T o d a y , a p o p t o s i s a n d n e c r o s i s a r e s e e n a s the  be two  p r i m a r y m o d e s of cell d e a t h , a n d a l t h o u g h a large g r e y a r e a e x i s t s b e t w e e n the t w o , e a c h e m b o d i e s a distinct set of cell b e h a v i o r s that d e l i n e a t e t h e s e p r o c e s s e s f r o m o n e a n o t h e r  (Table 1.3).  N e c r o s i s is a n i n c i d e n t a l a n d p a s s i v e form of cell d e a t h that i n v o l v e s the c a t a s t r o p h i c failure of cellular h o m e o s t a s i s , a n d it m a y result f r o m s o u r c e s s u c h a s the d e p l e t i o n of nutrients e x p o s u r e to toxins or c y t o t o x i c c h e m i c a l s , or m e c h a n i c a l injury.  or o x y g e n ,  T h e s e influences generally c a u s e a  p r o g r e s s i v e d e g r a d a t i o n in e n z y m e s , a l o s s of o s m o t i c a n d m e m b r a n e integrity, a n d e v e n t u a l rupture or  9 bursting  of the  cell.  Release  of cellular c o n t e n t s  may damage  neighboring  cells and  incite  an  inflammatory r e s p o n s e , a c o m m o n occurrence w h e n widespread necrosis o c c u r s . 2  In c o n t r a s t to n e c r o s i s , the e v e n t s l e a d i n g to c e l l u l a r d e a t h v i a a p o p t o s i s a r e both p r o g r a m m e d and regulated.  It m a y b e d i s t i n g u i s h e d from n e c r o s i s in that it r e q u i r e s e n e r g y , i n v o l v e s a s e r i e s of  e x c l u s i v e e v e n t s , a n d incites the p r o d u c t i o n of novej proteins. S e v e r a l f e a t u r e s c h a r a c t e r i z e cell d e a t h v i a a p o p t o s i s : 1) l o s s of p l a s m a m e m b r a n e a s y m m e t r y ; 2) c o n d e n s a t i o n of the c y t o p l a s m a n d n u c l e u s ; 3) c l e a v a g e of D N A ; a n d , 4) the b l e b b i n g of the p l a s m a m e m b r a n e a n d the f o r m a t i o n of a p o p t o t i c b o d i e s . T h e s e a p o p t o t i c b o d i e s s e q u e s t e r c e l l u l a r c o n t e n t s a n d a r e r e c o g n i z e d a n d e n g u l f e d b y m a c r o p h a g e s . In this w a y , c e l l u l a r p r o t e i n s , a n d s p e c i f i c a l l y p r o t e a s e s , a r e c o n t a i n e d a n d a n i m m u n e r e s p o n s e is a v o i d e d . 3  Table 1.3 Features of Apoptosis and Necrosis Necrosis Apoptosis 4  passive process cellular s w e l l i n g m e m b r a n e rupture inflammation  active p r o c e s s cellular s h r i n k a g e intact m e m b r a n e no i n f l a m m a t i o n  W h e n c o n s i d e r i n g the m i c r o e n v i r o n m e n t within a s o l i d t u m o r , n e c r o s i s a n d a p o p t o s i s a r e both p r e v a l e n t , with the d e g r e e a n d location of e a c h d e p e n d i n g o n m a n y f a c t o r s .  A n d w h i l e the p r e s e n c e of  n e c r o s i s certainly m a y affect the e f f i c a c y of c h e m o t h e r a p y d r u g s o r r a d i a t i o n u s e d to treat a tumor, a p o p t o s i s is e s p e c i a l l y significant in this r e g a r d a s t h e s e t h e r a p i e s kill c e l l s b y triggering the a p o p t o t i c cascade . 5  1.2.2  SOURCES AND MECHANISMS  OF APOPTOSIS  A p o p t o s i s is a n effective a n d c o m p r e h e n s i v e m e a n s of d e s t r o y i n g c e l l s , but it is not a r a n d o m p r o c e s s — it r e q u i r e s a s t i m u l u s , either internal o r e x t e r n a l , in o r d e r to b e i n s t i g a t e d . may  Numerous sources  trigger a p o p t o s i s , including h y p o x i a , h o r m o n e a c t i o n , l o s s of cell a d h e s i o n , activation b y i m m u n e  c e l l s , a s well a s c y t o t o x i c a g e n t s a n d radiation  (Figure 1.1).  O n c e the p r o c e s s of a p o p t o s i s h a s b e e n  initiated by o n e of t h e s e triggers, a s e t of distinct c e l l u l a r e v e n t s will inevitably unfold within the c e l l . T h e s e e v e n t s m a y b e d i v i d e d into t h o s e that o c c u r relatively e a r l y in a p o p t o s i s , a n d t h o s e that o c c u r later.  10 O n e of the earliest a p o p t o t i c e v e n t s is the l o s s of the m i t o c h o n d r i a l m e m b r a n e potential (Ay/). T h i s is f o l l o w e d b y the r e l e a s e of C a  2 +  a n d a p o p t o t i c p r o t e i n s f r o m the m i t o c h o n d r i a into the c y s t o l ,  i n c l u d i n g c y t o c h r o m e c, A p o p t o s i s Inducing F a c t o r (AIF), a n d A p o p t o s i s P r o t e a s e A c t i v a t i n g F a c t o r - 1 (APAF-1).  A d d i t i o n a l l y , the l o s s of p l a s m a m e m b r a n e a s y m m e t r y o c c u r s e a r l y in a p o p t o s i s .  This  h a p p e n s w h e n p h o s p h a t i d y l s e r i n e r e s i d u e s n o r m a l l y f o u n d o n l y o n the i n n e r leaflet of the m e m b r a n e a r e e x t e r n a l i z e d a n d b o u n d by the protein A n n e x i n V .  T h e s e e a r l y e v e n t s l e a d to a n a m p l i f i c a t i o n of the  a p o p t o t i c s i g n a l and the start of m u c h larger a n d w i d e - r e a c h i n g a p o p t o t i c p r o c e s s e s within the c e l l . s t a g e s of a p o p t o s i s b e g i n with the activation of c a s p a s e s , a s e r i e s of p r o t e a s e s within the c e l l .  Later These  p r o t e i n s affect a w i d e r a n g e of targets within the c e l l , i n c l u d i n g structural p r o t e i n s , D N A s y n t h e s i s a n d repair p r o t e i n s , p r o t e i n s i n v o l v e d in c e l l s i g n a l i n g a n d proliferation, a n d n u c l e a r p r o t e i n s . E v e n t u a l l y , D N A is c l e a v e d into s m a l l f r a g m e n t s a p p r o x i m a t e l y 200 b a s e p a i r s in s i z e a n d the p l a s m a m e m b r a n e b e g i n s to bleb and contort o u t w a r d .  T h e terminal p h a s e of a p o p t o s i s i n v o l v e s the f o r m a t i o n of a p o p t o t i c b o d i e s  f r o m the o n c e intact c e l l , w h i c h a r e then e n g u l f e d b y m a c r o p h a g e s . T h i s d e s c r i p t i o n of a p o p t o s i s is s o m e w h a t s i m p l i f i e d , but n o n e t h e l e s s e n c o m p a s s e s the salient e v e n t s of this p r o c e s s . In reality, h o w e v e r , a p o p t o s i s is a c o m p l e x a n d h i g h l y r e g u l a t e d p h e n o m e n o n a n d the p r e s e n c e of o n e o r m o r e a p o p t o t i c triggers d o e s not n e c e s s a r i l y e n s u r e that a p o p t o s i s will c o m e to fruition.  I n d e e d , the i n f l u e n c e of a n a p o p t o t i c trigger m a y not b e sufficient to tip the c e l l into a p o p t o s i s , or,  if the a p o p t o t i c m a c h i n e r y is d e f e c t i v e in s o m e w a y , the s i g n a l for c e l l d e a t h s i g n a l m a y b e e r r o n e o u s l y disregarded. T h e r e g u l a t i o n of t h e s e a p o p t o t i c e v e n t s is quite c o m p l e x , a n d their e x a c t s e q u e n c e m a y differ f r o m o n e a p o p t o t i c trigger to a n o t h e r . F o r e x a m p l e , a p o p t o s i s i n d u c e d b y d e a t h r e c e p t o r s s u c h a s F a s or Tumor  Necrosis Factor - a  ( T N F - a ) involve the activation of c a s p a s e s v e r y e a r l y o n a n d d o  n e c e s s a r i l y i n v o l v e d disruption of the m i t o c h o n d r i a l m e m b r a n e potential o r the r e l e a s e of f a c t o r s f r o m the m i t o c h o n d r i a .  not  apoptotic  T h i s is in c o n t r a s t to a p o p t o s i s i n s t i g a t e d b y d a m a g e to D N A , w h e r e  a p o p t o s i s is r e l a y e d v i a the p53 protein a n d i n v o l v e s a great d e g r e e of r e g u l a t i o n at the m i t o c h o n d r i a .  11  12  W h i l e the p a t h w a y s utilized within the c e l l to initiate a n d u n d e r t a k e a p o p t o s i s in r e s p o n s e to v a r i o u s a p o p t o t i c triggers a r e s l o w l y b e i n g r e v e a l e d , the o n e that is m o s t s i g n i f i c a n t in t h e c o n t e x t o f d r u g r e s i s t a n c e is the p 5 3 - m e d i a t e d a p o p t o t i c p a t h w a y .  p 5 3 is a t u m o r s u p p r e s s o r that m o n i t o r s the integrity  of g e n o m e a n d m a y i n d u c e a p o p t o s i s in r e s p o n s e to w i d e s p r e a d d a m a g e to D N A .  T h u s , it is t h r o u g h  this a r m of the a p o p t o t i c m a c h i n e r y that the e f f i c a c y of c h e m o t h e r a p e u t i c d r u g s a n d radiation is reliant s i n c e m a n y of t h e s e t r e a t m e n t s function b y incurring d a m a g e to the g e n o m e .  A n d although apoptosis  m a y b e m e d i a t e d by other a p o p t o t i c triggers s u c h a s h o r m o n e s or d e a t h r e c e p t o r s , it is b e y o n d the s c o p e of this t h e s i s to d i s c u s s t h e s e o t h e r w i s e f u n d a m e n t a l l y important s u b j e c t s .  1.2.3 P53 MEDIATED APOPTOSIS  Function of p53 p 5 3 is a n e x t r e m e l y important m o l e c u l e in c a n c e r a n d it inevitably s e e m s to b e i n v o l v e d in m a n y m e c h a n i s m s of o n c o g e n e s i s . p 5 3 w a s first d i s c o v e r e d a s a protein c o m p l e x e d with the l a r g e T - a n t i g e n of the S V 4 0 virus a n d it w a s later s h o w n to p r e v e n t t r a n s f o r m a t i o n of f i b r o b l a s t s . p 5 3 w a s a l s o s h o w n to b e 6  i n d u c e d u p o n t r e a t m e n t with radiation a n d it d i s p l a y e d a n ability to m o d u l a t e the c e l l c y c l e w h e n it w a s s h o w n to arrest c e l l s in p 5 3 , not m u t a n t s . 1 0  phase ' 7  8 , 9  ; significantly, this o n l y o c c u r r e d in c e l l s w h i c h c o n t a i n e d wild-type  p 5 3 is n o w r e c o g n i z e d a s a c l a s s i c t u m o r s u p p r e s s o r s i n c e l o s s of function o f  m u t a t e d f o r m s of this protein contributes to the t r a n s f o r m a t i o n of c e l l s  1 1 , 1 2  .  M o r e o v e r , its i m p o r t a n c e is  m a d e e v e n m o r e a p p a r e n t by the fact that it is m u t a t e d in o v e r fifty p e r c e n t of h u m a n t u m o r s . 1 3  S t u d i e s of p 5 3 a n d its role within the cell h a s r e v e a l e d that it r e g u l a t e s a w i d e variety of c e l l u l a r f u n c t i o n s : p 5 3 is intimately i n v o l v e d in c e l l c y c l e c h e c k p o i n t s a n d c a n i n d u c e g r o w t h a r r e s t at GJS;  it is  i n v o l v e d in D N A r e p a i r a n d m a i n t e n a n c e of g e n o m i c integrity;  and  it c o n t r i b u t e s  to the f o r m a t i o n  regulation of a n g i o g e n e s i s ; a n d , it is c a p a b l e of i n d u c i n g s e n e s c e n c e or a p o p t o s i s . 1 4  W h i l e p 5 3 m a y h a v e n u m e r o u s r o l e s in the d e v e l o p m e n t of o n c o g e n e s i s , o n e of the significant is its c o n t r o l of g e n o m i c integrity a n d r e s p o n s e to d a m a g e to D N A .  most  In this r e g a r d , p 5 3  g e n e r a l l y f o l l o w s o n e of two p a t h w a y s : it m a y arrest cell growth at the G , / S p h a s e of the c e l l c y c l e s u c h  13 that the D N A d a m a g e m a y b e r e p a i r e d , or, if the d e g r e e of d a m a g e is too great, it m a y i n d u c e the cell to undergo apoptosis. A s to w h e t h e r p 5 3 i n d u c e s growth arrest or a p o p t o s i s is d e p e n d e n t not o n l y o n t h e extent of cellular d a m a g e , but a l s o o n the state of other regulatory p r o t e i n s within t h e c e l l . F o r e x a m p l e , the s t a t u s of the r e t i n o b l a s t o m a protein ( R b ) m a y b e d e t e r m i n a t e in this r e s p e c t .  R b is a GJS  protein a n d inactivation of this g e n e r e l e a s e s the E 2 F - 1 t r a n s c r i p t i o n f a c t o r s .  c h e c k p o i n t control  p 5 3 i n d u c t i o n in c e l l s with  intact R b g e n e r a l l y l e a d s to growth arrest, w h i l e in c e l l s with m u t a t e d R b , it l e a d s to a p o p t o s i s . 1 5  f a c t o r s s u c h a s the p r e s e n c e of certain c y t o k i n e s or growth f a c t o r s , a s w e l l a s t h e t i s s u e type contribute to w h e t h e r the cell e n t e r s g r o w t h arrest or a p o p t o s i s  1 6 , 1 7  .  Other may  It is c l e a r f r o m s t u d i e s involving p 5 3  that this protein is e s p e c i a l l y s e n s i t i v e to d a m a g e d D N A a n d the state of the cell c y c l e , a n d p r o v i d e d that p 5 3 is intact, p e r t u r b a t i o n s to either of t h e s e e l e m e n t s c a u s e p 5 3 i n d u c t i o n . In addition to its c a p a b i l i t y of i n d u c i n g growth arrest, p 5 3 is a l s o the p r i m a r y arbiter of a p o p t o s i s in r e s p o n s e to d a m a g e to D N A , a s w e l l a s a n u m b e r of other triggers.  T h e c a p a c i t y of p 5 3 to i n d u c e  a p o p t o s i s w a s first s h o w n by Y a n o s h - R o u a c h et a l . , u s i n g a t e m p e r a t u r e s e n s i t i v e mutant in m y e l o i d l e u k e m i a cell l i n e s . A d d i t i o n a l s t u d i e s u s i n g p 5 3 k n o c k - o u t m i c e s h o w e d that t h y m o c y t e s f r o m p 5 3 null 1 8  m i c e w e r e r e s i s t a n t to r a d i a t i o n - i n d u c e d a p o p t o s i s , c o m p a r e d with p 5 3 +/+ m i c e . N o t a b l y h o w e v e r , p 5 3 /-  mice  retained  sensitivity  to  apoptosis  induced  by  glucocorticoids  and  T-cell-receptor  mediated  19 20  a p o p t o s i s , indicating the d i v e r g e n t c e l l u l a r p a t h w a y s u s e d b y t h e s e a p o p t o t i c t r i g g e r s  ' .  L i k e t h o s e treated with radiation, the r e s p o n s e of p 5 3 null m u t a n t s to c h e m o t h e r a p y p r o v i d e d a similar outcome.  A b s e n c e of f u n c t i o n a l p 5 3 incurred r e s i s t a n c e to c h e m o t h e r a p e u t i c a g e n t s a n d t u m o r s  f r o m p 5 3 +/+ c e l l s that b e c a m e resistant to c h e m o t h e r a p y w e r e f o u n d to h a v e a c q u i r e d p 5 3 m u t a t i o n s d u r i n g t u m o r p r o g r e s s i o n . M o s t n o t a b l e , h o w e v e r , w a s t h e fact that t h e t r a n s f e c t i o n o f p 5 3 into p 5 3 null c e l l s r e s t o r e d sensitivity to c h e m o t h e r a p y d r u g s . 2 1  p 5 3 is c l e a r l y c a p a b l e of i n d u c i n g a p o p t o s i s p r o v i d e d the c e l l u l a r c o n d i t i o n s a r e permitting.  And  although elements s u c h as ribonucleotide depletion, hypoxia, and oxidative stress m a y induce apoptosis v i a the p 5 3 p a t h w a y , the m o s t significant e v e n t in the c o n t e x t of c h e m o t h e r a p y d r u g s a n d radiation is d a m a g e to D N A . D a m a g e to D N A c a n o c c u r a s a result of U V a n d g a m m a r a d i a t i o n , a s w e l l a s e x p o s u r e to c h e m o t h e r a p y d r u g s .  T h i s i n c l u d e s t o p o i s o m e r a s e inhibitors s u c h a s e t o p o s i d e a n d c a m p t o t h e c i n s ,  alkylating a g e n t s s u c h a s m i t o m y c i n C , D N A intercalating a g e n t s s u c h a s a c t i n o m y c i n D a n d d o x o r u b i c i n , and even antimetabolites s u c h as methotrexate.  A s to h o w p 5 3 d e t e c t s this d a m a g e to D N A is not  14 a l t o g e t h e r c l e a r ; h o w e v e r , it m a y identify D N A d a m a g e directly, o r t h r o u g h a n u m b e r o f i n t e r m e d i a r i e s , s u c h a s A T M , R e f - 1 , T F l l - H , or M L H - 1 . 2 2  E a c h of t h e s e i n t e r m e d i a r i e s is s e n s i t i v e to different t y p e s of  D N A l e s i o n s , a n d t h u s m a y b e a c t i v a t e d in r e s p o n s e to t r e a t m e n t with a s p e c i f i c a g e n t or d r u g .  p53  a n d the  Bcl-2 F a m i l y of  Proteins  O n c e s u b s t a n t i a l g e n o m i c d a m a g e h a s b e e n s u s t a i n e d , a n d t h e b a l a n c e t i p p e d in f a v o r of a p o p t o s i s , p 5 3 will r e g u l a t e a n u m b e r of d o w n s t r e a m proteins to i n f l u e n c e p r o c e s s e s in the c e l l .  These  i n c l u d e G a d d 4 5 to inhibit D N A s y n t h e s i s , p21 to inhibit c y c l i n - d e p e n d e n t k i n a s e s a n d i n d u c e g r o w t h arrest, a n d finally a s s o c i a t i o n with the B a x p r o t e i n , w h i c h p r o v i d e s a link to the B c l - 2 f a m i l y of proteins, a n important family of a p o p t o t i c r e g u l a t o r s l o c a t e d primarily in the m i t o c h o n d r i a . T h e i m p o r t a n c e of B a x in p 5 3 m e d i a t e d a p o p t o s i s w a s first e s t a b l i s h e d by Y i n et a l . u s i n g L T A g m u t a n t T 1 2 1 t r a n s g e n i c m i c e that c o n t a i n e d l a r g e n u m b e r s of t u m o r s within the c h o r o i d p l e x u s ; m e d i a t e d a p o p t o s i s w a s d r a s t i c a l l y r e d u c e d w h e n t h e s e m i c e w e r e c r o s s e d with b a x -/- m i c e . 2 3  p53  Further  s t u d i e s i n d i c a t e d s i m i l a r results w h e n b a x - / - f i b r o b l a s t s s h o w e d that p 5 3 - m e d i a t e d a p o p t o s i s is r e d u c e d in the  a b s e n c e of  apoptosis  2 4 , 2 5  Bax and  that  i n c r e a s e s in  Bax  levels  occur  in  cells  undergoing  p53-mediated  A l t h o u g h the r e l a t i o n s h i p b e t w e e n B a x a n d p 5 3 s e e m s a p p a r e n t in s o m e s t u d i e s , this  relationship m a y b e c e l l type s p e c i f i c , a s p 5 3 - m e d i a t e d a p o p t o s i s o c c u r r e d in s o m e s t u d i e s without u p r e g u l a t i o n in the a m o u n t s of B a x or in m o d e l s with n o n - f u n c t i o n a l B a x  2 6 , 2 7  .  I n d e e d , it a p p e a r s a s t h o u g h  p53 mediated apoptosis m a y occur via Bax-dependent and B a x independent pathways. W i t h the a c t i v a t i o n of the p 5 3 - m e d i a t e d p a t h w a y of a p o p t o s i s b y c h e m o t h e r a p y d r u g s or other s o u r c e s , the a p o p t o t i c s i g n a l is p a s s e d f r o m p 5 3 to the B c l - 2 f a m i l y of p r o t e i n s at the m i t o c h o n d r i a .  This  s e r i e s of proteins p r o v i d e s a carefully t u n e d level of regulation of a p o p t o s i s . P r i o r to this point, a p o p t o s i s is by no m e a n s i r r e v e r s i b l e , a n d p r o v i d e d c e l l u l a r d a m a g e c a n b e r e p a i r e d , the a p o p t o t i c s i g n a l m a y b e attenuated.  Ultimately, this is m o s t likely to o c c u r , at least in part, at t h e b e h e s t of the B c l - 2 f a m i l y of  p r o t e i n s , s i n c e e v e n t s d o w n s t r e a m of t h e s e p r o t e i n s g e n e r a l l y r e p r e s e n t a point of n o return in the p r o c e s s of p 5 3 - m e d i a t e d a p o p t o s i s .  1.2.4 BCL-2 FAMILY OF PROTEINS  15 W i t h the t r a n s f e r r e n c e of the a p o p t o t i c s i g n a l f r o m p 5 3 to the B c l - 2 f a m i l y of p r o t e i n s v i a B a x , the p r o c e s s p r o g r e s s e s to the m i t o c h o n d r i a .  It is in this o r g a n e l l e that the majority of the B c l - 2 family  m e m b e r s r e s i d e , a n d o n e of the m o s t important m e m b e r s of this f a m i l y is the B c l - 2 protein itself.  Bcl-2  ( B - c e l l l y m p h o m a 2) a n d w a s first d i s c o v e r e d in a t(14:18) c h r o m o s o m a l t r a n s l o c a t i o n in n o n - H o d g k i n ' s 28 29  lymphomas  '  T h i s t r a n s l o c a t i o n r e s u l t e d in the c o d i n g r e g i o n of the B c l - 2 g e n e b e i n g p l a c e d u n d e r the  control of the e n h a n c e r for the i m m u n o g l o b i n h e a v y c h a i n with a r e s u l t i n g o v e r e x p r e s s i o n of B c l - 2 protein. T h i s m i s - r e g u l a t i o n of B c l - 2 led to its identification a s a n o n c o g e n e a n d g e n e r a t e d m u c h interest in its role within the c e l l . E v e n t u a l l y , B c l - 2 w o u l d e m e r g e a s o n e of the c e n t r a l r e g u l a t o r s of a p o p t o s i s . L o c a t e d primarily the in outer m e m b r a n e of m i t o c h o n d r i a , but a l s o in e n d o p l a s m i c reticulum a n d n u c l e u s , B c l - 2 m a y o c c u r in both c y s t o l i c a n d m e m b r a n e b o u n d f o r m s . 3 0  B c l - 2 ' s anti-apoptotic properties  a r e f a r - r e a c h i n g , a n d it c a n inhibit a p o p t o s i s d e r i v e d f r o m a w i d e v a r i e t y of s o u r c e s , i n c l u d i n g d e p r i v a t i o n of c y t o k i n e s , h e a t s h o c k , c h e m o t h e r a p y a g e n t s a n d r a d i a t i o n , c a l c i u m i o n o p h o r e s , h y p o x i a , a n d l o s s of substrate a t t a c h m e n t  3 1 , 3 2 , 3 3  Interestingly, B c l - 2 h a s little effect o n F a s - m e d i a t e d a p o p t o s i s , s u p p o r t i n g t h e  p r e s u m p t i o n that the m i t o c h o n d r i a l h a v e little control in d e a t h - r e c e p t o r - m e d i a t e d a p o p t o s i s  3 4 ,  3 5  .  The  p r i m a r y f u n c t i o n s of B c l - 2 a n d it's f a m i l y m e m b e r s a r e the r e g u l a t i o n a n d m a i n t e n a n c e of m i t o c h o n d r i a l m e m b r a n e potential (Ay/)  a n d the s e q u e s t r a t i o n of a p o p t o t i c p r o t e i n s in the m i t o c h o n d r i a ,  c y t o c h r o m e c, A P A F - 1 , a n d A I F , that u p o n r e l e a s e , w o u l d l e a d to c a s p a s e a c t i v a t i o n . o n l y the first in a w h o l e f a m i l y of r e l a t e d a p o p t o t i c p r o t e i n s with s i m i l a r f u n c t i o n s .  including  Bcl-2 represents  T h e s e proteins s h a r e  h o m o l o g y with B c l - 2 a n d a r e d i v i d e d into t h o s e that o p p o s e a p o p t o s i s a n d t h o s e that p r o m o t e a p o p t o s i s . T h e s e two g r o u p s a r e referred to a s a n t i - a p o p t o t i c proteins a n d p r o - a p o p t o t i c p r o t e i n s , r e s p e c t i v e l y  (Table 1.4)  16  Table 1.4 Pro- and Anti-apoptotic Bcl-2 Family Members Pro-Apoptotic Reference Anti-apoptotic Proteins Proteins  A1  40 42 44  Boo  46  37 39 41 43 45 47  Bax Bak Bok Bik  36 38  Bcl-2 BCI-XL Bcl-w Mcl-1  Reference  s  Bid Bim  48 49  Blk Bad Bcl-X Hrk (harikari)  50 51  S  Bcl-2 Rheostat Theory W i t h the d i s c o v e r y of Bcl-2 a n d the s u b s e q u e n t identification of a n u m b e r of related f a m i l y m e m b e r s , the q u e s t i o n t h e n r e m a i n e d : H o w d o t h e s e p r o t e i n s f u n c t i o n to r e g u l a t e a p o p t o s i s ? In  1993,  O l t v a i a n d K o r s m e y e r p r o p o s e d the Bcl-2 R h e o s t a t T h e o r y in a n a t t e m p t to e x p l a i n the m e c h a n i s m b y w h i c h t h e s e p r o t e i n s arbitrate the a p o p t o t i c s i g n a l at the m i t o c h o n d r i a . 5 2  fact that m a n y Bcl-2  T h i s t h e o r y is b a s e d u p o n the  f a m i l y m e m b e r s a r e c a p a b l e of h e t e r o - a n d h o m o d i m e r i z a t i o n , a n d that  the  d i m e r i z a t i o n state of t h e s e p r o t e i n s w o u l d d e t e r m i n e w h e t h e r a p o p t o s i s is o p p o s e d or p r o p a g a t e d . A n u m b e r of p r o - a p o p t o t i c p r o t e i n s , a n d s p e c i f i c a l l y the B a x p r o t e i n , a r e c a p a b l e of p r o m u l g a t i n g the a p o p t o t i c s i g n a l w h e n b o u n d to o n e a n o t h e r in a h o m o d i m e r i z e d c o m p l e x .  The  anti-apoptotic  p r o t e i n s , s p e c i f i c a l l y Bcl-2 a n d B c l - X , m a y bind p r o - a p o p t o t i c p r o t e i n s , t h e r e b y p r e v e n t i n g t h e m f r o m L  forming homodimerized c o m p l e x e s .  In this w a y , the a n t i - a p o p t o t i c p r o t e i n s s e q u e s t e r the p r o - a p o p t o t i c  p r o t e i n s a n d p r e v e n t the t r a n s m i s s i o n of the a p o p t o t i c s i g n a l b e y o n d the m i t o c h o n d r i a . E f f e c t i v e l y t h e n , it  is the stoichiometric ratios of anti-apoptotic proteins to pro-apoptotic proteins that determines whether apoptosis is favored or opposed. If g r e a t e r a m o u n t s o f a n t i - a p o p t o t i c p r o t e i n s a r e p r e s e n t , then the bulk of the p r o - a p o p t o t i c p r o t e i n s will b e b o u n d in h e t e r o d i m e r i z e d c o m p l e x e s a n d the a p o p t o t i c s i g n a l will b e h a l t e d . If the o p p o s i t e is true, p r o - a p o p t o t i c  Bcl-2 Bcl-2 Survival  Anti- Pro (Aptoptotic)  4  Bcl-2 LBax] Anti- Pro (Aptoptotic)  Cell Death  1.2  4 t  Bax  Anti- Pro (Aptoptotic)  FIGURE Bcl-2 Rheostat Thoery. P r o g r e s s i o n to a p o p t o s i s is d e t e r m i n e d b y t h e relative ratios of p r o - a p o p t o t i c to a n t i - a p o p t o t i c Bcl-2 f a m i l y m e m b e r s at t h e m i t o c h o n d r i a . A n t i - a p o p t o t i c p r o t e i n s , by h e t e r o d i m e r i z i n g with p r o - a p o p t o t i c p r o t e i n s , a c t a s foils to p r e v e n t the f o r m a t i o n of p r o - a p o p t o t i c h o m o d i m e r s w h i c h w o u l d p r o m o t e a p o p t o s i s .  18 m e m b e r s will c o a l e s c e to d i s s e m i n a t e the a p o p t o t i c s i g n a l a n d s u b s e q u e n t a p o p t o t i c e v e n t s will o c c u r . S u c h is the b a s i s of the B c l - 2 R h e o s t a t T h e o r y . 5 3  G i v e n this e v i d e n c e in s u p p o r t of this m o d e l , it is t h e n u n d e r s t a n d a b l e h o w o v e r e x p r e s s i o n of B c l - 2 , a s o c c u r r e d in the l y m p h o m a s in w h i c h it w a s first d i s c o v e r e d , c o u l d c o n t r i b u t e t o w a r d s the state of d r u g r e s i s t a n c e in c a n c e r c e l l s .  Structure  of Bcl-2 Family Members  T h e structure of the B c l - 2 f a m i l y of proteins is intimately a s s o c i a t e d with their f u n c t i o n a s the ability of t h e s e p r o t e i n s to d i m e r i z e a n d r e g u l a t e a p o p t o s i s is d u e to a n u m b e r of h o m o l o g o u s r e g i o n s f o u n d within e a c h protein.  T h e s e proteins s h a r e h o m o l o g y by m e a n s of a s e r i e s of four B H ( B c l - 2  homology) domains: B H 1 , B H 2 , B H 3 , B H 4  5 4 , 5 5  .  T h e s e d o m a i n s s e r v e to m e d i a t e i n t e r a c t i o n s b e t w e e n  p r o t e i n s a n d in s o d o i n g r e g u l a t e p r o - a n d a n t i - a p o p t o t i c f u n c t i o n s .  I n d e e d , the p r o t e i n s B c l - X , B c l - X , L  s  B c l - w , B a k w e r e all first identified b y s e a r c h i n g for t h e s e r e g i o n s with the p r o t e i n s . T h e m o s t significant of the B H d o m a i n s is B H 3 , s i n c e all m a m m a l i a n B c l - 2 f a m i l y p r o t e i n s , with the e x c e p t i o n of A 1 , c o n t a i n this d o m a i n ; in fact, this is the h o m o l o g o u s r e g i o n that d e f i n e s m e m b e r s h i p to this family of p r o t e i n s . Bid. B a d , B c l - X  s  -  F u r t h e r m o r e , a n u m b e r of the p r o - a p o p t o t i c p r o t e i n s — i n c l u d i n g B i k , B i m , Hrk,  contain  only  r e q u i r e d for p r o - a p o p t o t i c a c t i v i t y . 56  the B H 3 d o m a i n , r e v e a l i n g that, in s o m e c a s e s , only this d o m a i n is T h e r e m a i n i n g p r o - a p o p t o t i c p r o t e i n s ( B a x , B a k , B o k ) , a s well a s all  of the a n t i - a p o p t o t i c p r o t e i n s , c o n t a i n the highly c o n s e r v e d B H 1 a n d B H 2 d o m a i n s .  In the c a s e of B c l - 2  a n d B c l - X L , t h e s e d o m a i n s a r e r e q u i r e d for t h e s e p r o t e i n s to retain their f u n c t i o n .  T h e final a n d l e a s t  57  c o n s e r v e d of the d o m a i n s , B H 4 , is f o u n d in both p r o - a n d a n t i - a p o p t o t i c p r o t e i n s a n d in s o m e p r o t e i n s , s u c h a s B c l - 2 , this d o m a i n is r e q u i r e d in o r d e r to o p p o s e a p o p t o s i s . 5 8  In a d d i t i o n to the  B H d o m a i n s , m a n y of the  B c l - 2 f a m i l y of p r o t e i n s c o n t a i n a  t r a n s m e m b r a n e d o m a i n that a n c h o r s t h e m to the m i t o c h o n d r i a l m e m b r a n e .  C-terminal  T h i s d o m a i n is e s s e n t i a l in  l o c a l i z i n g t h e s e p r o t e i n s to the m i t o c h o n d r i a a n d d e l e t i o n of this r e g i o n m a y a b o l i s h t h e f u n c t i o n of s o m e of t h e s e p r o t e i n s . 59  19 Bcl-2 a n d t h e M i t o c h r o n d r i a T h e ability of B c l - 2 f a m i l y m e m b e r s to interact a n d d i m e r i z e r e g u l a t e s a p o p t o s i s r e g u l a t i o n v i a the f o r m a t i o n of c h a n n e l s in the o u t e r m i t o c h o n d r i a l m e m b r a n e or b y m o d u l a t i n g e x i s t i n g c h a n n e l s in this structure. T h e l o s s of m i t o c h o n d r i a l m e m b r a n e potential ( A ^ ) , a s w e l l a s the r e l e a s e of a p o p t o t i c f a c t o r s f r o m the m i t o c h o n d r i a ( A I F , A P A F - 1 , c y t o c h r o m e c , a n d C a ) , is a h a l l m a r k f e a t u r e in the a p o p t o t i c 2 +  p a t h w a y a n d is g e n e r a l l y r e g a r d e d a s the point of n o return. members  to  form  or regulate  p o r e activity  in this  structure  T h u s , the putative ability of B c l - 2 f a m i l y s e r v e s to  explain  how  this  may  be  a c c o m p l i s h e d a n d highlights the i m p o r t a n c e of t h e s e p r o t e i n s in m i t o c h o n d r i a l r e g u l a t i o n of a p o p t o s i s . T h e o r i e s p e r t a i n i n g to h o w B c l - 2 protein f a m i l y m e m b e r s r e g u l a t e Aiy fall into two c a t e g o r i e s : t h o s e i n v o l v i n g the B c l - 2 f a m i l y p r o t e i n s a s c h a n n e l f o r m i n g p r o t e i n s t h e m s e l v e s , o r s p e c u l a t i o n that t h e s e proteins m o d u l a t i n g existing c h a n n e l s within the outer m i t o c h o n d r i a l m e m b r a n e . T h e first s u g g e s t i o n s that B c l - 2 family p r o t e i n s m a y f o r m p o r e s in the m i t o c h o n d r i a l m e m b r a n e a r o s e f r o m the d i s c o v e r y that B c l - X , c l o s e l y related to B c l - 2 , h a d s i m i l a r structure to p o r e f o r m i n g r e g i o n s L  of b a c t e r i a l t o x i n s . L a t e r , B c l - X , B c l - 2 , a n d B a x , w e r e s h o w n to b e c a p a b l e of f o r m i n g ion c h a n n e l s in 6 0  L  synthetic m e m b r a n e s . 6 1  attributes.  T h e c h a n n e l s formed by Bcl-2 family m e m b e r s e n c o m p a s s a number  T h e y a r e g e n e r a l l y v o l t a g e s e n s i t i v e , d i s p l a y p o o r ion selectivity, a n d a r e p H s e n s i t i v e . A n t i -  apoptotic proteins Bcl-2 and B c l - X  L  f o r m p o r e s at low p H a n d a r e s e l e c t i v e for c a t i o n s w h i l e the pro-  a p o p t o t i c B a x f o r m s c h a n n e l s at neutral p H a n d is s e l e c t i v e for a n i o n s . 6 2  pertaining  of  to  the  mitochondrial  environment,  such as  the  lipid  F u r t h e r m o r e , other f a c t o r s  composition  of  the  mitochondrial  m e m b r a n e , m a y dictate w h e t h e r t h e s e p r o t e i n s a r e c a p a b l e of f o r m i n g c h a n n e l s . W h e t h e r proteins s u c h as Bcl-2, B c l - X  L  a n d B a x , c o n d u c t a p o p t o t i c f a c t o r s out of m i t o c h o n d r i a  directly, or w h e t h e r t h e y alter e x i s t i n g t r a n s p o r t e r s h a s not yet b e e n c l e a r l y e s t a b l i s h e d . A n u m b e r of t h e s e p r o t e i n s , h o w e v e r , h a v e b e e n l i n k e d to the P e r m e a b i l i t y T r a n s i t i o n P o r e ( P T P ) , a s c o m p l e x o f t r a n s p o r t e r s s p a n n i n g both m i t o c h o n d r i a l m e m b r a n e s that f o r m s  in c e l l s s u b j e c t e d to a p o p t o s i s or  n e c r o s i s . R e s u l t s of i n c r e a s e d p e r m e a b i l i t y of this p o r e result in m i t o c h o n d r i a l d e p o l a r i z a t i o n , u n c o u p l i n g of o x i d a t i v e p h o s p h o r y l a t i o n , a n d s w e l l i n g of m i t o c h o n d r i a . 63  T h e l i n k a g e of B c l - 2 f a m i l y proteins to this c o m p l e x is s u p p o r t e d both b y s t u d i e s involving the p h y s i c a l s t u d y of this c o m p l e x a s well a s the m o d u l a t i o n of its f u n c t i o n .  T h e p r o - a p o p t o t i c protein B a x  h a s b e e n s h o w n to b e part of the P T P c o m p l e x a n d it c o - p r e c i p i t a t e s with P T P c o m p o n e n t s , while the  20 a n t i - a p o p t o t i c p r o t e i n s Bcl-2 a n d B c l - X a r e p e r i p h e r a l c o m p o n e n t s . F u r t h e r m o r e , the p r o - a p o p t o t i c B a k 6 4  L  protein h a s a l s o b e e n i m p l i c a t e d in this c o m p l e x a n d m a y bind to P T P  6 5  .  O n a f u n c t i o n a l l e v e l , Bcl-2 m a y  p r e v e n t P T P o p e n i n g b y i n c r e a s i n g proton efflux from the m i t o c h o n d r i a a n d inhibiting respiration . 66  mitochondrial  In addition to s t a b i l i z i n g m i t o c h o n d r i a l m e m b r a n e p o t e n t i a l , this w o u l d a l s o result in a  d e c r e a s e in t h e f r e e r a d i c a l p r o d u c t i o n , p o s s i b l y e x p l a i n i n g s o m e o f t h e putative a n t i - o x i d a n t p r o p e r t i e s o f  bcl-2  67,68  . A n t i - o x i d a n t p r o p e r t i e s of Bcl-2 a n d it's ability to r e g u l a t e p r o t o n flux h a v e b e e n p r e v i o u s l y  r e p o r t e d , but a s to w h e t h e r or not it c a r r i e s out t h e s e f u n c t i o n s v i a the P T P is not k n o w n . A l t h o u g h for the s a k e of clarity the c a p a c i t y of Bcl-2 f a m i l y m e m b e r s to r e g u l a t e a p o p t o s i s h a s b e e n d i s c u s s e d in t h e c o n t e x t of either their ability to d i m e r i z e or f o r m c h a n n e l s , t h e s e two p r o c e s s i n g a r e m o r e than likely i n d i s s o l u b l e . T h e i d e a that d i m e r i z a t i o n of t h e s e p r o t e i n s a n d their p o r e - f o r m i n g faculties a r e not entirely s e p a r a t e is s u p p o r t e d b y the fact that s o m e m e m b e r s c a n o n l y f o r m d i m e r i z e d ; the B a x protein is o n e s u c h e x a m p l e . 6 9  pores when  F u r t h e r m o r e , the c h a n n e l f o r m i n g ability of B a x is  a n n u l l e d b y the p r e s e n c e of Bcl-2, thus l e n d i n g m o r e c r e d e n c e to the i d e a that d i m e r i z a t i o n a n d c h a n n e l f o r m i n g ability a r e l i n k e d . 7 0  T h i s link p r o v i d e s a m e c h a n i s m that is c o n s i s t e n t with the R h e o s t a t T h e o r y  outlined p r e v i o u s l y .  Phosphorylation of Bcl-2 U n d e n i a b l y , the l e v e l s of Bcl-2 proteins in the cell a n d s u b s e q u e n t d i m e r i z a t i o n , a s s u p p o r t e d b y the R h e o s t a t T h e o r y , h a v e a significant role in regulating a p o p t o s i s at the m i t o c h o n d r i a . second,  and  likely  equally  important,  level  of  regulation  by  these  proteins  may  Nevertheless, a occur  through  p h o s p h o r y l a t i o n . A d d i t i o n a l control r e l e g a t e d b y the p h o s p h o r y l a t i o n state of t h e s e p r o t e i n s m a y s e r v e to fine t u n e the r e g u l a t i o n of a p o p t o s i s at this s t a g e . A n a l y s i s of Bcl-2 r e v e a l e d that it c o n t a i n s s e v e n p o s s i b l e p h o s p h o r y l a t i o n s i t e s , of w h i c h o n e at position 70 a p p e a r s to b e significant in regulating f u n c t i o n ; s p e c i f i c a l l y , d e l e t i o n or substitution of this r e s i d u e d i m i n i s h e s the anti-apoptotic function of B c l - 2 . 71  A l t h o u g h distinct p a t h w a y s a r e u n c l e a r , Bcl-2  a p p e a r s to b e p h o s p h o r y l a t e d t h r o u g h a m e c h a n i s m involving P r o t e i n K i n a s e C ( P K C ) a s treatment of m y e l o i d c e l l s with b r y o s t a t i n , a n activator of P K C , r e s u l t e d in p h o s p h o r y l a t i o n of B c l - 2 . F u r t h e r m o r e , this 7 2  p h o s p h o r y l a t i o n m a y b e p r e v e n t e d b y the u s e of P K C inhibitors.  O t h e r s t u d i e s h a v e d o w n p l a y e d the  i m p o r t a n c e of P K C in p h o s p h o r y l a t i o n of Bcl-2, a s in vivo t r e a t m e n t with P K C b l o c k e r s t a u r o s p o r i n e d o e s not inhibit a n t i - a p o p t o t i c f u n c t i o n . 73  21 While  phosphorylation  of  B c l - 2 at  Serine-70 may  be  required  for  this  protein  to  function,  p h o s p h o r y l a t i o n h a s a l s o b e e n s h o w n to d i m i n i s h its f u n c t i o n . P h o s p h o r y l a t i o n a s a result of e x p o s u r e to the p h o s p h a t a s e inhibitor O k a d a i c A c i d or the c h e m o t h e r a p y d r u g T a x o l (both of w h i c h i n d u c e a p o p t o s i s ) c a u s e p h o s p h o r y l a t i o n of B c l - 2 a n d a b o l i s h a n t i - a p o p t o t i c b e h a v i o r . 7 4  produce a similar effect . 75  Other microtubule targeting agents  C o n s p i c u o u s l y , p h o s p h o r y l a t i o n of B c l - 2 h a s b e e n s h o w n to limit its ability to  b i n d to B a x , a n d this m a y e x p l a i n in part h o w B c l - 2 b e h a v e s in r e s p o n s e to t r e a t m e n t with t h e s e d r u g s . 7 6  T h e role of the p h o s p h o r y l a t i o n s t a t e of B c l - 2 in r e g u l a t i n g its f u n c t i o n in a p o p t o s i s is, at b e s t , quite u n c l e a r .  It r e m a i n s to b e s e e n h o w t r e a t m e n t  with c h e m o t h e r a p y d r u g s , a n d s p e c i f i c a l l y the  t a x a n e s , p r o d u c e s p h o s p h o r y l a t i o n of B c l - 2 . W h i l e the p a t h w a y s l e a d i n g to p h o s p h o r y l a t i o n of B c l - 2 a r e at this t i m e u n r e s o l v e d , u n q u e s t i o n a b l y c e r t a i n p l a y e r s within the  cell  s i g n a l i n g p a t h w a y s of a p o p t o s i s ,  s u c h a s P r o t e i n K i n a s e C , will b e p r o v e to b e significant.  1.2.5 DOWNSTREAM OF BCL-2  Release of Apoptotic Factors  O n c e the B c l - 2 f a m i l y of p r o t e i n s h a s r e g u l a t e d e v e n t s in the f a v o r of a p o p t o s i s , o n e of the e a r l i e s t e v e n t s is l o s s of the m i t o c h o n d r i a l m e m b r a n e p o t e n t i a l .  C o n c u r r e n t with this e v e n t is the r e l e a s e  of s e v e r a l f a c t o r s f r o m m i t o c h o n d r i a that will ultimately c a r r y the s i g n a l f r o m this o r g a n e l l e to the c a s p a s e p r o t e a s e c a s c a d e in the c y t o p l a s m ( a m o n g other targets).  T h r e e principle c o m p o n e n t s have been  identified w h i c h a r e r e l e a s e d f r o m the m i t o c h o n d r i a e a r l y in a p o p t o s i s - A I F , A P A F - 1 , a n d cytochrome  c-  a n d l o s s of A y / a n d the r e l e a s e of t h e s e f a c t o r s is g e n e r a l l y c o n s i d e r e d the point of n o return in a p o p t o s i s . A I F is a f l a v o p r o t e i n ( c o n t a i n s a flavin a d e n i n e d i n u c l e o t i d e p r o s t h e t i c g r o u p ) r e s e m b l i n g b a c t e r i a l o x i d o r e d u c t a s e s a n d is f o u n d e x c l u s i v e l y in the m i t o c h o n d r i a in intact, n o r m a l c e l l s . protein  during  a p o p t o s i s is c a p a b l e of  rendering  chromatin condensation and D N A fragmentation . 77  morphological  c h a n g e s to the  R e l e a s e of this  nucleus,  including  T h i s protein is a l s o r e l e a s e d in r e s p o n s e to t r e a t m e n t  with c h e m o t h e r a p e u t i c d r u g s i n c l u d i n g a l k y l a t i n g a g e n t s s u c h a s c i s p l a t i n or T o p o i s o m e r a s e II inhibitors s u c h a s e t o p o s i d e . F u r t h e r m o r e , o v e r - e x p r e s s i o n of B c l - 2 is k n o w n to inhibit t h e r e l e a s e of A I F f r o m the 7 8  m i t o c h o n d r i a . H o w e v e r , o v e r - e x p r e s s i o n of B c l - 2 w a s ineffective in inhibiting the e f f e c t s of A I F w h e n it 7 9  22 w a s directly injected into the c y t o p l a s m , indicating that the r e l e a s e of this factor m a y r e p r e s e n t a n irreversible e v e n t in the a p o p t o t i c p a t h w a y . O n c e r e l e a s e d f r o m the m i t o c h o n d r i a , A I F is initiates m e m b r a n e a s y m m e t r y disruption at the p l a s m a m e m b r a n e in the f o r m of the e x p o s u r e of p h o s p h a t i d y l s e r i n e r e s i d u e s to the exterior of the c e l l . 8 0  A l t h o u g h d e p e n d e n t u p o n l o s s of A y / f o r t r a n s l o c a t i o n to the c y s t o l , o n c e t h e r e it f u n c t i o n s in a c a s p a s e i n d e p e n d e n t m a n n e r s i n c e c a s p a s e inhibitors d o not inhibit its f u n c t i o n . 81  A P A F - 1 is a significant protein in that it r e p r e s e n t s a direct link b e t w e e n the m i t o c h o n d r i a a n d activation of the cell d e a t h p a t h w a y through its interaction with C a s p a s e 9 . 8 2  Specifically, A P A F - 1 binds  a n d c a u s e s the activation of C a s p a s e 9 t h r o u g h a m e c h a n i s m d e p e n d e n t u p o n d A T P a n d c y t o c h r o m e c . 8 3  O v e r - e x p r e s s i o n of A P A F - 1 h a s b e e n s h o w n to a u g m e n t a p o p t o s i s in r e s p o n s e to r a d i a t i o n , 84  etoposide and T a x o l and/or B c l - 2 . 8 6  X  L  8 5  a n d this protein m a y a l s o b e s e q u e s t e r e d at m i t o c h o n d r i a l m e m b r a n e by B c l - X  L  T h i s c o n c e p t is s o m e w h a t t e n u o u s s i n c e interactions b e t w e e n A P A F - 1 a n d B c l - 2 or B c l -  h a v e not b e e n e s t a b l i s h e d . F u r t h e r m o r e , e x p e r i m e n t s u s i n g h e t e r o z y g o u s a n d h o m o z y g o u s A P A F - 1  m u t a n t s i n d i c a t e d that protective effects of B c l - 2 m a y b e i n d e p e n d e n t of A P A F - 1 . primarily l o c a l i z e d in the c y s t o l , w h i l e B c l - 2 a n d B c l - X  L  B e c a u s e A P A F - 1 is  a r e m i t o c h o n d r i a l p r o t e i n s , activation of A P A F - 1  m a y not rely o n direct interactions with B c l - 2 , but rather that of c y t o c h r o m e c , w h o s e r e l e a s e is c o n t r o l l e d by Bcl-2 a n d B c l - X  8 7 L  . A l t h o u g h t h e s e p r o t e i n s a r e l i n k e d in r e g u l a t i n g a p o p t o s i s at t h e m i t o c h o n d r i a a n d  the t r a n s f e r r e n c e of the a p o p t o t i c s i g n a l to C a s p a s e 9, the e x a c t r e l a t i o n s h i p b e t w e e n A P A F - 1 a n d B c l - 2 a n d B c l - X r e m a i n s to b e e l u c i d a t e d . L  C y t o c h r o m e c is a n o t h e r protein that is r e l e a s e d f r o m the m i t o c h o n d r i a d u r i n g a p o p t o s i s a n d in addition to it's role in a p o p t o s i s , this protein f u n c t i o n s in n o r m a l c e l l u l a r m e t a b o l i s m a s w e l l . C y t o c h r o m e c is e s s e n t i a l in the transfer of e l e c t r o n s b e t w e e n C o m p l e x III (ubiquinol) a n d C o m p l e x IV ( c y t o c h r o m e o x i d a s e ) of the e l e c t r o n transport c h a i n .  In a p o p t o s i s , a n u m b e r of stimuli h a v e b e e n c o r r e l a t e d with  c y t o c h r o m e c r e l e a s e , i n c l u d i n g treatment with c h e m o t h e r a p y d r u g s s u c h a s c i s p l a t i n , e t o p o s i d e , a n d T a x o l , as well a s ionizing r a d i a t i o n  8 8 , 8 9  . A s s t a t e d p r e v i o u s l y , c y t o c h r o m e c is e s s e n t i a l to the activation of  C a s p a s e 9, the starting point in the c a s p a s e c a s c a d e in p 5 3 - m e d i a t e d a p o p t o s i s . R e l e a s e of c y t o c h r o m e c is a i d e d b y B a x w h i l e it is limited through o v e r - e x p r e s s i o n of B c l - 2 a n d B c l - X  9 0 , 9 1 , 9 2 L  .  However, Bcl-2  a n d B c l - X L m a y r e g u l a t e t h e a c t i o n of c y t o c h r o m e c b e y o n d controlling its r e l e a s e f r o m the m i t o c h o n d r i a , a s o v e r - e x p r e s s i o n of t h e s e p r o t e i n s a l s o s u p p r e s s e d a p o p t o s i s u p o n direct c y t o p l a s m i c injection of c y t o c h r o m e c.  23  Activation of Caspases  C a s p a s e s a r e a s e r i e s of proteolytic proteins in the cell that a r e the w o r k - h o r s e s of a p o p t o s i s a n d their n a m e d e r i v e s f r o m the fact that t h e y c o n t a i n a c y s t e i n a c t i v e site a n d c l e a v e targets after a n a s p a r t i c acid residue.  A c t i v a t i o n of t h e s e p r o t e a s e s is a u n i q u e i n d i c a t o r of a p o p t o s i s a n d inhibition of t h e s e  p r o t e i n s s e v e r e l y limits a p o p t o s i s .  K n o c k o u t m i c e for a n u m b e r of t h e s e c a s p a s e s d i e perinatally f r o m  p r o f o u n d d e f e c t s a s the result of i m p a i r e d d e v e l o p m e n t a l c e l l d e a t h  9 3  .  E x i s t i n g in the cell a s in a p r e c u r s o r or z y m o g e n f o r m , c a s p a s e s a r e a c t i v a t e d t h r o u g h p r o t e o l y s i s by other c a s p a s e s or through a u t o a c t i v a t i o n . 94  B e c a u s e t h e s e p r o t e i n s a r e c a p a b l e of activating o n e  a n o t h e r , c a s p a s e a c t i v a t i o n e x i s t s in a c a s c a d e in a p o p t o s i s , a n d this a l l o w s a m p l i f i c a t i o n of the a p o p t o t i c s i g n a l a s w e l l a s its d i s s e m i n a t i o n to all r e g i o n s within. A t the p r e s e n t t i m e , f o u r t e e n c a s p a s e s h a v e b e e n identified in m a m m a l i a n c e l l s a n d t h e s e fall into two c l a s s e s :  C l a s s I c a s p a s e s , w h i c h a r e c a s p a s e s that a r e a c t i v a t e d relatively e a r l y in a p o p t o s i s  a n d f u n c t i o n a s p r o g e n i t o r s in the c a s p a s e c a s c a d e ; a n d , C l a s s II c a s p a s e s , w h i c h a r e late o r t e r m i n a l l y activated c a s p a s e s .  C a s p a s e s 1, 2 , 4 , 5, 8, 9, 1 0 , 12, a n d 14 b e l o n g in the first c l a s s w h i l e C a s p a s e s 3,  6, 7, a n d 11 b e l o n g in the latter c l a s s . 9 5  In addition to their p o s i t i o n s in the c a s p a s e c a s c a d e , certain  c a s p a s e s a r e a c t i v a t e d in r e s p o n s e to different a r m s of the a p o p t o t i c p a t h w a y .  T h e best known example  is the e x c l u s i v e activation of C a s p a s e 8 b y d e a t h - r e c e p t o r - m e d i a t e d a p o p t o s i s ( F a s or T N F - a ) a n d the a c t i v a t i o n of C a s p a s e 9 in r e s p o n s e to p 5 3 - m e d i a t e d a p o p t o s i s a n d the u s e o f c h e m o t h e r a p y d r u g s o r radiation ( C a s p a s e 9 is the o n l y c a s p a s e that r e q u i r e s activation b y c y t o c h r o m e c ) . 9 6  However, these  p a t h w a y s e v e n t u a l l y c o n v e r g e at d o w n s t r e a m c a s p a s e s , a n d both C a s p a s e 8 a n d C a s p a s e 9 activate Caspase 3  9 7  .  O n c e the c a s p a s e c a s c a d e h a s b e e n a c t i v a t e d , t h e s e e n z y m e s will affect a h o s t of cellular targets to c o n c l u d e the a p o p t o t i c p r o c e s s . O v e r forty p r o t e i n s h a v e b e e n identified w h i c h a r e targets of c a s p a s e s , a n d i n v o l v e proteins with s u c h d i v e r s e f u n c t i o n s a s structural p r o t e i n s , D N A repair proteins, proteins i n v o l v e d in cell proliferation, a n d p r o t e i n s i n v o l v e d in c e l l s i g n a l i n g . T a b l e 1.5 s u m m a r i z e s s o m e of t h e s e t a r g e t s .  24 T a b l e 1.5  C e l l u l a r T a r g e t s of C a s p a s e s  98  Consequence of Cleavage  Target Nuclear Proteins PARP D N A Protein K i n a s e s H i s t o n e s H1 a n d H 2 T o p o i s o m e r a s e s I a n d II SAF-A L a m i n s A , B, a n d C RP-C Rb  ICAD/DFF Structural Proteins Actin Fodrin  Inhibition of D N A r e p a i r Inhibition of D N A r e p a i r L o s s of c h r o m a t i n a r c h i t e c t u r e Inhibition of replication a n d t r a n s c r i p t i o n L o s s of c h r o m a t i n a r c h i t e c t u r e L o s s of n u c l e a r e n v e l o p e structure Inhibition of D N A r e p l i c a t i o n L o s s of cell c y c l e control A c t i v a t i o n of D N a s e s L o s s of c e l l u l a r s t r u c t u r e ; c e l l r o u n d i n g Actin depolymerization Actin/membrane detachment Intermediate f i l a m e n t b r e a k d o w n Microvilli b r e a k d o w n D e r e g u l a t i o n of a c t i n p o l y m e r i z a t i o n D e r e g u l a t i o n of a c t i n p o l y m e r i z a t i o n Inactivation of a p o p t o t i c s u p p r e s s o r function Induction of cell d e a t h  /?-catenin Keratin ERM D4-GDI Gas2 FAK Gelsolin Protein K i n a s e s  Induction Induction Induction Induction  PKC6 PKC6 Mekkl PAK-2/hPak-65 Caspase Activators Bcl-2 Bcl-XL p 2 8 B a p 31 Others Rabaptin-5 SREBP kB  of of of of  cell cell cell cell  death death death death  A m p l i f i c a t i o n of c a s p a s e a c t i v a t i o n A m p l i f i c a t i o n of c a s p a s e a c t i v a t i o n Induction of c e l l d e a t h Inhibits e n d o s o m e f u s i o n D e r e g u l a t i o n of c h o l e s t e r o l s y n t h e s i s P r o d u c t i o n of inhibitor of N F - K B  It is important to note that c a s p a s e s not o n l y target p r o t e i n s with d i v e r s e f u n c t i o n s in the c e l l , but a l s o other c a s p a s e s (which t h e y a c t i v a t e through p r o t e o l y s i s ) a n d inhibitors of c a s p a s e s ( s u c h a s B c l - 2 and  Bcl-X ). L  T h i s f u n c t i o n s a s a p o s i t i v e f e e d b a c k loop a n d s e r v e s to a m p l i f y t h e a p o p t o t i c s i g n a l  t h r o u g h o u t the c e l l Through  9 9 , 1 0 0  the  .  eventual destruction  of c e l l u l a r p r o t e i n s  by c a s p a s e s  the  later  p r o c e s s e s of  a p o p t o s i s o c c u r to bring cell d e a t h to fruition: D N A replication a n d t r a n s c r i p t i o n is p r e v e n t e d a n d cell c y c l e p r o g r e s s i o n is b l o c k e d ; structural p r o t e i n s a r e d e s t r o y e d a n d s i g n a l p a t h w a y s a r e d i s r u p t e d ; the n u c l e u s b r e a k s d o w n a n d D N A is c l e a v e d into s m a l l s e g m e n t s ; finally, the p l a s m a m e m b r a n e b l e b s a n d a p o p t o t i c b o d i e s f o r m . T h u s , c o m p l e t i n g the a p o p t o t i c p r o c e s s .  25  1.2.7 REFERENCES K e r r J F , W y l l i e A H , C u r r i e A R . 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FEBS Lett. 1997 S e p 2 2 ; 4 1 5 ( 1 ):29-32.  9 1  Y a n g J , Liu X , B h a l l a K, K i m C N , Ibrado A M , C a i J , P e n g TI, J o n e s D P , W a n g X . P r e v e n t i o n of a p o p t o s i s by B c l - 2 : r e l e a s e of c y t o c h r o m e c f r o m m i t o c h o n d r i a b l o c k e d . Science. 1 9 9 7 F e b 21 ; 2 7 5 ( 5 3 0 3 ) : 1 1 2 9 - 3 2 .  9 2  V a r f o l o m e e v E E , S c h u c h m a n n M , L u r i a V , C h i a n n i l k u l c h a i N , B e c k m a n n J S , Mett IL, R e b r i k o v D, B r o d i a n s k i V M , K e m p e r O C , Kollet O , L a p i d o t T , S o f t e r D, S o b e T , A v r a h a m K B , G o n c h a r o v T , H o l t m a n n H, L o n a i P , W a l l a c h D. T a r g e t e d disruption of the m o u s e C a s p a s e 8 g e n e a b l a t e s c e l l d e a t h induction b y the T N F r e c e p t o r s , F a s / A p o 1 , a n d D R 3 a n d is lethal prenatally. Immunity. 1 9 9 8 A u g ; 9 ( 2 ) : 2 6 7 - 7 6 . 9 3  9 4  W o l f B B , G r e e n D R . S u i c i d a l t e n d e n c i e s : a p o p t o t i c cell d e a t h b y c a s p a s e f a m i l y p r o t e i n a s e s . 1999 J u l 1 6 ; 2 7 4 ( 2 9 ) : 2 0 0 4 9 - 5 2 .  J Biol Chem.  K u m a r S . R e g u l a t i o n of c a s p a s e activation in a p o p t o s i s : i m p l i c a t i o n s in p a t h o g e n e s i s a n d treatment of d i s e a s e . Clin Exp Pharmacol Physiol. 1 9 9 9 A p r ; 2 6 ( 4 ) : 2 9 5 - 3 0 3  9 5  9 6  P a n G , H u m k e E W , Dixit V M . A c t i v a t i o n of c a s p a s e s triggered b y c y t o c h r o m e c in vitro.  FEBS Lett. 1 9 9 8 A p r 10;426(1 ):151-4. Li P , N i j h a w a n D, B u d i h a r d j o I, S r i n i v a s u l a S M , A h m a d M , A l n e m r i E S , W a n g X . C y t o c h r o m e c a n d d A T P - d e p e n d e n t f o r m a t i o n of A p a f - 1 / c a s p a s e - 9 c o m p l e x initiates a n a p o p t o t i c p r o t e a s e c a s c a d e . Cell. 1997 N o v 14;91(4):479-89.  9 7  D e p r a e t e r e V , G o l s t e i n P . D i s m a n t l i n g in cell d e a t h : m o l e c u l a r m e c h a n i s m s a n d r e l a t i o n s h i p to c a s p a s e activation. Scand J Immunol. 1 9 9 8 J u n ; 4 7 ( 6 ) : 5 2 3 - 3 1 . 9 8  C h e n g E H , K i r s c h D G , C l e m R J , R a v i R, K a s t a n M B , B e d i A , U e n o K, H a r d w i c k J M . C o n v e r s i o n o f B c l 2 to a B a x - l i k e d e a t h effector by c a s p a s e s . Science. 1 9 9 7 D e c 1 2 ; 2 7 8 ( 5 3 4 5 ) : 1 9 6 6 - 8 . 9 9  C l e m R J , C h e n g E H , K a r p C L , K i r s c h D G , U e n o K, T a k a h a s h i A , K a s t a n M B , Griffin D E , E a r n s h a w W C , V e l i u o n a M A , H a r d w i c k J M . M o d u l a t i o n of cell d e a t h by B c l - X L t h r o u g h c a s p a s e interaction. Proc Natl Acad Sci. 1 9 9 8 J a n 2 0 ; 9 5 ( 2 ) : 5 5 4 - 9 . 1 0 0  31  1.3 B C L - 2 A S A N A R B I T E R O F D R U G R E S I S T A N C E  W i t h further a d v a n c e s in r e s e a r c h into the p r o c e s s of a p o p t o s i s , the Bcl-2 f a m i l y of proteins is i n c r e a s i n g l y t a k i n g a central role in the regulation of this p r o c e s s . T h e Bcl-2 protein a n d the m y r i a d of related proteins f o r m a c o r e c o m p l e x that a p p e a r s to b e i n v o l v e d in a p o p t o s i s t r i g g e r e d b y w i d e variety of sources, including chemotherapy drugs.  It is not s u r p r i s i n g , t h e r e f o r e , that Bcl-2, a s well a s other  h o m o l o g o u s p r o t e i n s , are significant e l e m e n t s in the d e v e l o p m e n t of r e s i s t a n c e to c h e m o t h e r a p y d r u g s . In addition to a t t e m p t s to u n d e r s t a n d h o w t h e s e proteins m a y c o n f e r d r u g r e s i s t a n t p h e n o t y p e s to c e l l s , t h e s e p r o t e i n s h a v e a l s o b e e n s i n g l e d out a s t h e r a p e u t i c targets in o r d e r to i n c r e a s e the e f f i c a c y of v a r i o u s c h e m o t h e r a p y treatment r e g i m e n s . It is with this c o n s i d e r a t i o n in m i n d that the following s e c t i o n c o n s i d e r s the role of Bcl-2, a s well a s the related p r o - a p o p t o t i c proteins B a x a n d B a k , in the d e v e l o p m e n t of drug r e s i s t a n c e .  1.3.1 NORMAL TISSUE DISTRIBUTION OF BCL-2, BAX AND BAK  A l t h o u g h Bcl-2 a n d its a n c i l l a r y proteins m a y contribute to d r u g r e s i s t a n c e within t u m o r s , t h e s e proteins are a l s o f o u n d in significant l e v e l s in n o r m a l t i s s u e .  I m m u n o h i s t o c h e m i c a l a n a l y s i s of v a r i o u s  m u r i n e t i s s u e s r e v e a l e d the p r e s e n c e of B a x , but not Bcl-2, in the h e p a t o c y t e s of the liver, the c e l l s of the p a n c r e a s , a n d tubule epithelial c e l l s of the k i d n e y . T i s s u e s in w h i c h both p r o t e i n s w e r e f o u n d i n c l u d e the epithelia of the s m a l l i n t e s t i n e s , c o l o n , b r e a s t , p r o s t a t e , r e s p i r a t o r y tract, a n d s k i n . 1  Bax expression  t e n d e d to b e s t r o n g e s t in the outer l a y e r s of epithelia in the s m a l l intestine w h i l e Bcl-2 w a s s t r o n g e s t in the inner l a y e r s , a n d not s u r p r i s i n g l y , this c o r r e l a t e s with the natural p r o g r e s s i o n of t h e s e c e l l s w h e r e i n outer l a y e r s of epithelial c e l l s die v i a a p o p t o s i s . Interestingly, Bcl-2 is a l s o f o u n d in n o n - c y c l i n g c e l l s s u c h a s n e u r o n s , a l t h o u g h it is not p r e s e n t in m u s c l e (or a n y other t i s s u e of m e s o d e r m a l origin); g e n e r a l l y , it is f o u n d in the h i g h e s t a m o u n t s in p e r i p h e r a l l y m p h o c y t e s , a n d for the m o s t part, the p r e s e n c e of this protein is c o r r e l a t e d with the a p o p t o t i c potential of the c e l l . 2  In t e r m s of B a k e x p r e s s i o n , i m m u n o h i s t o c h e m i c a l a n a l y s i s of different h u m a n t i s s u e s r e v e a l e d significant B a k e x p r e s s i o n in epithelia of the n a s o p h a r y n x , e s o p h a g u s , c o l o n a n d b l a d d e r , a n d like its proa p o p t o t i c h o m o l o g u e B a x , the h i g h e s t l e v e l s e x i s t e d in the o u t e r m o s t l a y e r s of the e p i t h e l i u m .  Other  32 significant  tissues  in  relation  to  Bak  expression  were  terminally  differentiated  granulocytes,  c a r d i o m y o c y t e s , s m o o t h m u s c l e c e l l s , b a s a l c e l l s of the p r o s t a t e , m y o e p i t h e l i a l c e l l s of the g l a n d s , distal c o n v o l u t e d  t u b u l e s of the  kidney, epidermal  keratinocytes, enterocytes  mammary  of the  small  intestine, S e r t o l i a n d L e i d i g c e l l s of the t e s t e s , t h e c a interna c e l l s of the o v a r y , a n d the a d r e n a l cortex. N o t a b l y h o w e v e r , B a k e x p r e s s i o n w a s low or a b s e n t in the c e n t r a l n e r v o u s s y s t e m a n d in p e r i p h e r a l lymphocytes . 3  A s the l e v e l s of t h e s e three proteins v a r y b e t w e e n different t i s s u e s within the b o d y in the n o r m a l s t a t e , the s a m e is true for a m o u n t s of t h e s e p r o t e i n s f o u n d in m a l i g n a n c i e s of different o r i g i n s . T h e t i s s u e specificity of t h e s e proteins c o m p l i c a t e s attempts to a s s e s s their role in d r u g r e s i s t a n c e . i n v e s t i g a t i o n s into e l e v a t e d l e v e l s of t h e s e proteins in v a r i o u s t u m o r s , a s well a s  Nonetheless,  in vitro  and  in vivo  a n a l y s i s u s i n g h i g h - B c l - 2 e x p r e s s i n g c e l l s , h a s y i e l d e d interesting r e s u l t s .  1.3.2 BCL-2, APOPTOSIS AND DRUG RESISTANCE  T h e c h r o m o s o m a l t r a n s l o c a t i o n that led to the d i s c o v e r y of B c l - 2 r e s u l t e d in the o v e r - e x p r e s s i o n of this protein after the c o d i n g r e g i o n of B c l - 2 w a s p l a c e d u n d e r the control of the i m m u n o g l o b u l i n h e a v y chain promotor.  S i n c e that time, the ability of B c l - 2 to p r o m o t e r e s i s t a n c e w h e n o v e r e x p r e s s e d  h a s b e e n s t u d i e d u n d e r v a r i o u s c i r c u m s t a n c e s a n d in m a n y different cell l i n e s .  in vitro  S t u d i e s to d a t e indicate  that the o v e r e x p r e s s i o n of B c l - 2 is c a p a b l e of p r o l o n g i n g s u r v i v a l of c e l l s in r e s p o n s e to a n u m b e r of apoptotic  stimuli,  including  cytokine  deprivation  from  hematopoietic  cells,  glucocorticoid  treatment,  g a m m a irradiation, a n d growth factor deprivation U V radiation, heat s h o c k , o x i d a t i v e s t r e s s , c a l c i u m ionophores  4 , 5  ' ' 6  drugs as well.  7 , 8  .  S i m i l a r l y , B c l - 2 h a s b e e n linked to d r u g r e s i s t a n c e to a w i d e r a n g e of c h e m o t h e r a p y O v e r e x p r e s s i o n of B c l - 2 h a s r e s u l t e d in r e s i s t a n c e to p a c l i t a x e l in m y e l o m a c e l l s , to  cisplatin in b l a d d e r c e l l s , to d o x o r u b i c i n a n d c i s p l a t i n in l u n g c a n c e r c e l l s , to p a c l i t a x e l in m y e l o m a c e l l s , a n d to d o x o r u b i c i n a n d cisplatin in n e u r o b l a s t o m a s  9 , 1 0 , 1 1 , 1 2  .  multiple  T h e L N C a P h u m a n prostate  cell line h a s a l s o b e e n p o p u l a r in r e s e a r c h involving the high l e v e l s of B c l - 2 a n d o v e r - e x p r e s s i o n of B c l - 2 in this cell line, t h r o u g h t r a n s f e c t i o n with a B c l - 2 - c o n t a i n i n g p l a s m i d , c o n f e r r e d c e l l s with a n i n c r e a s e d r e s i s t a n c e to s e r u m - s t a r v a t i o n a n d a n d r o g e n - w i t h d r a w a l . 13  In t e r m s of its i n v o l v e m e n t in r e s i s t a n c e in  b r e a s t c a n c e r c e l l s l i n e s , the t i s s u e s t u d i e d in this t h e s i s , B c l - 2 l e v e l s w e r e c o r r e l a t e d to r e s i s t a n c e to m i t o m y c i n C , 5-fluorouracil, d o x o r u b i c i n , c i s p l a t i n , a n d c y c l o p h o s p h a m i d e . A s w e l l , in a s t u d y a n a l y z i n g 1 4  33 the eight p r i n c i p l e b r e a s t c a n c e r c e l l l i n e s f r o m N C I (MCF-7, MCF-7/ADR, T47D, HS578T, BT549, MDA-N, MDAMB231, and MDA 435) in r e s p o n s e to c a m p t o t h e c i n s , t o p o i s o m e r a s e I inhibitors, s t a u r o s p o r i n e s , a n d P K C inhibitors, B c l - 2 e x p r e s s i o n w a s f o u n d to b e i n v e r s e l y p r o p o r t i o n a l to a p o p t o t i c r e s p o n s e . 1 5  W h i l e the majority of s t u d i e s h a v e i n d i c a t e d a n i n c r e a s e in d r u g r e s i s t a n c e with high l e v e l s of B c l 2, a few h a v e found contradictory findings.  In o v a r i a n c a n c e r c e l l s , h i g h B c l - 2 e x p r e s s i o n led to sensitivity  to c i s p l a t i n ; this m a y , h o w e v e r , b e r e l a t e d to f a c t o r s s u c h a s g r o w t h rate, a s B c l - 2 h a s b e e n s h o w n in 1 6  s o m e c e l l s to limit proliferation, or to r e c i p r i c a l d o w n r e g u l a t i o n in o t h e r a n t i - a p o p t o t i c p r o t e i n s s u c h a s B c l - X , w h i c h h a s o c c u r r e d in o t h e r c e l l l i n e s  1 7 , 1 8  L  .  S t u d i e s involving B c l - 2 i n d i c a t e , that u n d e r c e r t a i n c i r c u m s t a n c e s , it m a y c o n f e r d r u g r e s i s t a n c e to c e l l s w h i c h e x p r e s s high l e v e l s of this protein. T h e s e s t u d i e s u n d o u b t e d l y p r o v i d e u s e f u l information in a s s e s s i n g the role of B c l - 2 in d r u g r e s i s t a n c e ; h o w e v e r i n v e s t i g a t i o n s into the f u n c t i o n of B c l - 2  in vivo  p r o v i d e a m o r e realistic m o d e l for the d e t e r m i n a t i o n of the i n f l u e n c e B c l - 2 m a y h a v e u p o n r e s i s t a n c e to chemotherapy drugs. S o m e of the m o s t r e v e a l i n g which  in vivo  s t u d i e s c o n c e r n i n g B c l - 2 i n v o l v e d t h e u s e of t r a n s g e n i c m i c e  h a v e b e e n d e v e l o p e d to o v e r - e x p r e s s B c l - 2 in a h o s t of c e l l l i n e s .  displayed increased  in vitro  and  in vivo  Predictably, these  mice  c e l l s u r v i v a l in the t i s s u e in w h i c h the o v e r - e x p r e s s e d B c l - 2  e x i s t e d , a n d this cell s u r v i v a l w a s in r e s p o n s e to m a n y c y t o t o x i c s t i m u l i , i n c l u d i n g c y t o k i n e d e p r i v a t i o n , g a m m a radiation, glucocorticoids and calcium ionophores.  T h i s s u g g e s t s that B c l - 2 m a y r e s i d e at a  c o m m o n c h e c k p o i n t in a p o p t o s i s i r r e s p e c t i v e of the a p o p t o t i c t r i g g e r . W h i l e B c l - 2 o v e r - e x p r e s s i o n in 19  itself m a y not h a v e led to t u m o r f o r m a t i o n in t r a n s g e n i c m i c e , in the p r e s e n c e of a c t i v a t e d o n c o g e n e s , 20 21  s u c h a s c - m y c , or m u t a n t t u m o r s u p p r e s s o r s , s u c h a s p 5 3 , i n c r e a s e d t u m o r o g e n i c i t y o c c u r r e d  '  A l t h o u g h B c l - 2 c o n f e r r e d a m o r e r o b u s t p h e n o t y p e o n v a r i o u s c e l l s in t r a n s g e n i c m i c e , B c l - 2 k n o c k o u t m i c e did not c o n t a i n the w i d e s p r e a d a p o p t o s i s or lethal e f f e c t s that m a y h a v e b e e n p r e d i c t e d — B c l - 2 -/- m i c e w e r e v i a b l e , albeit d i s p l a y i n g h i g h e r d e g r e e s of c e l l d e a t h in the k i d n e y , ' a n d d e a t h of m a t u r e  lymphocytes  2 2 , 2 3  .  T h i s , h o w e v e r , s t a n d s in c o n t r a s t to the situation  premature  involving  the  s i m i l a r l y - r e l a t e d B c l - X p r o t e i n , w h e r e B c l - X k n o c k - o u t m i c e a r e e m b r y o n i c lethal at d a y 1 3 a n d s h o w i n c r e a s e d a p o p t o s i s in the c e n t r a l n e r v o u s s y s t e m a n d the l i v e r . 24  In s p i t e of t h e s e f i n d i n g s , the a p p a r e n t  d i f f e r e n c e s b e t w e e n t h e s e two p r o t e i n s in this r e g a r d m a y b e d u e to their t i s s u e distribution a s m u c h a s their b i o c h e m i c a l f u n c t i o n .  34 T h e majority of e x p e r i m e n t s investigating the B c l - 2 f u n c t i o n in vivo, a n d all of t h o s e i n v o l v i n g B c l 2 o v e r - e x p r e s s i o n a n d r e s p o n s e to c h e m o t h e r a p y in vivo, i n v o l v e the u s e of x e n o t r a n s p l a n t s . A s with in vitro s t u d i e s , the L N C a P cell line w a s w i d e l y u s e d in a n i m a l s t u d i e s a s w e l l . L N C a P c e l l s o v e r - e x p r e s s i n g B c l - 2 g r e w at a f a s t e r rate in m i c e than c o n t r o l s a n d s h o w e d r e s i s t a n c e u p o n c a s t r a t i o n , s o m e t h i n g that did not o c c u r with c e l l s u n t r a n s f e c t e d with B c l - 2 . 2 5  F u r t h e r m o r e , a d d i t i o n a l s t u d i e s s h o w e d that o n l y  L N C a P c e l l s t r a n s f e c t e d with B c l - 2 w e r e c a p a b l e of f o r m i n g t u m o r s in c a s t r a t e d n u d e m i c e . 2 6  O v e r - e x p r e s s i o n of B c l - 2 not o n l y i n c r e a s e d c e l l u l a r g r o w t h r a t e s a n d a p o p t o t i c r e s i s t a n c e , but it a l s o a p p e a r e d to a m p l i f y the m e t a s t a t i c potential  in a n u m b e r of c e l l l i n e s .  Bladder c a n c e r cells  t r a n s f e c t e d with B c l - 2 p r o d u c e d m o r e lung t u m o r s , a n d e n h a n c e d the m e t a s t a t i c potential of t h e s e c e l l s . 2 7  L i k e w i s e , in b r e a s t t i s s u e , B c l - 2 o v e r - e x p r e s s i o n i n c r e a s e d t u m o r o g e n i c i t y of M C F - 7 A D R c e l l s a s w e l l their ability to f o r m m e t a s t a s e s . 2 8  A s with the f i n d i n g s in in vitro s t u d i e s , B c l - 2 a p p e a r e d to i n c r e a s e the viability of c e l l s w h e n a s s a u l t e d with c y t o t o x i c a g e n t s in vivo; w h e t h e r high B c l - 2 l e v e l s on their own m a y i n c r e a s e the potential for t u m o r f o r m a t i o n  r e m a i n s to b e s e e n , a l t h o u g h p r e s e n t e v i d e n c e s u g g e s t s that it d o e s not.  An  important finding u s i n g a n i m a l m o d e l s is the putative ability of high B c l - 2 l e v e l s to i n c r e a s e the m e t a s t a t i c potential of t u m o r c e l l s .  In a c l i n i c a l c o n t e x t , a l t h o u g h B c l - 2 m a y b e s i g n i f i c a n t a s a n arbiter of d r u g  r e s i s t a n c e , this ability to i n c r e a s e the f o r m a t i o n of m e t a s t a s e s is n o n e t h e l e s s a l s o quite important. the  ability of B c l - 2 to d e c r e a s e sensitivity to c h e m o t h e r a p y d r u g s is e v i d e n t , a l t h o u g h  not  c o m p l e t e l y c o n v i n c i n g , f r o m in vitro a n d in vivo s t u d i e s ; the s a m e c a n n o t b e s a i d , h o w e v e r , a b o u t clinical f i n d i n g s i n v o l v i n g B c l - 2 a n d r e s p o n s e to c h e m o t h e r a p y .  A t t e m p t s to c o r r e l a t e t h e s t a t u s of B c l - 2 with  patient p r o g n o s i s a n d r e s p o n s e to c h e m o t h e r a p y h a v e b e e n p e r p l e x i n g at b e s t .  Although Bcl-2 has  s h o w n itself to b e a n important o n c o g e n e in certain c a n c e r s , a c r o s s the s p e c t r u m of m a l i g n a n t d i s e a s e s , the c l i n i c a l i m p o r t a n c e of B c l - 2 r e m a i n s to b e p r o v e n . E x p r e s s i o n of B c l - 2 is f o u n d in m a n y t u m o r s , i n c l u d i n g b r e a s t , l u n g , c o l o n c a r c i n o m a s , a n d r o g e n 29  receptor negative prostate c a n c e r , l y m p h o m a s , l e u k e m i a s , n e u r o b l a s t o m a s , and gliomas  ; h o w e v e r , in  only a s e g m e n t of t h e s e t u m o u r t y p e s d o e s B c l - 2 c o n f e r a c h e m o r e s i s t a n t p h e n o t y p e .  Interestingly,  these c a n c e r s tend towards hematopoietic malignancies, including acute m y e l o g e n o u s leukemia ( A M L ) , acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphomas, and 30 31 32 33 34  multiple m y e l o m a , but this c o n d i t i o n m a y a l s o b e f o u n d in p r o s t a t e , b r e a s t , a n d o v a r i a n c a n c e r  ' ' ' ' .  F u r t h e r m o r e , in c o m p a r i n g t i s s u e b e f o r e a n d after c h e m o t h e r a p y , c e l l s t e n d e d to h a v e low e x p r e s s i o n s of  35 Bcl-2 prior to drug treatment, a n d r e m a i n i n g c e l l s after e x p o s u r e to c h e m o t h e r a p y g e n e r a l l y c o n t a i n e d h i g h e r l e v e l s of Bcl-2, indicating that drug treatment m a y s e l e c t of c e l l s with high Bcl-2 c o n t e n t . 35  In  c o n t r a s t to t h e c i r c u m s t a n c e s with t h e s e d i s e a s e s , h o w e v e r , Bcl-2 h a s a l s o b e e n r e p o r t e d a s a g o o d p r o g n o s t i c m a r k e r in other c a n c e r s , i n c l u d i n g m a l i g n a n c i e s of t h e b r e a s t , c o l o n , a n d n o n - s m a l l cell lung 36 37 38  cancer  ' T h e r e a s o n s for s u c h conflicting results m a y lie in the t i s s u e s p e c i f i c i t y a n d i n f l u e n c e of Bcl-2 of  different cell t y p e s , t h o u g h other r e a s o n s a l s o p e r s i s t .  O n e e x p l a n a t i o n m a y b e d u e to t h e variability of  treatment of s a m p l e g r o u p s a n d s m a l l s a m p l e s i z e , a s w e l l a s t h e a s s e s s m e n t of clinical s a m p l e s for B c l 2.  Invariably,  tissue  samples  received  for  the  determination  of  Bcl-2  status  through  i m m u n o h i s t o c h e m i s t r y a n d flow c y t o m e t r y m a y c o m e from patients w h o h a v e r e c e i v e d different t y p e s a n d m o d a l i t i e s of treatment, a n d c o m p a r i s o n s of Bcl-2 l e v e l s in n e o p l a s t i c t i s s u e is c o n f o u n d e d b y t h e l a c k of information r e g a r d i n g Bcl-2 l e v e l s in n o r m a l t i s s u e A n o t h e r e x p l a n a t i o n that m a y p o s s i b l y clarify w h y Bcl-2 is a g o o d p r o g n o s t i c m a r k e r in s o m e cancers  a n d not others  suppressors.  is t h e correlation  of Bcl-2 with t h e status  of other  o n c o g e n e s or t u m o r  In t h e c a s e of solid t u m o r s , s u c h a s b r e a s t c a r c i n o m a s , Bcl-2 e x p r e s s i o n c o r r e l a t e d well  with positive e s t r o g e n r e c e p t o r status a n d well a s intact  p53 , 39  40,41  ' . T h u s , it m a y not b e the influence of 42  Bcl-2 p e r s a y , but the status of t h e s e other g e n e s that dictate t h e clinical o u t c o m e in t h e d i s e a s e that is investigated. W h i l e the status of Bcl-2 in a m a l i g n a n c y m a y h a v e a n i m p a c t u p o n patient s u r v i v a l a n d r e s p o n s e to c h e m o t h e r a p y , additional Bcl-2 family m e m b e r s m a y a l s o contribute in this r e g a r d . T h e p r o - a p o p t o t i c proteins B a x a n d B a k a r e k n o w n to p l a y a role in c h e m o r e s i s t a n c e a s w e l l , a n d their role in this p r o c e s s is outlined b e l o w .  1.3.3 BAX, APOPTOSIS AND DRUG RESISTANCE  B a x is c o n s i d e r e d t h e prototypical p r o - a p o p t o t i c m e m b e r o f t h e Bcl-2 f a m i l y of proteins, both b e c a u s e of its a s s o c i a t i o n with p53,  but a l s o b e c a u s e it w a s o n e of t h e first p r o - a p o p t o t i c  d i s c o v e r e d a n d r e m a i n s o n e of the m o s t s t u d i e d .  proteins  B e c a u s e of its p o w e r f u l ability to instigate a p o p t o s i s ,  the m i s - r e g u l a t i o n of B a x is c a p a b l e of i n d u c i n g drug r e s i s t a n c e . I n d e e d , n u m e r o u s s t u d i e s h a v e s h o w n that d e f i c i e n c i e s in this protein p r o m o t e drug r e s i s t a n c e w h i l e o v e r - e x p r e s s i o n of B a x m a y s e n s i t i z e c e l l s  36 to c h e m o t h e r a p y a g e n t s a n d i n c r e a s e cell d e a t h u p o n t r e a t m e n t with t h e s e a g e n t s a n a l y z i n g e n d o g e n o u s l e v e l s of B a x levels fell Moreover,  in response  4 3 , 4 4 , 4 5 , 4 6  to c h e m o t h e r a p y s h o w e d that B a x l e v e l s r o s e a n d B c l - 2  in b r e a s t c a n c e r c e l l s in r e s p o n s e to t r e a t m e n t with e t o p o s i d e a n d pretreatment  with  etoposide,  . Other studies  followed  by  treatment  with  the  cyclophosphamide.  active  metabolite  of  c y c l o p h o s p h a m i d e i n c u r r e d the g r e a t e s t result in this r e g a r d . 4 7  B a x a l s o r e m a i n s the o n l y B c l - 2 f a m i l y m e m b e r that h a s links to p 5 3 , a n d thus m u t a t i o n s affecting the s t a t u s or e x p r e s s i o n level of this protein m a y b e e s p e c i a l l y a d e p t in p r o m o t i n g d r u g r e s i s t a n c e . B a x is a direct transcriptional target of p 5 3 a n d c o n t a i n s p 5 3 b i n d i n g s i t e s in its p r o m o t o r . 48  In r e g a r d  to  treatment with c h e m o t h e r a p y d r u g s , B a x h a s b e e n s h o w n to f o r m c o m p l e x e s with p 5 3 u p o n t r e a t m e n t with c i s p l a t i n a n d m i g r a t e f r o m its primary location in the c y t o p l a s m to the m i t o c h o n d r i a l m e m b r a n e u p o n e x p o s u r e to c y t o t o x i c a g e n t s  In vivo  4 9 , 5 0  .  s t u d i e s involving the B a x protein r e v e a l e d s i m i l a r f i n d i n g s .  Transgenic mice showed  i n c r e a s e d t h y m o c y t e a p o p t o s i s in r e s p o n s e to c y t o t o x i c s t r e s s but not in t h e a b s e n c e of a n a p o p t o t i c trigger .  In m i c e deficient for B a x , k n o c k o u t s t u d i e s s h o w e d that B a x -/- m i c e w e r e v i a b l e , a l t h o u g h t h e y  51  d i s p l a y e d h y p o p l a s i a in the t e s t e s a n d w e r e sterile, a n d c o n t a i n e d s i m i l a r l e v e l s of a p o p t o s i s a s B a x +/+ mice  5 2 , 5 3  .  F u r t h e r m o r e , a p o p t o t i c triggers, s u c h a s y-radiation, p r o d u c e n o r m a l l e v e l s of cell d e a t h in  t h e s e m i c e a n d p 5 3 -/- m i c e e x p r e s s i n g a b a x t r a n s g e n e in the t h y m u s d i s p l a y e d identical r e s i s t a n c e to yradiation a s m i c e l a c k i n g the t r a n s g e n e . 5 4  T h u s , a l t h o u g h B a x m a y i n f l u e n c e d r u g r e s i s t a n c e in s o m e  m a n n e r , it is neither n e c e s s a r y nor sufficient for a p o p t o s i s , at l e a s t that i n d u c e d b y D N A d a m a g e v i a yradiation. In t e r m s of clinical a n a l y s i s of B a x , t u m o r s a m p l e s i n d i c a t e d that e n d o g e n o u s B a x l e v e l s w e r e c o r r e l a t e d with sensitivity to D o x o r u b i c i n a n d A c t i n o m y c i n D in a n u m b e r of t u m o r s . 5 5  M o r e o v e r , like the  original mutation that first identified B c l - 2 , c h r o m o s o m a l a b n o r m a l i t i e s i n v o l v i n g B a x m a y a l s o contribute to o n c o g e n e s i s a s f r a m e shift m u t a t i o n s resulting in n o n - f u n c t i o n a l B a x h a v e b e e n i m p l i c a t e d in c o l o n 56  cancer  . T a k e n t o g e t h e r , t h e s e results imply a role for B a x in the sensitivity of c e l l s to c h e m o t h e r a p y , a n d  therefore the s t a t u s of this protein w a s i n c l u d e d in the e x p e r i m e n t s c o n d u c t e d for this t h e s i s . T h e status of a s i m i l a r protein, B a k , a l s o i n v e s t i g a t e d , a s it m a y contribute to the c h e m o r e s p o n s i v e n e s s of c e l l s a s well.  37  1.3.4 BAK, APOPTOSIS AND DRUG RESISTANCE  T h e B a k protein s h a r e s m a n y similarities to B a x in t e r m s of its i n v o l v e m e n t in d r u g r e s i s t a n c e a n d it is o n e of the m o r e c o m m o n l y s t u d i e d p r o - a p o p t o t i c proteins a l o n g with B a x , B i d a n d B a d .  Like Bax,  o v e r - e x p r e s s i o n of B a k results in cell d e a t h in a n u m b e r of cell l i n e s , i n c l u d i n g b r e a s t a n d c o l o n c a n c e r cells, and f i b r o b l a s t s  5 7 , 5 8 , 5 9  , w h i l e B a k deficient c e l l s s h o w e d c h e m o r e s i s t a n c e to a w i d e r a n g e of s o u r c e s ,  i n c l u d i n g U V radiation, s t a u r o s p o r i n , e t o p o s i d e , b l e o m y c i n , a n d c i s p l a t i n .  This resistance has been  60  putatively attributed to its arbitration of c y t o c h r o m e c r e l e a s e , w h i c h d o e s not o c c u r in B a k deficient c e l l s . S e n s i t i v i t y to s o m e of the c a n c e r d r u g s m a y b e r e s t o r e d b y t r a n s f e c t i o n of B a k , a s is the p r e s e n c e of c y t o c h r o m e c r e l e a s e . M o r e o v e r , t h e s e e v e n t s a r e i n d e p e n d e n t of B a x a n d B i d , s i n c e t r a n s f e c t i o n of B a k is c a p a b l e of s e n s i t i z i n g c e l l s that a r e B a x -/- a n d B i d - / - . 61  A n a l y s i s of B a k in v a r i o u s t u m o r s r e v e a l e d this protein to b e a b s e n t o r exist in l o w a m o u n t s , w h i l e in other t u m o r s its p r e s e n c e w a s m o r e c o n s p i c u o u s , e s p e c i a l l y u p o n t r e a t m e n t with c h e m o t h e r a p y d r u g s s u c h as paclitaxel and c i s p l a t i n  6 2 , 6 3  .  In b r e a s t c a n c e r , s t u d i e s s h o w e d that B a k e x p r e s s i o n , like that of  Bcl-2, is c o r r e l a t e d with e s t r o g e n r e c e p t o r p o s i t i v i t y . 64  In g e n e r a l , the s i g n i f i c a n c e of the B a k protein in  the clinic is not well e s t a b l i s h e d , d u e in part, n o doubt, to its relatively r e c e n t d i s c o v e r y .  Hopefully,  information r e g a r d i n g the clinical r e l e v a n c e of B a k will b e f o r t h c o m i n g .  1.3.5. CONCLUSION  T h e g e n e r a l c o n s e n s u s thus far c o n c e r n i n g Bcl-2 a s a d e t e r m i n a t e in d r u g r e s i s t a n c e is that in elevated  levels, and  in certain  cell t y p e s ,  it is c a p a b l e of  c h e m o t h e r a p y d r u g s , a n d i n d e e d , other a p o p t o t i c triggers. B a k m a y a l s o contribute to the drug resistant state. Bak  appear  to m o d u l a t e  sensitivity  conferring  a d e c r e a s e d sensitivity  to  L i k e w i s e , d e f i c i e n c i e s in a m o u n t s B a x a n d  H o w e v e r , it is important to note that Bcl-2, B a x a n d  in s o m e cell lines to  chemotherapy  drugs,  but  not  increase  t u m o r o g e n i c i t y b y t h e m s e l v e s . A s with m o s t c a n c e r s , additional m u t a t i o n s in o t h e r s a l i e n t g e n e s , s u c h a s  p53 or  c-myc, are  r e q u i r e d in o r d e r to t r a n s f o r m c e l l s a n d d e v e l o p n e o p l a s i a s . W h i l e Bcl-2, B a x a n d B a k  38 m a y affect rates of proliferation d u e to their i n f l u e n c e in a p o p t o s i s , t h e y r e p r e s e n t o n l y a s e g m e n t of the r e q u i r e d m u t a t i o n s r e q u i r e d to d e v e l o p m a l i g n a n c i e s . W h i l e the ability of B c l - 2 to contribute to d r u g r e s i s t a n c e h a s b e e n s h o w n in m a n y s t u d i e s , there a r e s o m e e x c e p t i o n s , a n d the c o n t r a d i c t o r y  nature  of the f i n d i n g s  q u e s t i o n i n g of its viability a s a m o d u l a t o r of d r u g r e s i s t a n c e .  surrounding  B c l - 2 h a v e led  to  A s stated previously, s o m e explanations  m a y lie not only in the g e n e r a l b i o c h e m i c a l function of B c l - 2 , but a l s o in the a p p a r e n t t i s s u e specificity of this protein.  F u r t h e r m o r e , difficulties  in vitro, in vivo,  between  in a s s e s s i n g the i m p o r t a n c e of B c l - 2 t o w a r d s d r u g  sensitivity  a n d clinical s t u d i e s m a y b e d u e a n u m b e r of r e a s o n s p e r t a i n i n g to the m o d e l  s y s t e m e m p l o y e d . T h e s e i n c l u d e the o b v i o u s e l e m e n t s s u c h a s t u m o r e n v i r o n m e n t , w h i c h is not a factor in  in vitro  a b s e n t in  s t u d i e s , a n d other c o n s i d e r a t i o n s s u c h a s i m m u n e e f f e c t s , w h i c h a r e significant in the clinic, but  in vitro  s t u d i e s , a n d limited in  in vivo  experiments.  A s w e l l , a n e x t r e m e l y important factor that  v a r i e s highly b e t w e e n s t u d i e s , is the type of d r u g u s e d a n d the l e v e l s of d r u g e x p o s u r e . In c o n t r a s t to in  vitro  e x p e r i m e n t s w h i c h utilize c o n s t a n t d r u g e x p o s u r e at fixed c o n c e n t r a t i o n s , c l i n i c a l a n d  e x p o s u r e t e n d s to b e m o r e c y c l i c a l in nature a n d d r u g c o n c e n t r a t i o n s m o r e v a r i a b l e . a m o u n t s of d r u g e x p o s u r e  in vivo  in vivo  drug  V a r i a b i l i t y of  or in c l i n i c a l s t u d i e s will b e a f f e c t e d b y f a c t o r s s u c h a s b l o o d flow to  different o r g a n s , d r u g diffusion f r o m the b l o o d v e s s e l s , protein b i n d i n g of t h e d r u g , lipid solubility of the d r u g , a n d rates of d r u g m e t a b o l i s m in the liver.  T h i s c o n t r i b u t e s a d e g r e e of uncertainty to s t u d i e s  p e r f o r m e d u s i n g clinical t u m o r s a m p l e s or a n i m a l m o d e l s . T h u s , d i f f e r e n c e s in t i s s u e t y p e , a s w e l l a s f a c t o r s p e r t a i n i n g to c e l l u l a r e n v i r o n m e n t s a n d d r u g e x p o s u r e , s e r v e to c o m p l i c a t e the interpretation of results f r o m e x p e r i m e n t s d e s i g n e d to d e t e r m i n e the function of B c l - 2 in r e s p o n s e to a p o p t o t i c a g e n t s .  In spite of t h e s e d i s c r e p a n c i e s , h i g h l e v e l s of B c l - 2  h a v e b e e n s h o w n to protect c e l l s f r o m a p o p t o s i s a n d i n c r e a s e cell s u r v i v a l in a m y r i a d of cell l i n e s , a n d e x p r e s s i o n level r e m a i n s a strong indicator of c h e m o r e s p o n s i v e n e s s . therefore antibodies  b e e n the target of t h e r a p e u t i c directed  E x p e r i m e n t s outlined  against  Bcl-2,  and  intervention antisense  in this t h e s i s a p p l y two  W i t h this in m i n d , B c l - 2 h a s  using such approaches as hormone oligonucleotides  s p e c i f i c for  of t h e s e a p p r o a c h e s in a t t e m p t s  r e s i s t a n c e , a n d further e l u c i d a t e the i m p o r t a n c e of this s o m e w h a t e n i g m a t i c g e n e .  the  bcl-2  ablation, mRNA.  to a t t e n u a t e  drug  39  1.3.6 REFERENCES Krajewski S , Krajewska M , S h a b a i k A , Miyashita T, W a n g H G , R e e d J C . d e t e r m i n a t i o n of in vivo distribution of B a x , a d o m i n a n t inhibitor of B c l - 2 . Am J Pathol. 1 9 9 4 D e c ; 1 4 5 ( 6 ) : 1 3 2 3 - 3 6 . 1  Immunohistochemical  B a r g o u R C , D a n i e l P T , M a p a r a M Y , B o m m e r t K, W a g e n e r C , K a l l i n i c h B, R o y e r H D , D o r k e n B . E x p r e s s i o n of the b c l - 2 g e n e family in n o r m a l a n d m a l i g n a n t b r e a s t t i s s u e : l o w b a x - a l p h a e x p r e s s i o n in t u m o r c e l l s c o r r e l a t e s with r e s i s t a n c e t o w a r d s a p o p t o s i s . IntJ Cancer. 1 9 9 5 M a r 1 6 ; 6 0 ( 6 ) : 8 5 4 - 9 . 2  K r a j e w s k i S , K r a j e w s k a M , R e e d J C . I m m u n o h i s t o c h e m i c a l a n a l y s i s of in v i v o patterns of B a k e x p r e s s i o n , a p r o a p o p t o t i c m e m b e r of the B c l - 2 protein family. Cancer Res. 1 9 9 6 J u n 1 5 ; 5 6 ( 1 2 ) : 2 8 4 9 55.  3  R e e d J C . Bcl-2 a n d the regulation of p r o g r a m m e d cell d e a t h . J Cell Biol. 1 9 9 4 J a n ; 1 2 4 ( 1 - 2 ) : 1 - 6 . Review.  4  T s u j i m o t o Y . S t r e s s - r e s i s t a n c e c o n f e r r e d by high level of bcl-2 a l p h a protein in h u m a n B l y m p h o b l a s t o i d c e l l . Oncogene. 1 9 8 9 N o v ; 4 ( 1 1 ) : 1 3 3 1 - 6 . 5  H o c k e n b e r y D M , O l t v a i Z N , Y i n X M , M i l l i m a n C L , K o r s m e y e r S J . B c l - 2 f u n c t i o n s in a n antioxidant p a t h w a y to p r e v e n t a p o p t o s i s . Cell. 1 9 9 3 O c t 22;75(2):241 - 5 1 .  6  S t r a s s e r A , Harris A W , J a c k s T , C o r y S . D N A d a m a g e c a n i n d u c e a p o p t o s i s in proliferating cells v i a p 5 3 - i n d e p e n d e n t m e c h a n i s m s inhibitable by B c l - 2 . Cell. 1 9 9 4 O c t 1;79(2):329-39. 7  lymphoid  S t r a s s e r A , A n d e r s o n R L . B c l - 2 a n d t h e r m o t o l e r a n c e c o o p e r a t e in cell s u r v i v a l . C e l l G r o w t h Differ. 1 9 9 5 Jul;6(7):799-805. 8  G a z i t t Y , R o t h e n b e r g M L , H i l s e n b e c k S G , F e y V , T h o m a s C , M o n t e g o m r e y W . B c l - 2 o v e r e x p r e s s i o n is a s s o c i a t e d with r e s i s t a n c e to p a c l i t a x e l , but not g e m c i t a b i n e , in multiple m y e l o m a c e l l s . Int J Oncol. 1 9 9 8 Oct;13(4):839-48.  9  M i y a k e H , H a r a I, Y a m a n a k a K, A r a k a w a S , K a m i d o n o S . S y n e r g i s t i c e n h a n c e m e n t of r e s i s t a n c e to cisplatin in h u m a n b l a d d e r c a n c e r c e l l s b y o v e r e x p r e s s i o n of m u t a n t - t y p e p 5 3 a n d B c l - 2 . J Urol. 1 9 9 9 1 0  Dec;162(6):2176-81. M i y a k e H , H a n a d a N , N a k a m u r a H , K a g a w a S , F u j i w a r a T , H a r a I, E t o H , G o h j i K, A r a k a w a S , K a m i d o n o S , S a y a H . O v e r e x p r e s s i o n of B c l - 2 in b l a d d e r c a n c e r c e l l s inhibits a p o p t o s i s i n d u c e d b y cisplatin a n d a d e n o v i r a l - m e d i a t e d p 5 3 g e n e transfer. Oncogene. 1 9 9 8 F e b 1 9 ; 1 6 ( 7 ) : 9 3 3 - 4 3 . 1 1  L a s o r e l l a A , l a v a r o n e A , Israel M A . Differentiation of n e u r o b l a s t o m a e n h a n c e s B c l - 2 e x p r e s s i o n a n d i n d u c e s alterations of a p o p t o s i s a n d d r u g r e s i s t a n c e . Cancer Res. 1 9 9 5 O c t 1 5 ; 5 5 ( 2 0 ) : 4 7 1 1 - 6 . 1 2  K a j i w a r a T , T a k e u c h i T , U e k i T , M o r i y a m a N , U e k i K, K a k i z o e T , K a w a b e K. Effect of B c l - 2 o v e r e x p r e s s i o n in h u m a n prostate c a n c e r c e l l s in vitro a n d in v i v o . Int J Urol. 1 9 9 9 c t ; 6 ( 1 0 ) : 5 2 0 - 5 . 1 3  Y a n g Q F , S a k u r a i T , Y o s h i m u r a G , S h a n L, S u z u m a T , T a m a k i T , U m e m u r a T , K o k a w a Y , N a k a m u r a Y , N a k a m u r a M , T a n g W , U t s u n o m i y a H , M o r i I, K a k u d o K. E x p r e s s i o n o f B c l - 2 but not B a x o r p 5 3 c o r r e l a t e s with in vitro r e s i s t a n c e to a s e r i e s of a n t i c a n c e r d r u g s in b r e a s t c a r c i n o m a . Breast Cancer Res Treat. 2 0 0 0 J u n ; 6 1 ( 3 ) : 2 1 1 - 6 1 4  G i b s o n L F , F o r t n e y J , M a g r o G , E r i c s o n S G , L y n c h J P , L a n d r e t h K S . R e g u l a t i o n of B A X a n d B C L - 2 e x p r e s s i o n in b r e a s t c a n c e r c e l l s by c h e m o t h e r a p y . Breast Cancer Res Treat. 1 9 9 9 M a y ; 5 5 ( 2 ) : 1 0 7 - 1 7 . 1 5  40  B e a l e P J , R o g e r s P , B o x a l l F, S h a r p S Y , K e l l a n d L R . B C L - 2 f a m i l y protein e x p r e s s i o n a n d platinum d r u g r e s i s t a n c e in o v a r i a n c a r c i n o m a . BrJ Cancer. 2 0 0 0 J a n ; 8 2 ( 2 ) : 4 3 6 - 4 0 . 1 6  M u r p h y K L , Kittrell F S , G a y J P , J a g e r R, M e d i n a D, R o s e n J M . B c l - 2 e x p r e s s i o n d e l a y s m a m m a r y t u m o r d e v e l o p m e n t in d i m e t h y l b e n z ( a ) a n t h r a c e n e - t r e a t e d t r a n s g e n i c m i c e . Oncogene. 1 9 9 9 N o v 11;18(47):6597-604. 1 7  A r r i o l a E L , R o d r i g u e z - L o p e z A M , H i c k m a n J A , C h r e s t a C M . B c l - 2 o v e r e x p r e s s i o n results in r e c i p r o c a l d o w n r e g u l a t i o n of B c l - X ( L ) a n d s e n s i t i z e s h u m a n testicular g e r m cell t u m o u r s to c h e m o t h e r a p y - i n d u c e d a p o p t o s i s . Oncogene. 1 9 9 9 F e b 1 8 ; 1 8 ( 7 ) : 1 4 5 7 - 6 4 . 1 8  R e v i e w e d in K u m a r , S Apoptosis: Biology and Mechanisms. H e i d e l b e r g , G e r m a n y : S p r i n g e r P r e s s . 1999. p.185  1 9  J a g e r R, H e r z e r U , S c h e n k e l J , W e i h e r H . O v e r e x p r e s s i o n o f B c l - 2 inhibits a l v e o l a r cell a p o p t o s i s d u r i n g involution a n d a c c e l e r a t e s c - m y c - i n d u c e d t u m o r i g e n e s i s o f the m a m m a r y g l a n d in t r a n s g e n i c m i c e . Oncogene. 1 9 9 7 O c t 9; 15(15): 1 7 8 7 - 9 5 . 2 0  M i y a k e H , H a n a d a N , N a k a m u r a H , K a g a w a S , F u j i w a r a T , H a r a I, E t o H , G o h j i K, A r a k a w a S , K a m i d o n o S , S a y a H . O v e r e x p r e s s i o n of B c l - 2 in b l a d d e r c a n c e r c e l l s inhibits a p o p t o s i s i n d u c e d b y cisplatin a n d a d e n o v i r a l - m e d i a t e d p 5 3 g e n e transfer. Oncogene. 1 9 9 8 F e b 1 9 ; 1 6 ( 7 ) : 9 3 3 - 4 3 .  2 1  N a k a y a m a K, N a k a y a m a K, N e g i s h i I, K u i d a K, S a w a H , L o h D Y . T a r g e t e d d i s r u p t i o n o f B c l - 2 a l p h a b e t a in m i c e : o c c u r r e n c e of g r a y hair, p o l y c y s t i c k i d n e y d i s e a s e , a n d l y m p h o c y t o p e n i a .  2 2  Proc Natl Acad Sci USA. 1 9 9 4 A p r 2 6 ; 9 1 ( 9 ) : 3 7 0 0 - 4 . N a k a y a m a K, N a k a y a m a K, N e g i s h i I, K u i d a K, S h i n k a i Y , L o u i e M C , F i e l d s L E , L u c a s P J , S t e w a r t V , Alt F W , et a l . D i s a p p e a r a n c e o f the l y m p h o i d s y s t e m in B c l - 2 h o m o z y g o u s m u t a n t c h i m e r i c m i c e . Science. 1 9 9 3 S e p 1 7 ; 2 6 1 ( 5 1 2 8 ) : 1 5 8 4 - 8 . 2 3  M o t o y a m a N , W a n g F, R o t h K A , S a w a H , N a k a y a m a K, N a k a y a m a K, N e g i s h i I, S e n j u S , Z h a n g Q , Fujii S , et a l . M a s s i v e cell d e a t h of i m m a t u r e h e m a t o p o i e t i c c e l l s a n d n e u r o n s in B c l - x - d e f i c i e n t m i c e . Science. 1 9 9 5 M a r 1 0 ; 2 6 7 ( 5 2 0 3 ) : 1 5 0 6 - 1 0 .  2 4  K a j i w a r a T , T a k e u c h i T , U e k i T , M o r i y a m a N , U e k i K, K a k i z o e T , K a w a b e K. E f f e c t o f B c l - 2 o v e r e x p r e s s i o n in h u m a n prostate c a n c e r c e l l s in vitro a n d in v i v o . Int J Urol. 1 9 9 9 c t ; 6 ( 1 0 ) : 5 2 0 - 5 . 2 5  R a f f o A J , P e r l m a n H , C h e n M W , D a y M L , S t r e i t m a n J S , B u t t y a n R . O v e r e x p r e s s i o n o f b c l - 2 protects prostate c a n c e r c e l l s f r o m a p o p t o s i s in vitro a n d c o n f e r s r e s i s t a n c e to a n d r o g e n d e p l e t i o n in v i v o . Cancer Res. 1 9 9 5 O c t 1 ; 5 5 ( 1 9 ) : 4 4 3 8 - 4 5 .  2 6  M i y a k e H , H a r a I, Y a m a n a k a K, G o h j i K, A r a k a w a S , K a m i d o n o S . O v e r e x p r e s s i o n o f B c l - 2 e n h a n c e s m e t a s t a t i c potential of h u m a n b l a d d e r c a n c e r c e l l s . BrJ Cancer. 1 9 9 9 A p r ; 7 9 ( 1 1 - 1 2 ) : 1 6 5 1 - 6 .  2 7  D e l B u f a l o D , B i r o c c i o A , Leonetti C , Z u p i G . B c l - 2 o v e r e x p r e s s i o n e n h a n c e s t h e m e t a s t a t i c potential o f a h u m a n b r e a s t c a n c e r line. F A S E B J . 1 9 9 7 O c t ; 1 1 ( 1 2 ) : 9 4 7 - 5 3 . , 2 8  P e z z e l l a F , G a t t e r K. W h a t is t h e v a l u e o f b c l - 2 protein d e t e c t i o n for h i s t o p a t h o l o g i s t s ? Histopathology. 1 9 9 5 J a n ; 2 6 ( 1 ) : 8 9 - 9 3 . R e v i e w  2 9  R e e d J C . Bcl-2: prevention of apoptosis a s a m e c h a n i s m of drug resistance. Hematol Oncol Clin North Am. 1 9 9 5 A p r ; 9 ( 2 ) : 4 5 1 - 7 3 . R e v i e w .  3 0  R e e d J C . R e g u l a t i o n of a p o p t o s i s b y b c l - 2 family proteins a n d its role in c a n c e r a n d c h e m o r e s i s t a n c e . CurrOpin Oncol. 1 9 9 5 N o v ; 7 ( 6 ) : 5 4 1 - 6 . R e v i e w .  3 1  S t r a s s e r A , H u a n g D C , V a u x D L . T h e role o f the b c l - 2 / c e d - 9 g e n e f a m i l y in c a n c e r a n d g e n e r a l i m p l i c a t i o n s of d e f e c t s in cell d e a t h control for t u m o u r i g e n e s i s a n d r e s i s t a n c e to c h e m o t h e r a p y . Biochim  3 2  41  Biophys Acta. 1 9 9 7 O c t 2 4 ; 1 3 3 3 ( 2 ) : F 1 5 1 - 7 8 . R e v i e w . K a s s i m S K , Ali H S , S a l l a m M M , F a y e d S T , S e a d a L S , abd-Elkawy E, S e a d a M A , Khalifa A . Increased bcl-2 e x p r e s s i o n is a s s o c i a t e d with p r i m a r y r e s i s t a n c e to c h e m o t h e r a p y in h u m a n epithelial o v a r i a n c a n c e r . Clin Biochem. 1 9 9 9 J u l ; 3 2 ( 5 ) : 3 3 3 - 8 . 3 3  Bonetti A , Z a n i n e l l i M , L e o n e R, C e t t o G L , P e l o s i G , B i o l o S , M e n g h i A , M a n f r i n E , B o n e t t i F , P i u b e l l o Q . b c l - 2 but not p 5 3 e x p r e s s i o n is a s s o c i a t e d with r e s i s t a n c e to c h e m o t h e r a p y in a d v a n c e d b r e a s t c a n c e r . Clin Cancer Res. 1 9 9 8 O c t ; 4 ( 1 0 ) : 2 3 3 1 - 6 .  3 4  H a a r m a n E G , K a s p e r s G J , P i e t e r s R, v a n Zantwijk C H , B r o e k e m a G J , H a h l e n K, V e e r m a n A J . B C L - 2 e x p r e s s i o n in c h i l d h o o d l e u k e m i a v e r s u s s p o n t a n e o u s a p o p t o s i s , d r u g i n d u c e d a p o p t o s i s , a n d in vitro drug r e s i s t a n c e . Adv Exp Med Biol. 1 9 9 9 ; 4 5 7 : 3 2 5 - 3 3 .  3 5  S i l v e s t r i n i R, V e n e r o n i S , D a i d o n e M G , B e n i n i E , B o r a c c h i P , M e z z e t t i M , D i F r o n z o G , R i l k e F , V e r o n e s i U . T h e B c l - 2 protein: a p r o g n o s t i c indicator s t r o n g l y related to p 5 3 protein in l y m p h n o d e n e g a t i v e b r e a s t c a n c e r patients. J Natl Cancer Inst. 1 9 9 4 A p r 6 ; 8 6 ( 7 ) : 4 9 9 - 5 0 4 . 3 5  M a n n e U , M y e r s R B , M o r o n C , P o c z a t e k R B , Dillard S , W e i s s H , B r o w n D , S r i v a s t a v a S , G r i z z l e W E . P r o g n o s t i c s i g n i f i c a n c e o f B c l - 2 e x p r e s s i o n a n d p 5 3 n u c l e a r a c c u m u l a t i o n in c o l o r e c t a l a d e n o c a r c i n o m a . Int J Cancer. 1 9 9 7 J u n 2 0 ; 7 4 ( 3 ) : 3 4 6 - 5 8 . R e v i e w .  3 7  P e z z e l l a F, T u r l e y H , K u z u I, T u n g e k a r M F , Dunnill M S , P i e r c e C B , H a r r i s A , G a t t e r K C , M a s o n D Y . bcl-2 protein in n o n - s m a l l - c e l l lung c a r c i n o m a . N Engl J Med. 1 9 9 3 S e p 2 ; 3 2 9 ( 1 0 ) : 6 9 0 - 4 .  3 8  R o c h a i x P , K r a j e w s k i S , R e e d J C , B o n n e t F , V o i g t J J , B r o u s s e t P . In v i v o patterns o f B c l - 2 f a m i l y protein e x p r e s s i o n in b r e a s t c a r c i n o m a s in relation to a p o p t o s i s . J Pathol. 1 9 9 9 M a r ; 1 8 7 ( 4 ) : 4 1 0 - 5 .  3 9  H e l l e m a n s P , v a n D a m P A , W e y l e r J , v a n O o s t e r o m A T , Buytaert P , V a n M a r c k E . Prognostic value of b c l - 2 e x p r e s s i o n in i n v a s i v e b r e a s t c a n c e r . 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Cell Death Differ. 2 0 0 0 M a y ; 7 ( 5 ) : 4 3 9 - 4 6 .  4 2  M c C u r r a c h M E , C o n n o r T M , K n u d s o n C M , K o r s m e y e r S J , L o w e S W . b a x - d e f i c i e n c y p r o m o t e s drug r e s i s t a n c e a n d o n c o g e n i c t r a n s f o r m a t i o n b y attenuating p 5 3 - d e p e n d e n t a p o p t o s i s .  4 3  Proc Natl Acad Sci USA. 1 9 9 7 M a r 18;94(6):2345-9. B a r g o u R C , D a n i e l P T , M a p a r a M Y , B o m m e r t K, W a g e n e r C , K a l l i n i c h B , R o y e r H D , D o r k e n B . E x p r e s s i o n of the b c l - 2 g e n e f a m i l y in n o r m a l a n d m a l i g n a n t b r e a s t t i s s u e : l o w b a x - a l p h a e x p r e s s i o n in t u m o r c e l l s c o r r e l a t e s with r e s i s t a n c e t o w a r d s a p o p t o s i s . Int J Cancer. 1 9 9 5 M a r 6 ; 6 0 ( 6 ) : 8 5 4 - 9 .  4 4  S c h m i t t E , S t e y a e r t A , C i m o l i G , B e r t r a n d R. B a x - a l p h a p r o m o t e s a p o p t o s i s i n d u c e d b y c a n c e r c h e m o t h e r a p y a n d a c c e l e r a t e s t h e activation of c a s p a s e 3-like c y s t e i n e p r o t e a s e s in p 5 3 d o u b l e mutant B l y m p h o m a N a m a l w a c e l l s . Cell Death Differ. 1 9 9 8 J u n ; 5 ( 6 ) : 5 0 6 - 1 6 .  4 5  S h i n o u r a N , Y o s h i d a Y , A s a i A , Kirino T , H a m a d a H . R e l a t i v e level o f e x p r e s s i o n of B a x a n d B c l - X L d e t e r m i n e s t h e c e l l u l a r fate o f a p o p t o s i s / n e c r o s i s i n d u c e d b y the o v e r e x p r e s s i o n o f B a x . Oncogene. 1 9 9 9 O c t 7 ; 1 8 ( 4 1 ) : 5 7 0 3 - 1 3 .  4 6  G i b s o n L F , Fortney J , M a g r o G , Ericson S G , Lynch J P , Landreth K S . Regulation of B A X and B C L - 2 e x p r e s s i o n in b r e a s t c a n c e r c e l l s b y c h e m o t h e r a p y . Breast Cancer Res Treat. 1 9 9 9 M a y ; 5 5 ( 2 ) : 1 0 7 - 1 7 .  4 7  42  M i y a s h i t a T , R e e d J C . T u m o r s u p p r e s s o r p 5 3 is a direct transcriptional a c t i v a t o r o f the h u m a n b a x g e n e . Cell. 1 9 9 5 J a n 2 7 ; 8 0 ( 2 ) : 2 9 3 - 9 . R a f f o A J , K i m A L , F i n e R L . F o r m a t i o n o f n u c l e a r B a x / p 5 3 c o m p l e x e s is a s s o c i a t e d with c h e m o t h e r a p y i n d u c e d a p o p t o s i s . Oncogene. 2 0 0 0 D e c 1 4 ; 1 9 ( 5 4 ) : 6 2 1 6 - 2 8 .  4 9  H s u Y T , W o l t e r K G , Y o u l e R J . C y t o s o l - t o - m e m b r a n e redistribution o f B a x a n d B c l - X ( L ) during a p o p t o s i s . Proc Natl Acad Sci U S A. 1 9 9 7 A p r 1 5 ; 9 4 ( 8 ) : 3 6 6 8 - 7 2 .  5 0  Brady H J , S a l o m o n s G S , Bobeldijk R C , Berns A J . T cells from baxalpha transgenic mice s h o w a c c e l e r a t e d a p o p t o s i s in r e s p o n s e to stimuli but d o not s h o w r e s t o r e d D N A d a m a g e - i n d u c e d cell d e a t h in the a b s e n c e of p 5 3 . g e n e p r o d u c t i n d u c e d cell d e a t h in the a b s e n c e o f p 5 3 EMBOJ. 1 9 9 6 M a r 15;15(6): 1 2 2 1 - 3 0 . 5 1  K n u d s o n C M , T u n g K S , Tourtellotte W G , B r o w n G A , K o r s m e y e r S J . B a x - d e f i c i e n t m i c e with l y m p h o i d h y p e r p l a s i a a n d m a l e g e r m cell d e a t h . Science. 1 9 9 5 O c t 6 ; 2 7 0 ( 5 2 3 3 ) : 9 6 - 9 .  5 2  K n u d s o n C M , J o h n s o n G M , L i n Y , K o r s m e y e r S J . B a x a c c e l e r a t e s t u m o r i g e n e s i s in p 5 3 - d e f i c i e n t m i c e . Cancer Res. 2001 J a n 1 5 ; 6 1 ( 2 ) : 6 5 9 - 6 5 .  5 3  Brady H J , S a l o m o n s G S , Bobeldijk R C , Berns A J . T cells from baxalpha transgenic mice s h o w a c c e l e r a t e d a p o p t o s i s in r e s p o n s e to stimuli but d o not s h o w r e s t o r e d D N A d a m a g e - i n d u c e d cell d e a t h in the a b s e n c e o f p 5 3 . g e n e p r o d u c t i n d u c e d cell d e a t h in the a b s e n c e o f p 5 3 EMBOJ. 1 9 9 6 M a r 1 5 ; 1 5 ( 6 ) : 1 2 2 1 - 3 0 . 5 4  M c P a k e C R , T i l l m a n D M , P o q u e t t e C A , G e o r g e E O , H o u g h t o n J A , H a r r i s L C . B a x is a n important d e t e r m i n a n t o f c h e m o s e n s i t i v i t y in pediatric t u m o r cell lines i n d e p e n d e n t o f B c l - 2 e x p r e s s i o n a n d p 5 3 s t a t u s . Oncol Res. 1 9 9 8 ; 1 0 ( 5 ) : 2 3 5 - 4 4 .  5 5  R a m p i n o N , Y a m a m o t o H , l o n o v Y , Li Y , S a w a i H , R e e d J C , P e r u c h o M . S o m a t i c f r a m e s h i f t m u t a t i o n s in the B A X g e n e in c o l o n c a n c e r s of the microsatellite m u t a t o r p h e n o t y p e . Science. 1 9 9 7 F e b 14;275(5302):967-9.  5 6  P a t a e r A , F a n g B , Y u R, K a g a w a S , H u n t K K , M c D o n n e l l T J , R o t h J A , S w i s h e r S G . A d e n o v i r a l B a k o v e r e x p r e s s i o n m e d i a t e s c a s p a s e - d e p e n d e n t t u m o r killing. Cancer Res. 2 0 0 0 F e b 1 5 ; 6 0 ( 4 ) : 7 8 8 - 9 2 . 5 7  M o s s S F , A g a r w a l B , A r b e r N , G u a n R J , K r a j e w s k a M , K r a j e w s k i S , R e e d J C , Holt P R . I n c r e a s e d intestinal B a k e x p r e s s i o n results in a p o p t o s i s . Biochem Biophys Res Commun. 1 9 9 6 J u n 5 ; 2 2 3 ( 1 ) : 1 9 9 203.  5 8  C h i t t e n d e n T , H a r r i n g t o n E A , O ' C o n n o r R, F l e m i n g t o n C , L u t z R J , E v a n G I , G u i l d B C . Induction o f a p o p t o s i s b y the B c l - 2 h o m o l o g u e B a k . Nature. 1 9 9 5 A p r 2 0 ; 3 7 4 ( 6 5 2 4 ) : 7 3 3 - 6 . 5 9  W a n g G Q , G a s t m a n B R , W i e c k o w s k i E, Goldstein L A , Gambotto A , K i m T H , F a n g B, Rabinovitz A , Y i n X M , R a b i n o w i c h H . A R o l e for M i t o c h o n d r i a l B a k in A p o p t o t i c R e s p o n s e t o A n t i c a n c e r D r u g s . J Biol Chem. 2001 S e p 7 ; 2 7 6 ( 3 6 ) : 3 4 3 0 7 - 3 4 3 1 7 . 6 0  . W a n g G Q , G a s t m a n B R , W i e c k o w s k i E, Goldstein L A , Gambotto A , K i m T H , F a n g B, Rabinovitz A , Y i n X M , R a b i n o w i c h H . A R o l e for M i t o c h o n d r i a l B a k in A p o p t o t i c R e s p o n s e t o A n t i c a n c e r D r u g s . J Biol Chem. 2001 S e p 7 ; 2 7 6 ( 3 6 ) : 3 4 3 0 7 - 3 4 3 1 7 . 6 1  G r a b e r HL), F r i e s s H , Z i m m e r m a n n A , K o r c M , A d l e r G , S c h m i d R, B u c h l e r M W . B a k e x p r e s s i o n a n d cell d e a t h o c c u r in p e r i t u m o r o u s t i s s u e but not in p a n c r e a t i c c a n c e r c e l l s . J Gastrointest Surg. 1 9 9 9 J a n - F e b ; 3 ( 1 ) : 7 4 - 8 0 ;  6 2  J o n e s N A , T u r n e r J , M c l l w r a t h A J , B r o w n R, D i v e C . C i s p l a t i n - a n d p a c l i t a x e l - i n d u c e d a p o p t o s i s o f o v a r i a n c a r c i n o m a c e l l s a n d t h e relationship b e t w e e n b a x a n d b a k u p - r e g u l a t i o n a n d t h e f u n c t i o n a l s t a t u s 6 3  43 o f p 5 3 . Mol Pharmacol. 1 9 9 8 M a y ; 5 3 ( 5 ) : 8 1 9 - 2 6 . E g u c h i H , S u g a K, S a j i H , T o i M , N a k a c h i K, H a y a s h i S I . Different e x p r e s s i o n patterns of B c l - 2 f a m i l y g e n e s in b r e a s t c a n c e r by e s t r o g e n r e c e p t o r s t a t u s with s p e c i a l r e f e r e n c e to p r o - a p o p t o t i c B a k g e n e . Cell Death Differ. 2 0 0 0 M a y ; 7 ( 5 ) : 4 3 9 - 4 6 .  6 4  44  1.4 ANTISENSE THERAPEUTICS 1.4.1 BASIC PRINCIPLES OF ANTISENSE OLIGONUCLEOTIDES  A n t i s e n s e o l i g o n u c l e o t i d e s a r e short s e g m e n t s of s y n t h e s i z e d D N A d e s i g n e d to b i n d to, a n d i n d u c e the d e g r a d a t i o n of, the m R N A of a target g e n e .  S t e a d y s t a t e c o n d i t i o n s of the c e l l  e n s u r e that the r e d u c t i o n or a b s e n c e of significant l e v e l s of m R N A for this g e n e result in the down-regulation  of c o m p l i m e n t a r y  protein  l e v e l s a n d the  b i o l o g i c a l effects that m a y  result.  A l t h o u g h o n e might s u r m i s e that d e s i g n of a n t i s e n s e m o l e c u l e s m a y b e d e t e r m i n e d a priori b a s e d o n g e n e s e q u e n c e s , the s e q u e n c e of t h e s e m o l e c u l e s a r e inevitably d e t e r m i n e d e m p i r i c a l l y d u e to the c o m p l e x a n d s o m e w h a t i n s c r u t a b l e t h r e e - d i m e n s i o n a l structure of target m R N A s . T h e r e a r e s e v e r a l w a y s in w h i c h a n t i s e n s e o l i g o n u c l e o t i d e s m a y p r e v e n t m R N A from b e i n g t r a n s l a t e d into protein.  T h i s i n c l u d e s the s u p p r e s s i o n of R N A s p l i c i n g , t r a n s l a t i o n arrest,  a n d interruption of R N A s e c o n d a r y structure, i n c l u d i n g 5 ' c a p s a n d 3' p o l y a d e n y l a t i o n s i t e s  1 , 2 , 3  .  W h i l e m a n y of t h e s e m e c h a n i s m s r e m a i n to b e c l e a r l y c h a r a c t e r i z e d , the o n e w e l l - c h a r a c t e r i z e d m e c h a n i s m by w h i c h a n t i s e n s e o l i g o n u c l e o t i d e s inhibit m R N A t r a n s l a t i o n is t h e a c t i v a t i o n  of  R N a s e H (Figure 1.3). R N a s e H is a u b i q u i t o u s e n z y m e that d e g r a d e s the R N A s t r a n d of a n R N A - D N A d u p l e x , s u c h a s that f o r m e d w h e n a n t i s e n s e o l i g o n u c l e o t i d e s bind to m R N A . 4  Its natural function in the  cell is in D N A replication a n d it is p r i m a r y f o u n d in the n u c l e u s , a l t h o u g h cytoplasm as well . 5  it o c c u r s in the  A l t h o u g h the e x a c t m e a n s by w h i c h this e n z y m e r e c o g n i z e s , b i n d s , a n d  s u b s e q u e n t l y d e g r a d e s the R N A s t r a n d of the R N A - D N A c o m p l e x is still b e i n g i n v e s t i g a t e d , it is k n o w n that D N A - R N A d u p l e x e s a s short a s four b a s e - p a i r s l o n g m a y b e r e c o g n i z e d b y R N a s e H  6 , 7  .  T h i s finding l e a d s into a c h a r a c t e r i s t i c of o l i g o n u c l e o t i d e s w h i c h is v e r y important to their  function: length.  G e n e r a l l y , a n t i s e n s e m o l e c u l e s u s e d for t h e r a p e u t i c p u r p o s e s c o n t a i n o n the  o r d e r of 1 5 - 2 0 b a s e s .  I n c r e a s i n g the length of o l i g o n u c l e o t i d e s m a y affect their u p t a k e b y the  c e l l , d u e in l a r g e part to i n c r e a s e s in c h a r g e . O l i g o n u c l e o t i d e c h a r g e is d e t e r m i n e d b y s e t 8  45  FIGURE 1.3 Mechanism of Action of Oligonucleotides. A n t i s e n s e m o l e c u l e s bind m R N A in a s e q u e n c e specific m a n n e r and induce the R N a s e degradation of the R N A strand of the d u p l e x , thereby preventing translation of the m R N A .  46  c h a r g e p e r unit length -  the l o n g e r the m o l e c u l e , the l a r g e r the c h a r g e .  Conversely, an  o l i g o n u c l e o t i d e that is too s m a l l d o e s not h a v e the s p e c i f i c i t y of a l a r g e r m o l e c u l e a n d i n c r e a s e s the d a n g e r of irrelevant c l e a v a g e a n d the d e s t r u c t i o n of m R N A s other t h a n the d e s i r e d target. T h o u g h the s p e c i f i c i t y of a n t i s e n s e m o l e c u l e s h a s l o n g b e e n t h o u g h t of a s o n e of its a d v a n t a g e s , the d e g r e e to w h i c h this is true is s o m e w h a t d e b a t a b l e .  A s p r e v i o u s l y m e n t i o n e d , R N a s e H is  c a p a b l e of r e c o g n i z i n g R N A - D N A d u p l e x e s a s short a s four b a s e - p a i r s in l e n g t h . T h e r e f o r e , a n y g i v e n a n t i s e n s e m o l e c u l e m a y c o n t a i n a n u m b e r of s e q u e n c e s c a p a b l e of i n d u c i n g R N a s e H d e g r a d a t i o n in a h o s t of irrelevant t a r g e t s . 9  A l t h o u g h this factor m a y b e c o m b a t e d by c a r e f u l  s e q u e n c e s e l e c t i o n a n d m o d i f i c a t i o n to the m o l e c u l e ( d i s c u s s e d b e l o w ) , it is s o m e t h i n g w h i c h should  be  considered  oligonucleotides.  whenever  evaluating  data  from  studies  involving  antisense  In g e n e r a l , a n t i s e n s e m o l e c u l e s s h o u l d b e u s e d in c o n j u n c t i o n with control  s e q u e n c e s in o r d e r to d e l i n e a t e s e q u e n c e - s p e c i f i c e f f e c t s .  Controls m a y include scrambled  s e q u e n c e s that d o not c o r r e s p o n d to a n y k n o w n g e n e s e g m e n t but retain the s a m e b a s e c o m p o s i t i o n , m i s m a t c h s e q u e n c e s w h e r e o n e or m o r e b a s e s a r e a l t e r e d in c o m p a r i s o n to the a n t i s e n s e s e q u e n c e , or r e v e r s e polarity  or s e n s e s e q u e n c e s , w h i c h  are complimentary  to  antisense sequences. While  early  antisense  oligonucleotides  (phosphodiester  oligonucleotides)  were  s y n t h e s i z e d with s t r u c t u r e s identical to natural D N A , t h e s e m o l e c u l e s w e r e s o o n f o u n d to b e s u s c e p t i b l e to d e g r a d a t i o n b y n u c l e a s e s , primarily in the 3' to 5' d i r e c t i o n . 10  Therefore, attempts  w e r e m a d e to m o d i f y the o l i g o n u c l e o t i d e s - primarily in the s u g a r m o i e t i e s or p h o s p h a t e g r o u p s in o r d e r to m a k e t h e m resistant to n u c l e a s e s . O n e of the first, a n d m o s t effective, m o d i f i c a t i o n s w a s the r e p l a c e m e n t of a n o n - b r i d g i n g o x y g e n in the p h o s p h a t e g r o u p with a sulfur to yield phosphorothioate oligonucleotides. o x y g e n with a m e t h y l  O t h e r m o d i f i c a t i o n s i n v o l v e d the r e p l a c e m e n t o f this s a m e  g r o u p to p r o d u c e  methyl  p h o s p h o n a t e ( M P ) o l i g o n u c l e o t i d e s or  the  substitution of the entire p h o s p h a t e g r o u p with a p e p t i d e b o n d to c r e a t e p e p t i d e n u c l e i c a c i d s ( P N A ) . L a t e r g e n e r a t i o n m o l e c u l e s i n c o r p o r a t e d c o m b i n a t i o n s of b a c k b o n e m o d i f i c a t i o n s , u s u a l l y in the f o r m of o l i g o n u c l e o t i d e s with m o d i f i e d e n d s , s u g a r m o d i f i c a t i o n s to i n c r e a s e stability o n c e b o u n d to R N A , a n d e v e n o l i g o n u c l e o t i d e s i n c l u d i n g s e g m e n t s of R N A . It s h o u l d b e n o t e d ,  47  h o w e v e r , that a n y alteration  of t h e structure  of t h e o l i g o n u c l e o t i d e will affect a n u m b e r of  c h a r a c t e r i s t i c s of t h e o l i g o n u c l e o t i d e . T h e s e i n c l u d e e l e m e n t s s u c h a s t h e ability of the m o d i f i e d o l i g o n u c l e o t i d e to i n d u c e R N a s e melting  point,  or chirality;  toxicity  H degradation; c h e m i c a l properties of the m o l e c u l e ;  cellular  uptake  s u c h a s the solubility, a n d the ability  of the  o l i g o n u c l e o t i d e to c r o s s m e m b r a n e s ; a n d protein b i n d i n g , distribution a n d m e t a b o l i s m in vivo. A l l of t h e s e of c h a r a c t e r i s t i c s m u s t b e t a k e n into a c c o u n t w h e n d e s i g n i n g a n o l i g o n u c l e o t i d e for therapeutic purposes. A l t h o u g h significant in the c o n t e x t of the e f f i c a c y of o l i g o n u c l e o t i d e s t h e r a p e u t i c s , t h e different t y p e s of m o d i f i c a t i o n s a n d the a d v a n t a g e s a n d d i s a d v a n t a g e s to e a c h a r e m u c h too e x t e n s i v e to d i s c u s s h e r e . T h e B c l - 2 a n t i s e n s e m o l e c u l e u s e d in o u r e x p e r i m e n t s , G e n a s e n s e ™ ( G 3 1 3 9 ) , is a p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e , a n d therefore further d i s c u s s i o n will b e limited to the attributes of this t y p e of o l i g o n u c l e o t i d e .  1.4.2 PHOSPHOTHIOATE ANTISENSE OLIGONUCLEOTIDES IN VITRO  Uptake and Extracellular Stability  T h e r e p l a c e m e n t of a n o n - b r i d g i n g o x y g e n with a sulfur a t o m in t h e b a c k b o n e of a n o l i g o n u c l e o t i d e c r e a t e s a n a n t i s e n s e m o l e c u l e that r e m a i n s c h a r g e d a n d h y d r o p h i l i c , but is a l s o chiral at e a c h r e s i d u e a n d highly resistant to n u c l e a s e s . M o r e importantly, h o w e v e r , it retains its ability to i n v o k e R N a s e  H d e g r a d a t i o n of R N A . 1 1  O n c e p l a c e d in t i s s u e culture m e d i a for  t r a n s f e c t i o n into c e l l s , t h e s e n e g a t i v e l y - c h a r g e d o l i g o n u c l e o t i d e s rapidly b i n d s e r u m primarily a l b u m i n .  proteins,  P r o t e i n s a c t a s c o m p e t i t i v e inhibitors for n u c l e a s e s p r e s e n t in t h e s e r u m , a n d  this, in c o m b i n a t i o n  with their chirality,  m a k e s them  s t a b l e in culture  media  1 2 , 1 3  .  Although  o l i g o n u c l e o t i d e s will bind proteins in a m a n n e r that m a y b e n o n - s p e c i f i c , s e q u e n c e s p e c i f i c a n d structure s p e c i f i c (all w h i c h m a y affect their stability in m e d i a ) , t h e half-life of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s a p p e a r s to b e a p p r o x i m a t e l y 2 4 h o u r s in t h e p r e s e n c e of fetal calf s e r u m  1 4 , 1 5  .  48  T h e u p t a k e of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s is t h o u g h t to b e b r o u g h t a b o u t t h r o u g h receptor-mediated  endocytosis, although  the degree  to w h i c h  o l i g o n u c l e o t i d e to o l i g o n u c l e o t i d e a n d cell type to cell t y p e . 1 6  this  occurs  may  vary  from  T h e uptake of these antisense  m o l e c u l e s is t i m e a n d t e m p e r a t u r e d e p e n d e n t , a n d other f a c t o r s g r e a t l y contribute to variability in a b s o r p t i o n o f p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s , i n c l u d i n g e l e m e n t s s u c h a s cell t y p e , t y p e o f m e d i u m , d e g r e e of c o n f l u e n c e , a n d the p r e s e n c e of s e r u m .  Furthermore, uptake m a y also b e  i n f l u e n c e d by the structure of the o l i g o n u c l e o t i d e itself s u c h a s c h e m i c a l m o d i f i c a t i o n s , the length of the oligo, a n d e v e n the s e q u e n c e . 1 7  A s m e n t i o n e d p r e v i o u s l y , the b i n d i n g o f s e r u m  protein  a d d s to the stability o f o l i g o n u c l e o t i d e s in culture, but it a l s o a p p e a r s to e n h a n c e the a b s o r p t i o n a s w e l l ; larger a m o u n t s o f n a k e d o l i g o n u c l e o t i d e (without uptake enhancement reagents) a r e a b s o r b e d with i n c r e a s e s in the level of fetal calf s e r u m in the culture m e d i a . 1 8  T h e a b s o r p t i o n of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s u s u a l l y r e a c h e s s t e a d y state within several  hours  and  oligonucleotides  tend  to a c c u m u l a t e  within t h e c e l l  (in c o m p a r i s o n to  c o n c e n t r a t i o n s in the e x t r a c e l l u l a r m e d i u m ) . A l t h o u g h s e r u m p r o t e i n s m a y a s s i s t s o m e w h a t in 1 9  the u p t a k e o f o l i g o n u c l e o t i d e s , the total a b s o r p t i o n o f o l i g o n u c l e o t i d e s u n a i d e d b y a n  artificial  carrier (e.g. c a t i o n i c lipids) is quite p o o r , g e n e r a l l y r a n g i n g b e t w e e n 0 . 5 % a n d 2 4 % o f the total a m o u n t of o l i g o n u c l e o t i d e s d e p e n d i n g o n cell t y p e . 2 0  in this  thesis, only  1.6%  In the c a s e o f M C F - 7 c e l l s , a c e l l line u s e d  to 2 . 2 % of total o l i g o n u c l e o t i d e s  used was  taken  up when the  o l i g o n u c l e o t i d e s a l o n e w e r e a p p l i e d to c e l l s . 2 1  W i t h s u c h a m e a g e r a n d ineffective a b s o r p t i o n rate of o l i g o n u c l e o t i d e s in vitro, a n u m b e r of t e c h n i q u e s a n d r e a g e n t s h a v e b e e n e m p l o y e d to i n c r e a s e the d e g r e e o f u p t a k e . include using strepsolysin D, electroporation, calcium phosphate precipitation,  These  microinjection,  a s s o c i a t i o n with p o l y c a t i o n s , p o l y a m i d o a m i n e d e n d r i m e r s , l i p o s o m e s a n d c a t i o n i c lipids. T h e m o s t p o p u l a r of t h e s e transfection r e a g e n t s o r t e c h n i q u e s a r e c a t i o n i c lipids, a n d m a n y c o m m e r c i a l f o r m s of t h e s e carrier m o l e c u l e s exist.  Generally, these preparations combine  a f u s o g e n i c lipid that is a d e p t a t b i n d i n g with the p l a s m a m e m b r a n e , with a c a t i o n i c lipid that b i n d s the o l i g o n u c l e o t i d e ; saturate  p r o t o c o l s e m p l o y i n g t h e s e lipids utilize a n e x c e s s positive c h a r g e to  the negatively-charged  antisense  molecule.  Unfortunately,  the efficacy  of t h e s e  49  p r e p a r a t i o n s v a r i e s , a n d a p p e a r s to b e highly s p e c i f i c to cell t y p e a n d a n t i s e n s e m o l e c u l e u s e d . Furthermore, themselves  while  the phosphorothioate  are even  more  toxic,  and  oligonucleotide  it is t h e s e  itself  is toxic,  compounds  which  the cationic  lipids  limit t h e a m o u n t o f  o l i g o n u c l e o t i d e s that m a y b e u s e d , . 2 2  2 3  Cellular Distribution of Oligonucleotides and Efflux  Once  i n s i d e t h e c e l l , t h e majority o f o l i g o n u c l e o t i d e s  a p p e a r to a c c u m u l a t e  c y t o p l a s m , with s o m e in the n u c l e u s , l y s o s o m e s , a n d m i t o c h o n d r i a however,  seems  temporally  dependent,  as some  studies  have  2 4 , 2 5 , 2 6  .  indicated  This  in t h e  distribution,  a c c u m u l a t i o n of  o l i g o n u c l e o t i d e s in t h e n u c l e u s o v e r t i m e . 2 7  T h e efflux o f o l i g o n u c l e o t i d e s f r o m c e l l s , like u p t a k e , is a l s o t i m e a n d dependent.  temperature  S t u d i e s u s i n g r a d i o l a b e l e d o l i g o n u c l e o t i d e s indicate that efflux t e n d s to b e b i p h a s i c ,  with half-life of the initial p h a s e b e i n g a b o u t 1 0 - 3 0 m i n u t e s , a n d the final p h a s e m u c h s l o w e r . 2 8  A l t h o u g h m o r e resistant to n u c l e a s e s than p h o s p h o d i e s t e r s , p h o s p h o r o t h i o a t e s a r e n o n e t h e l e s s a l s o d e g r a d e d by n u c l e a s e s , a n d the intracellular half life o f p h o s p h o r o t h i o a t e s in v a r i o u s cell lines h a s b e e n  reported  to b e b e t w e e n  1 2 to 2 4 h o u r s , a l t h o u g h  significant  amounts of  29 30  o l i g o n u c l e o t i d e h a v e a l s o b e e n reported up to 7 2 h o u r s after i n c u b a t i o n  , .  T h e a p p l i c a t i o n of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s in vitro m a y p r e s e n t p r o b l e m s , d u e primarily to p o o r u p t a k e of the m o l e c u l e , a n d efforts to transfect c e r t a i n cell lines with e n o u g h o l i g o n u c l e o t i d e in o r d e r to b e e f f i c a c i o u s m a y p r o v e difficult if not i m p o s s i b l e . however, the use of phosphorothioate  Surprisingly,  o l i g o n u c l e o t i d e s in vivo is g e n e r a l l y e a s i e r , with n o  r e q u i r e m e n t of a d d i t i o n a l d e l i v e r y m e t h o d s to e n h a n c e c e l l u l a r u p t a k e .  A l t h o u g h not c o m p l e t e l y  u n d e r s t o o d , introduction o f p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s into c e l l s in vivo is a i d e d b y the b i n d i n g of p l a s m p r o t e i n s . the  in vivo  W h i l e this a s p e c t o f the in vitro e n v i r o n m e n t m a y b e a d v a n t a g e o u s ,  e n v i r o n m e n t is m u c h m o r e c o m p l e x a n d other e l e m e n t s m u s t b e c o n s i d e r e d .  50  1.4.3 PHOSPHOTHIOATE ANTISENSE OLIGONUCLEOTIDES IN VIVO  T h e a p p l i c a t i o n of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s to a n i m a l m o d e l s p r o v i d e s a m o r e a c c u r a t e a s s e s s m e n t of their e f f i c a c y a n d u s e f u l n e s s , but a l s o i n t r o d u c e s a w h o l e n e w h o s t of factors  not p r e s e n t  oligonucleotides  in the in  include  vitro m o d e l .  nuclease  Factors  degradation,  influencing  protein  binding,  the pharmacokinetics plasma  clearance,  of  tissue  distribution, a n d c e l l u l a r u p t a k e . O n c e i n s i d e t h e c e l l , the e l e m e n t s w h i c h affect o l i g o n u c l e o t i d e s in vitro a l s o exist, i n c l u d i n g s u b c e l l u l a r distribution, availability of target m R N A , affinity, selectivity, efflux a n d n u c l e a s e d i g e s t i o n .  F u r t h e r m o r e , in vivo m o d e l s h a v e t h e a d d e d c o m p l i c a t i o n s of  v a r i a t i o n s in o l i g o n u c l e o t i d e e x p o s u r e to target c e l l s a s w e l l a s u n f o r e s e e n  immunostimulatory  effects.  Blood and Tissue Distribution of Phosphorothioate Oligonucleotides  P h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s a r e u s u a l l y rapidly c l e a r e d f r o m t h e b l o o d , d u e l a r g e l y to  tissue  distribution  oligonucleotide  used  and  not  metabolism  in e x p e r i m e n t s  in this  3 1 , 3 2 , 3 3 , 3 4  .  thesis,  Kinetics  of  the  phosphorothioate  G e n a s e n s e (G3139),  in t h e v a s c u l a r  c o m p a r t m e n t is t r i p h a s i c , with half-lives of 5 m i n u t e s , 3 7 m i n u t e s , a n d 11 h o u r s . 3 5  T i s s u e distribution of p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s is w i d e s p r e a d , with significant levels of this m o l e c u l e f o u n d in t h e s p l e e n , p a n c r e a s , a d r e n a l g l a n d , s a l i v a r y g l a n d , m e s e n t e r i c l y m p h n o d e s , d u o d e n u m a n d i l e u m , a n d m o s t strikingly in t h e k i d n e y a n d liver, a n d s u g g e s t s high d e g r e e s of u p t a k e of o l i g o n u c l e o t i d e s in t h e s e o r g a n s ; l e v e l s w e r e all but a b s e n t f r o m t h e brain and  fat  3 6 , 3 7  .  Interestingly,  some  studies  have  shown  that t i s s u e  distribution  m a y also be  d e p e n d e n t u p o n s e q u e n c e , a s a n t i s e n s e m o l e c u l e s of the s a m e length but differing s e q u e n c e s 38  d i s p l a y e d v a r y i n g t i s s u e distributions  .  T h e s e results w e r e born out with s t u d i e s involving G e n a s e n s e w h e r e this m o l e c u l e w a s d e t e c t e d in significant a m o u n t s  in the l u n g s , s p l e e n , heart, m u s c l e , t e s t e s , large a n d s m a l l  intestine, a n d at high l e v e l s in the k i d n e y a n d liver; it w a s a l m o s t a b s e n t f r o m t h e b r a i n . L e v e l s of  51  radioactivity f r o m  S - l a b e l e d G e n a s e n s e r e v e a l e d t i s s u e to p l a s m a ratios of 8 7 for k i d n e y , 17 for  liver, 5 for s p l e e n , 2 . 5 for heart a n d l u n g , a n d 3.5 for gut. initially  bound  by  proteins  (98%  at  5 minutes  M o s t of the a n t i s e n s e m o l e c u l e w a s  post-injection),  although  this  declined  upon  39  distribution to the t i s s u e s a s o n l y 2 3 % w a s protein b o u n d at 2 4 h o u r s .  Metabolism Metabolism  of  phosphorothioate  oligonucleotides  as  measured  by  plasma  content  a p p e a r s to o c c u r quite rapidly at first f o l l o w e d by a r e d u c e d rate of d e g r a d a t i o n , with d e g r a d a t i o n initially o c c u r r i n g f r o m the 3 ' e n d of the m o l e c u l e through p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s is d e t e r m i n a t e  3'exonucleases.  T h e chirality  of  in their rate of d i g e s t i o n b y n u c l e a s e s , a s  different e n a n t i o m e r s h a v e different affinities for n u c l e a s e s . 4 0  T h i s is s i g n i f i c a n t s i n c e e a c h  r e s i d u e of a p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e is c h i r a l , y i e l d i n g a r a c e m i c m i x t u r e of m o l e c u l e s with 2  n  d i a s t e r e o m e r s w h e r e n is the n u m b e r of r e s i d u e s ( e . g . a 18 b a s e pair o l i g o n u c l e o t i d e  w o u l d c o n t a i n o v e r 2 6 0 , 0 0 0 individual d i a s t e r e o m e r s ) .  M e t a b o l i s m m a y a l s o o c c u r t h r o u g h the  e x c h a n g e of the sulfur in p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s with a n o x y g e n , w h i c h w o u l d in effect revert the r e s i d u e b a c k to a p h o s p h o d i e s t e r l i n k a g e , m a k i n g it m u c h m o r e s u s c e p t i b l e to e n d o nucleases . 4 1  P h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s a p p e a r b e quite s t a b l e in m o s t t i s s u e s , e x c e p t for the k i d n e y a n d liver, w h i c h is indicative of a h i g h e r rate of m e t a b o l i s m in t h e s e o r g a n s .  Furthermore,  the n u m b e r of m e t a b o l i c p r o d u c t s in t h e s e o r g a n s is a l s o g r e a t e r , p o s s i b l y indicating a larger r a n g e of n u c l e a s e s p r e s e n t .  S t u d i e s i n d i c a t e that in m o s t t i s s u e s , the rate of d e g r a d a t i o n of  p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s is a b o u t 5 0 % at 4 8 h o u r s  4 2  G e n a s e n s e is m e t a b o l i z e d into three different p r o d u c t s , all of w h i c h a r e f o u n d in the p l a s m a , k i d n e y a n d liver.  T h e parent c o m p o u n d is m e t a b o l i z e d r a p i d l y at first, with o n l y 3 6 % of  the p a r e n t c o m p o u n d p r e s e n t in the p l a s m a after 6 h o u r s (similar r e s u l t s f o u n d in the liver); this rate of m e t a b o l i s m d e c l i n e s h o w e v e r , a s 2 5 % of the parent c o m p o u n d is still p r e s e n t after 4 8 h o u r s . F u r t h e r m o r e , the m e t a b o l i c p r o d u c t s elute earlier than the p a r e n t a l c o m p o u n d . 4 3  52  Elimination  While distribution of phosphorothioate oligonucleotides to various tissues and clearance from the vascular compartments is rapid, elimination of these molecules from the tissue is slow. Studies involving an  35  S - l a b e l e d 25 base-pair phosphorothioate oligonucleotide administered  intravenously revealed low levels remaining in tissues up to ten days after injection, particularly in the kidney . 44  Other studies involving whole body tissue-associated radioactivity indicated the  presence of low levels of radioactivity in tissues after seven days, also primarily in the liver and kidney . 45  In terms of the elimination rates of G e n a s e n s e , studies indicated that 33% of total radiation was excreted via the urine by day 1 post-injection and 56% by day 3 and 17% of total radiation by day 1 through the feces and 38% by day 3.  Furthermore, primarily metabolites, and  virtually no parent compound, are secreted in the feces and the urine . 46  1.4.4  IMMUNE EFFECTS  One attribute of phosphorothioate oligonucleotides that must be taken into consideration  in vivo  when using these molecules  is their ability to stimulate the immune system.  Immune  stimulation may occur due to the poly-anion nature of these molecules, and phosphorothioate oligonucleotides  have  been  shown to  induce cytokine  release . 47  However,  the  primary  immunostimulatory mechanism of phosphorothioate oligonucleotides, and antisense molecules in general, is the presence of C p G motifs.  C p G motifs are cytosine-guanine sequences that occur  in unmethylated form in bacterial D N A , while in vertebrate D N A they contain a methyl group on the 5 position of the cytosine ; moreover, the C G sequences themselves occur much more often 48  in bacterial  D N A than  in vertebrate  DNA.  The vertebrate  immune system is capable of  recognizing the unmethylated sequences that occur in bacterial D N A and the presence of these  53  motifs is effective  in activating a n u m b e r of c e l l s in t h e i m m u n e s y s t e m , i n c l u d i n g B - c e l l s ,  m a c r o p h a g e s , dendritic c e l l s , a n d natural killer c e l l s . 4 9  In t h e c o n t e x t of a n t i s e n s e t h e r a p e u t i c s  a n d their u s e in vivo, t h e s e motifs m a y p r e s e n t s o m e c o m p l i c a t i o n s in e v a l u a t i n g d a t a d e r i v e d f r o m t h e u s e of o l i g o n u c l e o t i d e s w h i c h c o n t a i n C G s e q u e n c e s  5 0 , 5 1  ; in e s s e n c e , this r e l a t e s to t h e  d e t e r m i n a t i o n of w h e t h e r a s p e c i f i c result is d u e to s e q u e n c e s p e c i f i c i t y o r i m m u n e e f f e c t s .  In  o r d e r to s o l v e this d i l e m m a , a p p r o p r i a t e o l i g o n u c l e o t i d e c o n t r o l s s h o u l d b e u s e d w h i c h c o n t a i n identical n u m b e r s of C p G motifs a s t h e a n t i s e n s e m o l e c u l e itself, s u c h that effects o c c u r r i n g in r e s p o n s e to t h e a n t i s e n s e m o l e c u l e a n d not t h e control c a n b e attributed to s e q u e n c e s p e c i f i c i t y a n d not i m m u n e s t i m u l a t i o n .  1.4.5 BCL-2 ANTISENSE  A s m e n t i o n e d p r e v i o u s l y , t h e a n t i s e n s e m o l e c u l e e m p l o y e d in o u r e x p e r i m e n t s w a s Genta's  Genasense™  (G3139).  This  molecule  is  an  18  base-pair  phosphorothioate  o l i g o n u c l e o t i d e s p e c i f i c f o r the first six c o d o n s of the o p e n r e a d i n g f r a m e of the b c l - 2 m R N A .  This  m o l e c u l e h a s b e e n u s e d e x t e n s i v e l y in in vitro a n d in vivo s t u d i e s , a s w e l l a s t w o c l i n i c a l trials. T h e following s u r v e y s s o m e of the results of t h e s e s t u d i e s a n d p r o v i d e s a b a s i s f o r the e v a l u a t i o n of this c o m p o u n d a s a v i a b l e t h e r a p e u t i c c o m p o u n d .  Efficacy of Bcl-2 Antisense in vitro  T h e majority of the s t u d i e s in vitro a s s e s s e d t h e ability of B c l - 2 a n t i s e n s e to i n d u c e a p o p t o s i s a n d s e n s i t i z e c e l l s to a p o p t o t i c a g e n t s .  T h e d o w n - r e g u l a t i o n o f B c l - 2 with a n t i s e n s e  o l i g o d e o x y n u c l e o t i d e s h a s b e e n s h o w n to inhibit t h e g r o w t h of p r o s t a t e t u m o u r  5 2  and non-  H o d g k i n ' s cell l i n e s , a s w e l l a s d e c r e a s e c e l l viability a n d i n d u c e a p o p t o s i s in g l i o m a s , B-cell 5 3  chronic  lymphocytic  5 4  leukemia c e l l s , acute myelogenous l e u k e m i a , Jurkat c e l l s , a n d a  n u m b e r of lung c a n c e r c e l l l i n e s  5 5  5 8 , 5 9 , 6 0  5 6  5 7  . A l t h o u g h B c l - 2 d o w n - r e g u l a t i o n h a s p r o v e n s u c c e s s f u l in  54 i n d u c i n g a p o p t o s i s in m a n y cell l i n e s , in f e w e r s t u d i e s h a s B c l - 2 a n t i s e n s e b e e n s h o w n c a p a b l e of c o n f e r r i n g sensitivity to c h e m o t h e r a p y d r u g s  6 1 , 6 2  '  6 3 , 6 4  ' . 6 5  T h e s e s t u d i e s i n v e s t i g a t e d the effect of B c l - 2 a n t i s e n s e o n c e l l u l a r c h a r a c t e r i s t i c s s u c h a s g r o w t h , viability, a n d drug sensitivity, a n d a l t h o u g h t h e y h a d s i m i l a r g o a l s in m i n d , different a p p r o a c h e s w e r e utilized to r e a c h this e n d . Variability in cell viability a s s a y s , a p o p t o t i c e n d p o i n t s , m R N A or protein a n a l y s i s , a n d t e c h n i q u e s a n d p r o t o c o l s utilized to a p p l y the a n t i s e n s e o c c u r r e d from o n e s t u d y to a n o t h e r , a n d the validity of different a p p r o a c h e s is s u b j e c t to c o m m e n t . O n e of the first d i f f e r e n c e s that is a p p a r e n t w h e n a s s e s s i n g different s t u d i e s is w h e t h e r the a u t h o r s a n a l y z e d m R N A or protein l e v e l s in c e l l s in r e s p o n s e to a n t i s e n s e treatment.  mRNA  w a s m o r e c o m m o n l y a n a l y z e d d u e to the fact that it is the direct s u b s t r a t e of the a n t i s e n s e molecule being applied.  A l t h o u g h this in itself p r o v i d e s a d v a n t a g e s , the d e g r e e of d o w n -  regulation of m R N A a n d s u b s e q u e n t d e c r e a s e s in the a m o u n t of B c l - 2 protein d o not a l w a y s c o r r e l a t e w e l l , a n d the i n c l u s i o n of W e s t e r n a n a l y s i s of protein l e v e l s is a l w a y s a d v i s a b l e . T h i s , h o w e v e r , in itself p r e s e n t s p r o b l e m s , e s p e c i a l l y in cell p o p u l a t i o n s w h e r e B c l - 2 treatment results in rapid a p o p t o s i s .  T h e o c c u r r e n c e of w i d e s p r e a d a p o p t o s i s p r o v i d e s o n l y a short  window  b e t w e e n the initial d o w n - r e g u l a t i o n of Bcl-2 a n d cell d e a t h , a n d t h e r e f o r e c a n c r e a t e difficulties in o b t a i n i n g s a m p l e s at the p r o p e r time. In t e r m s of a p o p t o t i c m a r k e r s , the m o s t c o m m o n l y a s s e s s e d e n d p o i n t s w e r e c a s p a s e activation, A n n e x i n V b i n d i n g , a n d T U N E L a s s a y s to m e a s u r e the d e g r e e of D N A c l e a v a g e . T h e i n c l u s i o n of t h e s e a s s a y s in a n y e x p e r i m e n t s s e e k i n g to e s t a b l i s h the p r e s e n c e of a p o p t o s i s is n e c e s s a r y a s cell viability a s s a y s s u c h a s trypan blue or M T T a s s a y s d o not d i s t i n g u i s h b e t w e e n n e c r o t i c a n d a p o p t o t i c c e l l s . F u r t h e r m o r e , it is prudent to divide t h e s e a s s a y s into e a r l y a p o p t o t i c e v e n t s , s u c h a s l o s s of m i t o c h o n d r i a l m e m b r a n e potential a n d A n n e x i n V b i n d i n g , a n d later a p o p t o t i c e v e n t s , s u c h a s D N A c l e a v a g e a n d c a s p a s e activation. Finally, in attempts to d e t e r m i n e c h e m o s e n s i t i v i t y of v a r i o u s cell lines in c o n j u n c t i o n with Bcl-2 a n t i s e n s e treatment,  several considerations must be resolved beforehand.  The  most  important of t h e s e a r e the interpretation of c h e m o s e n s i t i v i t y d a t a w h e n the B c l - 2 a n t i s e n s e itself is c y t o t o x i c or g r o w t h inhibitory.  C e l l p o p u l a t i o n s treated with a n t i s e n s e a n d d r u g m u s t  be  55  compared to controls which have received antisense treatment.  Comparison with completely  untreated control cells leads to an inability to delineate increased cytotoxicity as the result of the abrogation of drug resistance mechanisms from cytotoxicity induced by the sensitizing agent itself (i.e. Bcl-2 antisense).  Essentially, this condition will determine whether cytotoxicity from Bcl-2  antisense is simply additive when combined with chemotherapy drugs, or whether it is synergistic.  Efficacy of Bcl-2 Antisense in vivo  With the promising results emerging from in vitro studies, the efficacy of Bcl-2 antisense to inhibit tumor growth or formation in mice has also been investigated.  In addition, studies  involving the effectiveness of Bcl-2 antisense as an adjuvant to chemotherapy have been completed. Bcl-2 antisense, in the form of Genasense, has been shown to delay the progress of Bcell lymphomas in mice and even to completely eradicate lymphoma cells and cure mice when applied in conjunction with cyclophosphamide  66,67  . It has also been effective in the treatment of  melanoma cells, where Genasense was shown to sensitize cells to dacarbazine treatment in SCID m i c e . Some of the most extensive research, however, involves the use of Bcl-2 antisense 68  in the treatment of prostate cells. With Shinogi cells, a murine cell line, Bcl-2 antisense delays the emergence of androgen-independent tumors in mice, and this delay only increases when combined with chemotherapy treatment  69,70  .  Interestingly, the combination of Bcl-2 antisense  with an antisense molecule of another Bcl-2 family member, Bcl-X , also delays the emergence of L  androgen-independence in mice, and this effect is increased even further when these agents are combined with chemotherapy . Likewise, studies involving the L N C a P human prostate cell line 71  indicated similar results; Bcl-2 antisense delayed the formation of androgen-independent tumors in nude mice, and this effect was increased when combined with chemotherapy  72,73  .  Lastly,  studies done with human breast carcinoma cell lines in vivo indicated the ability of Bcl-2 antisense to impair the growth of tumors in comparison to untreated or mismatch controls, and  56  this g r o w t h i m p a i r m e n t w a s s u b s t a n t i a l l y i n c r e a s e d w h e n c o m b i n e d with p a c l i t a x e l , d o c e t a x e l , d o x o r u b i c i n or c i s p l a t i n  7 4 , 7 5  .  Bcl-2 Antisense in Clinical Studies T w o c l i n i c a l trials utilizing G e n a s e n s e h a v e p r e s e n t l y b e e n p u b l i s h e d - o n e in m e l a n o m a a n d o n e in l y m p h o m a - a n d m a n y others a r e u n d e r w a y . being  conducted  using  this  molecule  in  malignant  P r e s e n t l y , p h a s e III c l i n i c a l trials a r e  melanoma,  multiple  myeloma,  chronic  l y m p h o c y t i c l e u k e m i a ; p h a s e II in a c u t e m y e l o i d l e u k e m i a , c h r o n i c l y m p h o c y t i c l e u k e m i a , a n d prostate c a n c e r ; a n d other s t u d i e s in c o l o n c a n c e r , s m a l l cell l u n g c a n c e r , a n d a c u t e l e u k e m i a . 7 6  The  study  involving  malignant  melanoma  entailed  sub-cutaneous  G e n s a s e n s e in c o m b i n a t i o n with s t a n d a r d d a c a r b a z i n e ( D T I C ) t r e a t m e n t . m g / k g w e r e a d m i n i s t e r e d with up to 1 0 0 0 m g / m well t o l e r a t e d .  2  injections  of  D o s e s a s high a s 6 . 5  of D T I C a n d the c o m p o u n d w a s s h o w n to b e  M i l d h e m a t o l o g i c toxicities o c c u r r e d , s p e c i f i c a l l y l y m p h o p e n i a , a n d a s s e s s m e n t of  t u m o r r e s p o n s e s i n d i c a t e d 1 c o m p l e t e r e s p o n s e , 2 partial r e s p o n s e s , a n d 3 m i n o r r e s p o n s e s out of a total of 14 patients t r e a t e d . 77  In the n o n - H o d g k i n ' s l y m p h o m a s t u d y , G e n a s e n s e w a s a l s o i n j e c t e d s u b - c u t a n e o u s l y u p to a m a x i m d o s e of 195.8 m g / m / d . 2  In this s t u d y , g r e a t e r toxicities w e r e r e p o r t e d ,  primarily  t h r o m b o c y t o p e n i a , h y p o t e n s i o n , f e v e r a n d a s t h e n i a , a n d the m a x i m u m - t o l e r a t e d d o s e w a s 1 4 7 . 2 mg/m /d. 2  T h e r e s p o n s e s f r o m a total of 16 patients treated w e r e 1 c o m p l e t e r e s p o n s e , 2 m i n o r  r e s p o n s e s , nine c a s e s of s t a b l e d i s e a s e , a n d nine c a s e s of p r o g r e s s i v e d i s e a s e . 7 8  A l t h o u g h c o m p l e t e reports of o n g o i n g s t u d i e s h a v e not b e e n r e l e a s e d , e a r l y indicate s i m i l a r finding a s t h o s e in the two p u b l i s h e d s t u d i e s .  reports  T o x i c i t y is not d o s e - l i m i t i n g a n d  p l a s m a l e v e l s f o u n d in p r e c l i n i c a l in vivo s t u d i e s a r e a c h i e v a b l e .  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A n t i s e n s e B c l - 2 o l i g o d e o x y n u c l e o t i d e s inhibit p r o g r e s s i o n to a n d r o g e n - i n d e p e n d e n c e after c a s t r a t i o n in the S h i o n o g i t u m o r m o d e l . Cancer Res. 1 9 9 9 A u g  7 0  15;59(16):4030-4. M i y a k e H , M o n i a B P , G l e a v e M E . Inhibition of p r o g r e s s i o n to a n d r o g e n - i n d e p e n d e n c e b y c o m b i n e d a d j u v a n t t r e a t m e n t with a n t i s e n s e B C L - X L a n d a n t i s e n s e B c l - 2 o l i g o n u c l e o t i d e s plus taxol after c a s t r a t i o n in the S h i o n o g i t u m o r m o d e l . Int J Cancer. 2 0 0 0 J u n 1 5 ; 8 6 ( 6 ) : 8 5 5 - 6 2 . 7 1  Leung S , Miyake H, Zellweger T, Tolcher A , Gleave M E . Synergistic chemosensitization and inhibition of p r o g r e s s i o n to a n d r o g e n i n d e p e n d e n c e b y a n t i s e n s e B c l - 2 o l i g o d e o x y n u c l e o t i d e a n d paclitaxel in the L N C a P prostate t u m o r m o d e l . Int J Cancer. 2 0 0 1 M a r 1 5 ; 9 1 ( 6 ) : 8 4 6 - 5 0 .  7 2  G l e a v e M , T o l c h e r A , M i y a k e H, N e l s o n C , B r o w n B, B e r a l d i E , G o l d i e J . P r o g r e s s i o n to a n d r o g e n i n d e p e n d e n c e is d e l a y e d b y a d j u v a n t t r e a t m e n t with a n t i s e n s e B c l - 2  7 3  62  o l i g o d e o x y n u c l e o t i d e s after c a s t r a t i o n in the L N C a P prostate t u m o r m o d e l . 1999 Oct;5(10):2891-8.  Clin Cancer Res.  L o p e s de M e n e z e s D E , Hudon N, Mcintosh N, Mayer L D . Molecular and pharmacokinetic p r o p e r t i e s a s s o c i a t e d with the t h e r a p e u t i c s of bcl-2 a n t i s e n s e o l i g o n u c l e o t i d e G 3 1 3 9 c o m b i n e d with free a n d l i p o s o m a l d o x o r u b i c i n . C l i n C a n c e r R e s . 2 0 0 0 J u l ; 6 ( 7 ) : 2 8 9 1 - 9 0 2 . 7 4  Y a n g , D., L i n g , Y . , A l m a z a n , M . , G u o , R., M u r r a y , A . , B r o w n , B., L i p p m a n , M . E . . T u m o r R e g r e s s i o n of h u m a n b r e a s t c a r c i n o m a s b y c o m b i n a t i o n t h e r a p y of anti-bcl-2 a n t i s e n s e o l i g o n u c l e o t i d e a n d c h e m o t h e r a p e u t i c d r u g s . P r o c A m e r A s s C a n R e s . 1 9 9 9 M a r c h ; (40) 7 2 9 . 7 5  7 6  G e n t a Inc. W e b s i t e , h t t p : / / w w w . g e n t a . c o m / p r o g r a m s / p r o q r a m s . h t m .  A u g u s t 5, 2 0 0 1 .  J a n s e n B, W a c h e c k V, H e e r e - R e s s E , S c h l a g b a u e r - W a d l H , H o e l l e r C , L u c a s T, H o e r m a n n M , H o l l e n s t e i n U, Wolff K, P e h a m b e r g e r H . C h e m o s e n s i t i s a t i o n of m a l i g n a n t m e l a n o m a by B C L 2 antisense therapy. Lancet. 2000 N o v 18;356(9243):1728-33.  7 7  W a t e r s J S , W e b b A , C u n n i n g h a m D, C l a r k e P A , R a y n a u d F, di S t e f a n o F, C o t t e r F E . P h a s e I clinical a n d p h a r m a c o k i n e t i c s t u d y of b c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e t h e r a p y in patients with n o n H o d g k i n ' s l y m p h o m a . J Clin Oncol. 2 0 0 0 M a y ; 1 8 ( 9 ) : 1 8 1 2 - 2 3 . 7 8  63  1.5 MATERIALS AND METHODS 1.5.1 MATERIALS  Chemotherapy Drugs In o r d e r to fully c h a r a c t e r i z e the effect of Bcl-2 o n d r u g r e s i s t a n c e a n d r e s p o n s e s to a p o p t o s i s , w e utilized c h e m o t h e r a p e u t i c a g e n t s with differing m e c h a n i s m s o f a c t i o n .  Cisplatin, doxorubicin, and  p a c l i t a x e l w e r e c h o s e n for this r e a s o n , a s well a s their w i d e s p r e a d u s e a n d practicality for in vitro experiments.  Cisplatin Cisplatin  1  f u n c t i o n s similar to c l a s s i c a l alkylating a g e n t s a n d w a s first identified w h e n growth in  bacterial culture w a s limited u p o n e x p o s u r e to platinum e l e c t r o d e s .  Its structure c o n s i s t s o f a platinum  m o l e c u l e b o u n d to two c h l o r i n e g r o u p s a n d t w o a m m o n i a g r o u p s (Figure 1.4) a n d it is primarily u s e d to treat solid t u m o r s a n d is e s p e c i a l l y effective a g a i n s t testicular c a n c e r . C y t o t o x i c i t y of cisplatin is m e d i a t e d b y its ability to form interstrand c r o s s - l i n k s a n d a d d u c t s in DNA.  T h e c h l o r i n e g r o u p s of cisplatin a r e d i s p l a c e d b y n u c l e o p h i l i c a t o m s in t h e D N A structure a n d  cisplatin preferentially b i n d s at the N-7 p o s i t i o n s of g a u n i n e a n d a d e n i n e . C i s p l a t i n is effective during all p h a s e s of the cell c y c l e . S t u d i e s e m p l o y i n g r a d i o a c t i v e platinum indicated that it is w i d e l y d i s t r i b u t e d , highly b o u n d b y p l a s m a proteins u p o n injection, a n d that its terminal half-life is 58 to 72 h o u r s .  A s w e l l , this d r u g is  primarily e x c r e t e d in t h e urine. T h e principal toxicities of cisplatin a r e h e a r i n g l o s s , neurotoxicity, s e v e r e n a u s e a a n d v o m i t i n g , a n d k i d n e y d a m a g e , the latter of w h i c h is m o s t likely d u e to the a c c u m u l a t i o n o f the d r u g in the k i d n e y prior to e x c r e t i o n .  Doxorubicin D o x o r u b i c i n ( a d r i a m y c i n ) is a n a n t h r a c y c l i n e that w a s first i s o l a t e d f r o m Streptomyces peucetius 2  variant casesius o v e r forty y e a r s a g o (Figure 1.4). Its first u s e w a s to treat a c u t e  A. C l \ ,ci  / N H  3  p t  \ NH,  FIGURE 1.4 Structure of Cisplatin, Doxorubicin, Paclitaxel, and C6-Ceramide. A. Elemental platinum, source of cisplatin; 2D and 3D structure of cisplatin. B . Source of doxorubicin, Streptomyces peucetius variant casesius; 2D and 3D structure of doxorubicin. C. The Pacific yew tree (Taxis brevifolia), source of paclitaxel;; 2D and 3D structure of paclitaxel D . 2D structure of C6-Ceramide.  65 l e u k e m i a s , a n d a l t h o u g h it r e m a i n s a s t a n d a r d treatment for A M L , it u s e h a s e x p a n d e d to i n c l u d e other cancers a s well. D o x o r u b i c i n is c y t o t o x i c b y n o l e s s than four m e c h a n i s m s , w h i c h i n c l u d e :  1) inhibition of  T o p o i s o m e r a s e II a n d t h e s u b s e q u e n t disruption of D N A a n d R N A s y n t h e s i s ; 2) intercalation of D N A ; 3) g e n e r a t i o n of s i n g l e a n d d o u b l e s t r a n d b r e a k s in D N A ; a n d 4) free r a d i c a l p r o d u c t i o n . doxorubicin  is a l s o k n o w n  to disrupt  protein  s y n t h e s i s a n d i n d u c e cytotoxicity  Furthermore,  b y b i n d i n g to cell  m e m b r a n e s . N o t a b l y , cytotoxicity of d o x o r u b i c i n is effective during all p h a s e s of the cell c y c l e . 3  D o x o r u b i c i n is highly b o u n d b y p l a s m a proteins a n d t i s s u e , a n d is c h a r a c t e r i z e d b y w i d e a n d rapid distribution a n d s l o w e l i m i n a t i o n ; s t u d i e s i n d i c a t e d that t h e s u c c e s s i v e half-lives of d o x o r u b i c i n in p l a s m a a r e 5 m i n u t e s , 1 hour, a n d 30 h o u r s , a n d t h e majority of the d r u g is e x c r e t e d in bile. T o x i c i t i e s of d o x o r u b i c i n i n c l u d e hair l o s s , n a u s e a , v o m i t i n g , m u c o s i t i s , l o c a l t i s s u e n e c r o s i s at the site of injections, a n d o n e of the m o s t limiting, m y e l o s u p p r e s s i o n . A s w e l l , a n o t h e r limiting factor in the u s e of d o x o r u b i c i n is its high d e g r e e of c a r d i a c toxicity, p r o b a b l y r e l a t e d to t h e o x i d a t i v e p r o p e r t i e s of the d r u g .  Paclitaxel P a c l i t a x e l ( T a x o l ™ ) is a plant a l k a l o i d i s o l a t e d f r o m the bark a n d n e e d l e s of the P a c i f i c Y e w tree 4  {Taxis brevifolia){Figure  1.3).  D i s c o v e r e d forty y e a r s a g o , t h e u s e of this d r u g a s a n a n t i - c a n c e r a g e n t  w a s not initially e x p l o i t e d b e c a u s e of limitations in a m o u n t s of drug that c o u l d b e o b t a i n e d . P a c l i t a x e l w a s initially a p p r o v e d for the treatment of o v a r i a n c a n c e r , a l t h o u g h it is n o w u s e d to treat b r e a s t a n d lung c a r c i n o m a s a s well a s s o m e s a r c o m a s . P a c l i t a x e l is c y t o t o x i c to c e l l s b y virtue of its ability to bind m i c r o t u b u l e s a n d p r e v e n t their dissassembly.  T h u s , it is cytotoxic to c e l l s u n d e r g o i n g cell d i v i s i o n a n d is m o s t effective during the G M 2  p h a s e of the cell c y c l e . P a c l i t a x e l is w i d e l y d i s t r u b u t e d a n d highly b o u n d b y p l a s m a proteins a n d is e l i m i n a t e d primarily in the bile; it h a s a half-life of a p p r o x i m a t e l y 10 to 12 h o u r s . Primary  toxicities  of  paclitaxel  myelosuppression, specifically neutropenia.  C6-ceramide  include  peripheral  sensory  neuropathy  and  severe  66 Ceramide  5  is a c y t o t o x i c a g e n t that is a p o w e r f u l i n d u c e r o f a p o p t o s i s .  It is c r e a t e d b y t h e  c l e a v a g e the p h o s p h o c h o l i n e h e a d g r o u p o f the lipid s p h i n g o m y e l i n v i a s p h i n g o m y e l i n a s e . T h e resulting h y d r o p h o b i c m o l e c u l e , c e r a m i d e , c o n s i s t s of two a l i p h a t i c c h a i n s b o n d e d b y a a m i d e g r o u p C e r a m i d e production  (Figure 1.4)  in t h e cell m a y b e i n d u c e d b y a n u m b e r o f stimuli, i n c l u d i n g  ionizing  r a d i a t i o n , c y t o k i n e s , h e a t s h o c k , growth factor d e p r i v a t i o n , a n d t r e a t m e n t with c h e m o t h e r a p y d r u g s . A l t h o u g h the e x a c t p a t h w a y s b y w h i c h c e r a m i d e i n d u c e s a p o p t o s i s a r e still b e i n g i n v e s t i g a t e d , c e r a m i d e p r o d u c t i o n is intimately a s s o c i a t e d with the a p o p t o t i c p r o c e s s .  Anti-estrogen Compounds Initial e x p e r i m e n t s c o n c e r n i n g the d o w n - r e g u l a t i o n o f B c l - 2 t h r o u g h h o r m o n e w i t h d r a w a l i n v o l v e d media containing charcoal-stripped s e r u m .  A l t h o u g h r e m o v i n g e s t r o g e n f r o m s e r u m with c h a r c o a l w a s  effective, t h e p r o c e d u r e a l s o r e m o v e d f a c t o r s f r o m t h e s e r u m w h i c h a f f e c t e d t h e survivability o f cell culture, e s p e c i a l l y in the f a c e of a n t i s e n s e treatment. T h u s , a m o r e s p e c i f i c t e c h n i q u e u s i n g a n t i - e s t r o g e n c o m p o u n d s w a s utilized to a b r o g a t e the effect of e s t r o g e n s o n c e l l c u l t u r e s .  Faslodex™ (ICI 182780) F a s l o d e x ™ (ICI 1 8 2 7 8 0 ) is a p u r e s t e r o i d a l a n t i - e s t r o g e n c o m p o u n d c o n s i s t i n g o f a substitution at the 7 a - p o s i t i o n o f e s t r a d i o l with a n i n e - c a r b o n p e n t a f l u o r o p e n t y l s u l f i n y l g r o u p that w a s first s y n t h e s i z e d in 1991  (Figure 1.5) . It 6  is a p u r e a n t i e s t r o g e n c o m p o u n d in that it l a c k s a n y a g o n i s t i c tropic e s t r o g e n - l i k e  effects f o u n d with c o m p o u n d s s u c h a s T a m o x i f e n , a n d c o m p l e t e l y b l o c k s a l l s t i m u l a t o r y a c t i o n s o f estrogens.  T h e a n t i - e s t r o g e n effect o f F a s l o d e x is a c h i e v e d b y p r e v e n t i n g t h e n u c l e a r u p t a k e o f the  e s t r o g e n r e c e p t o r w h e n b o u n d to this c o m p o u n d . 7  67  FIGURE 1.5 Structure of 170 Estradiol and Antiestrogen Compound Faslodex. A. Structure of 17(3 Estradiol . B. Structure of the pure anti-estrogen compound Faslodex (ICI 182780) with a subsititution at the 7a position.  68  Cell Lines W e c h o s e to f o c u s o u r r e s e a r c h o n the role of B c l - 2 in b r e a s t t i s s u e . c o m m o n l y u s e d b r e a s t c a r c i n o m a c e l l s lines r e c e p t o r positive - that c o u l d b e u s e d in both  one estrogen-receptor  in vitro  and  in vivo  Therefore, we chose t w o  negative, and one estrogen-  experiments.  MDA-MB-435/LCC6 T h e M D A - M B - 4 3 5 cell line, f r o m w h i c h the M D A - M B - 4 3 5 / L C C 6 cell line w a s d e r i v e d , w a s t a k e n f r o m the pleural effusion of a 31 y e a r - o l d C a u c a s i a n f e m a l e with d u c t a l c a r c i n o m a of the m a m m a r y g l a n d w h o h a d r e c e i v e d no prior t r e a t m e n t . T h e M D A - M B - 4 3 5 / L C C 6 cell line w a s g e n e r a t e d f r o m the a s c i t e s 8  of n u d e m i c e injected with M D A - M B - 4 3 5 c e l l s , a n d is c a p a b l e of g r o w t h a s c i t e s a n d solid t u m o r s  in vivo  with a n i n c i d e n c e of a l m o s t  in vitro  a s w e l l a s f o r m i n g both  100%. M D A - M B - 4 3 5 / L C C 6  cells are  e s t r o g e n - r e c e p t o r - n e g a t i v e s p i n d l e - s h a p e d a d h e r e n t c e l l s w h i c h d o u b l e in t u m o r s i z e a p p r o x i m a t e l y 2 . 3 ± 0.7 d a y s w h e n injected into the m a m m a r y fat p a d of n u d e m i c e .  Furthermore, these cells  tested  were  with a h o s t of c h e m o t h e r a p y d r u g s , i n c l u d i n g p a c l i t a x e l , d o x o r u b i c i n , a n d c i s p l a t i n , a n d s h o w e d sensitivity that c o r r e l a t e d well with c l i n i c a l d a t a , s u g g e s t i n g that t h e s e c e l l s c o n t a i n n o e n d o g e n o u s r e s i s t a n c e mechanisms. MCF-7 T h e M C F - 7 cell line w a s e s t a b l i s h e d f r o m the pleural e f f u s i o n of a 6 9 - y e a r - o l d  female  Caucasian  with a d e n o c a r c i n o m a of the m a m m a r y g l a n d . M C F - 7 c e l l s a r e differentiated a d h e r e n t e s t r o g e n - r e c e p t o r 9  positive epithelial c e l l s that readily f o r m m o n o l a y e r s  in vitro  and form solid tumors  in vivo  if s u p p l e m e n t e d  with h u m a n 17(3-estradiol.  A n i m a l Models S t u d i e s e v a l u a t i n g B c l - 2 a n d drug r e s i s t a n c e b e l o w . T h e m o s t c o m m o n l y u s e d a n i m a l s u s e d for  in vivo  in vivo  in t h e s e m i c e that result in B a n d T cell d e f i c i e n c y a l s o Therefore,  since  chemotherapy  determinations  treatment were  of  the  drug  appear  resistance  and  w e r e c a r r i e d out in R a g - 2 M m i c e d e s c r i b e d studies are  scid  m i c e , h o w e v e r the m u t a t i o n s  to b e i n v o l v e d in D N A repair m e c h a n i s m s . apoptotic  g o a l of o u r s t u d i e s , t h e s e  response  mice were  not  in  conjunction  appropriate  for  with this  a p p l i c a t i o n . Inevitably, d u e to c o m p r o m i s e d D N A repair m e c h a n i s m s , t h e s e m i c e a r e e s p e c i a l l y s e n s i t i v e to s o m e c h e m o t h e r a p y d r u g s a n d t h e s e a g e n t s incur g r e a t e r toxicity in m i c e c o m p a r e d to c o n t r o l s . 10  69  RAG2-M Mice RAG2-M  1 1  m i c e w e r e first c r e a t e d in 1 9 9 2 a n d h a v e a s e v e r e c o m b i n e i m m u n e deficient ( S C I D )  p h e n o t y p e o w i n g to the h o m o z y g o u s k n o c k o u t of a large portion of the recombination activating gene 2  (rag2) g e n e . T h i s g e n e is r e q u i r e d for the p r o p e r a s s e m b l y of v a r i a b l e (V), diversity (D), a n d j o i n i n g (J) g e n e s e g m e n t s that e n c o d e for the i m m u n o g l o b i n (Ig) m o l e c u l e s of B l y m p h o c y t e s a n d the T cell r e c e p t o r ( T C R ) of T l y m p h o c y t e s .  W i t h o u t t h e s e m o l e c u l e s , the m i c e lack f u n c t i o n a l B a n d T l y m p h o c y t e s a n d  contain no cell-mediated immunity a n d cannot recognize antigen. R A G 2 - M m i c e w e r e b r e d on a 1 2 9 / S v E v b a c k g r o u n d a n d h o m o z y g o u s m u t a n t s a n a l y z e d for i m m u n e function.  R A G 2 -/- m i c e c o n t a i n e d u n d e r d e v e l o p e d p r i m a r y l y m p h o i d o r g a n s ( s p l e e n a n d  t h y m u s ) a n d l a c k e d l g G + c e l l s in the b o n e m a r r o w a n d b l o o d a n d thymus.  CD8+ or CD4+ ( T C R ) c e l l s in the  H i s t o c h e m i c a l a n a l y s i s r e v e a l e d n o c h a n g e s in o r g a n s , other that the p r i m a r y l y m p h o i d o r g a n s ,  in c o m p a r i s o n with control m i c e ; this i n c l u d e d the liver, k i d n e y , p a n c r e a s , heart, l u n g , intestine a n d gonads.  F u r t h e r m o r e , a n a l y s e s of the viability of other i m m u n e c e l l s r e v e a l e d f u n c t i o n a l m y e l o i d c e l l s ,  including m a c r o p h a g e s a n d natural killer ( N K ) c e l l s .  1.5.2  METHODS  C e l l V i a b i l i t y and S u r v i v a l Assays In o r d e r to quantify  the effect  of c h e m o t h e r a p y d r u g s in c o n j u n c t i o n with s e v e r a l  putative  s e n s i t i z i n g a g e n t s , three different a s s a y s w e r e u s e d to d e t e r m i n e t h e relative a m o u n t of v i a b l e c e l l s a n d / o r their potential to f o r m n e w c e l l s .  MTT Assay T h e m e t h y l thiazolyl t e t r a z o l i u m ( M T T ) a s s a y is a c o m m o n n o n - c l o n o g e n i c in vitro a s s a y u s e d to a s s e s s drug sensitivity.  T h i s c o l o r o m e t r i c a s s a y m e a s u r e s the a m o u n t of v i a b l e c e l l s in a g i v e n s a m p l e  by their ability to c o n v e r t a t e t r a z o l i u m salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into a c o l o r e d f o r m a z a n p r o d u c t w h i c h m a y b e r e a d o n a s p e c t r o m e t e r ( s e e Figure 1.5).  This assay has  the a d v a n t a g e of e a s e - o f - u s e a n d p r o d u c i n g rapid r e s u l t s , a n d p r o v i d e d a s s e s s m e n t of the d r u g toxicity is p e r f o r m e d s e v e r a l h o u r s to d a y s after the initial drug e x p o s u r e , the r e s u l t s of this a s s a y c o r r e l a t e w e l l with traditional c l o n o g e n i c a s s a y s  1 2 , 1 3  .  70 H o w e v e r , r e l i a n c e u p o n c e l l s u r v i v a l a s a indicator of d r u g sensitivity m a y not a l w a y s b e a c c u r a t e , e s p e c i a l l y if m e a s u r e s of cell s u r v i v a l a r e d e t e r m i n e d too s o o n after d r u g e x p o s u r e . A s w e l l , cell viability in itself p r o v i d e s n o indication of the r e p r o d u c t i v e potential of a t u m o r c e l l p o p u l a t i o n - a m o r e relevant quality - a n d c h a n g e s in latent c l o n o g e n i c s u r v i v a l m a y not b e d e t e c t e d b y this a s s a y . T h e M T T protocol u s e d to a s s e s s d r u g sensitivity is outline in detail in F i g u r e 1.7.  O n d a y 1, cell  p o p u l a t i o n s in w h i c h the d r u g sensitivity is to b e a s s e s s e d a r e t r y p s i n i z e d , c o u n t e d o n a h e m o c y t o m e t e r , diluted to the d e s i r e d c o n c e n t r a t i o n , a n d t h e n p l a t e d in 9 6 - w e l l p l a t e s .  O n d a y 2 , s e r i a l dilutions of the  d e s i r e d d r u g c o n c e n t r a t i o n s a r e c r e a t e d a n d a d d e d to the c o r r e s p o n d i n g w e l l . M T T solution is a d d e d to the plates a n d a l l o w e d to i n c u b a t e for four h o u r s .  After 4 8 to 7 2 h o u r s , a n M T T a n d m e d i a a r e then  r e m o v e d a n d the f o r m a z a n p r o d u c t s o l u b i l i z e d in dimethyl s u l f o x i d e ( D M S O ) a n d r e a d o n a s p e c t r o m e t e r at  570 n m . T r y p a n Blue T r y p a n b l u e is a s t a i n u s e d in a d y e e x c l u s i o n a s s a y to a s s e s s c e l l viability;  live c e l l s e x c l u d e  t r y p a n b l u e w h i l e t r y p a n b l u e is t a k e n up by d e a d c e l l s . T h e relative n u m b e r of d e a d to live c e l l s in a population c a n then be determined by counting cells on a hemacytometer. W h i l e a v a l u a b l e a s s a y for indicating cell viability, s t a i n i n g with t r y p a n b l u e is a l s o p r o b l e m a t i c b e c a u s e it d o e s not d i s t i n g u i s h b e t w e e n n e c r o t i c a n d a p o p t o t i c c e l l s . F u r t h e r m o r e , it is o n l y c a p a b l e of identifying c e l l s that h a v e r e c e n t l y d i e d , limiting its u s e in d e t e r m i n i n g total c e l l d e a t h in a p o p u l a t i o n o v e r time.  71  // w //  NH  I  N  N  \\ / b  C H  ^CH  Tetrazolium  /  -N=N  '/  CH// S-CH 3  3  3  3  Formazan  FIGURE 1.6 Structure of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Conversion of tetrazolium salt to a formazan product during the MTT assay.  72  A. DAY 1 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0  Cells w a s h e d and trypsinized  Cells counted on a H e m a c y t o m e t e r  B L A N K  B. DAY 2  9 6 W e l l Plate  C O N T R O L  D R U G C O N C E N T R A T I O N S  (media o n l y ) (cells o n l y )  Water _  • Drug  Cells diluted to a desired concentration  Serial dilutions to produce  HIGH  *- LOW  ^©0 O O O O O O O O O 0 ttttttQOOO o t 0# O t 1 1 1 1 ©®0 0 o o ©tt«S©0OOO o t o  t  o  t  ©  t  t  t  t  t  t  o  o  o  @ttt®©©000 0 00 0 0 0 0 0 0 0 0 0  desired d r u g concentrations  C . DAY 5  9 6 W e l l Plate  000000000000 000000000000 000000000000 000 0 0 0 0 00 0 0 0 000000000000 000000000000 000000000000 000000000000 MTT Reagent  4 hours Incubation  +  DMSO  000 000 0«« 000 1 I 00 0 0 00 000 000 0 00 000 000 000  9 6 W e l l Plate  000  • •o • #0 #•0 • •0 • •0 •  t  000  9 6 W e l l Plate  of Drufl (uM)  Chart V a l u e s  FIGURE 1.7 Methyl Thiazolyl Tetrazolium (MTT) assay.  Cells are plated on D a y  1, e x p o s e d to d r u g o n D a y 2 , a n d t h e a m o u n t of v i a b l e c e l l s r e m a i n i n g a s s e s s e d after 7 2 h o u r s .  73 Clonogenicity  Assays  C l o n o g e n i c a s s a y s a s s e s s for the ability of s i n g l e c e l l s f r o m a p o p u l a t i o n to f o r m c o l o n i e s after the p o p u l a t i o n of c e l l s h a s b e e n e x p o s u r e to d r u g . B e c a u s e t h e y e v a l u a t e not o n l y c e l l viability, but a l s o the r e p r o d u c t i v e potential a n d g r o w t h rate of c e l l s , t h e y r e p r e s e n t the g o l d s t a n d a r d in a s s e s s i n g the sensitivity of cell p o p u l a t i o n s to d r u g s . the  g e n e r a l p r o t o c o l for c l o n o g e n i c a s s a y s involving c h e m o t h e r a p y d r u g s is to e x p o s e a n  identical n u m b e r of c e l l s to v a r i o u s d r u g c o n c e n t r a t i o n s for a fixed p e r i o d of t i m e  (Figure 1.8).  After  e x p o s u r e to the d r u g , s i n g l e cell s u s p e n s i o n s a r e c r e a t e d , p l a t e d o n t i s s u e c u l t u r e p l a t e s , a n d i n c u b a t e d for set p e r i o d of t i m e .  C e l l s a r e then s t a i n e d with m e t h y l e n e b l u e a n d the n u m b e r of c o l o n i e s c o u n t e d .  T h e n u m b e r of c o l o n i e s in the treated g r o u p is d i v i d e d b y the n u m b e r of c o l o n i e s f o r m e d by the u n t r e a t e d g r o u p to g e n e r a t e a plating efficiency.  E a c h plating e f f i c i e n c y c o e f f i c i e n t m a y t h e n b e g r a p h e d a g a i n s t  the d r u g c o n c e n t r a t i o n u s e d to g e n e r a t e a s u r v i v a l c u r v e . C l o n o g e n i c a s s a y s m a y a l s o b e u s e d c r e a t i n g s i n g l e cell s u s p e n s i o n s f r o m t u m o r s a n d following the s a m e p r o c e d u r e .  in vivo  by  74  Single cell suspentfon  Step 1. Treat mice or cells with select drug concentrations  Control  Ce\\s counted on a Hemacytometer  Step 2. Create single cell suspensions from tumors or tissue culture, count cells, and dilute to desired concentrations  Experimental  Plate Cells  Step 3. Plate cells and incubate.  Cells diluted to a desired concentrator  Control  Experimental  Stain Cells  Step 4. Stain cells with methylene blue to identify colonies  1  o5  x  irvival  o  \  _>  CO  0.0001 .001  1000  Drug Concentration (log)  Step 5. Determine the number of colonies in control and experimental groups  Step 6. Rote the survival (plating efficiency) versus drug concentration.  FIGURE 1.8 Clonogenicity Assay. S i n g l e cell s u s p e n s i o n s a r e c r e a t e d f r o m t u m o r s o r cell culture after d r u g e x p o s u r e a n d p l a t e d . T h e n u m b e r of c o l o n i e s f o r m e d i n d i c a t e s t h e long t e r m viability of cell p o p u l a t i o n .  75 Flow Cytometry F l o w c y t o m e t r y w a s u s e d w i d e l y in o u r e x p e r i m e n t s , primarily for t h e a c c u r a t e d e t e r m i n a t i o n protein  levels  in  cell  populations,  but  also  to  evaluate  apoptotic  markers  upon  treatment  of  with  c h e m o t h e r a p y d r u g s a n d a g e n t s d e s i g n e d to a t t e n u a t e d r u g r e s i s t a n c e . Flow  Cytometry  is  a  technology  capable  of  measuring  the  physical  and/or  chemical  c h a r a c t e r i s t i c s of c e l l s by m e a s u r i n g the optical qualities of c e l l s a s they p a s s s i n g l e file in the fluid p h a s e p a s t a m e a s u r i n g d e v i c e e m p l o y i n g a l a s e r . T h e o p t i c a l c h a r a c t e r i s t i c s of c e l l s w h i c h m a y b e m e a s u r e d by the flow c y t o m e t e r a r e the light s c a t t e r i n g p a r a m e t e r s (forward a n d s i d e s c a t t e r ) of the c e l l s or the f l u o r e s c e n c e g e n e r a t e d by f l u o r e s c e n t - l a b e l e d m o l e c u l e s .  T h e light s c a t t e r i n g p a r a m e t e r s of the cell  result f r o m the incident of light that is reflected f r o m the cell a s it p a s s e s t h r o u g h the l a s e r ;  forward  s c a t t e r (less than 2° a n g l e from the incident a x i s of the l a s e r ) i n d i c a t e s cell s i z e w h i l e s i d e s c a t t e r (90° a n g l e f r o m the incident a x i s of the laser) s p e c i f i e s the d e g r e e of granularity a n d s u r f a c e c o m p l e x i t y of the cell.  F l o w c y t o m e t r y c a n a l s o b e u s e d to d e t e c t the f l u o r e s c e n c e f r o m m o l e c u l e s or a n t i b o d i e s w h i c h  h a v e b e e n l a b e l e d with a f l u o r e s c e n t m a r k e r (probe) a n d u s e d to s t a i n the cell s a m p l e .  Antibodies  s p e c i f i c for a particular a n t i g e n (i.e. protein) or d y e s s p e c i f i c for c e l l u l a r s t r u c t u r e s s u c h a s D N A or o r g a n e l l e s s u c h a s the m i t o c h o n d r i a m a y b e c o n j u g a t e d to m o l e c u l e s that f l u o r e s c e ( p r o b e s ) .  Although  p r o b e s m a y a b s o r b a n d emit light o v e r a r a n g e of w a v e l e n g t h s , e a c h p r o b e will h a v e a s p e c i f i c p e a k exicitation w a v e l e n g t h (^excitation) a n d a p e a k w a v e l e n g t h at w h i c h light is e m i t t e d f r o m the which  (^emmission)  may  be subsequently detected  b y filters  s p e c i f i c for that w a v e l e n g t h  molecule  in the  flow  cytometer. E x p e r i m e n t s in this t h e s i s i n c o r p o r a t e d e x t e n s i v e flow c y t o m e t r y a n a l y s i s . O n e p r o b e u s e d quite often  to  assess  B c l - 2 content  in c e l l s w a s  Bcl-2-specific antibody  that w a s  bound  to  fluorescein  i s o t h i o c y a n a t e ( F I T C ) , w h i c h h a s a ^citation of 4 8 0 n m a n d a E m i s s i o n of 5 2 5 n m . C e l l s p r o b e d with B c l - 2 F I T C w e r e p e r m e a b l i z e d with 1% T w e e n - 2 0 a n d f i x e d with a 2 % f o r m a l d e h y d e s o l u t i o n in o r d e r to a l l o w the a n t i b o d i e s internal a c c e s s to B c l - 2 a n t i g e n . T w o p r o b e s u s e d a s s e s s the s t a t u s a n d quantity of D N A in the cell w e r e p r o p i d i u m iodide a n d H o e c h s t d y e s . P r o p i d i u m iodide is a c o m m o n p r o b e for D N A a n d b i n d s to this m o l e c u l e b y  76  77 intercalation b e t w e e n b a s e p a i r s , a n d it w a s u s e d in t h e s e e x p e r i m e n t s to d e t e c t f r a g m e n t a t i o n of D N A ; it's Excitation is 5 3 6 n m (blue) a n d it h a s a n E m i s s i o n of 6 2 0 n m . H o e c h s t 3 3 3 4 2 stain a l s o b i n d s D N A , a n d it w a s u s e d in t h e s e s t u d i e s to a n a l y z e the c e l l c y c l e profile in cell p o p u l a t i o n s ;  it b i n d s D N A at r e g i o n s  c o n t a i n i n g three c o n s e c u t i v e AT s e q u e n c e s a n d h a s a ^-excitation of 3 6 0 n m a n d a E m i s s i o n of 4 6 5 n m . A n o t h e r p r o b e e m p l o y e d to d e t e r m i n e m i t o c h o n d r i a l m e m b r a n e potential in c e l l s w a s  DiOC  6  ( d i m e t h y l o x a c a r b o c y a n i n e 6), a l i p h o p h i l i c c a t i o n i c d y e that a c c u m u l a t e s in the m i t o c h o n d r i a of c e l l s with intact m i t o c h o n d r i a l m e m b r a n e potential. It h a s a ^excitation of 4 8 3 n m a n d a ^-emmission of 6 0 0 n m . L a s t l y , N o n y l a c r i d i n e O r a n g e w a s u s e d to a s s e s s m i t o c h o n d r i a l c o n t e n t with c e l l s a s this d y e is not d e p e n d e n t u p o n m i t o c h o n d r i a l m e m b r a n e potential in o r d e r to stain t h e s e o r g a n e l l e s . It h a s a ^-excitation of 4 9 5 n m a n d a E m i s s i o n of 5 2 2  nm.  Fluorescence Microscopy F l u o r e s c e n c e m i c r o s c o p y w a s utilized in o u r s t u d i e s to d e t e r m i n e protein l o c a l i z a t i o n within c e l l s and  a l s o a s s e s s certain a p o p t o t i c e n d p o i n t s .  E s s e n t i a l l y u s i n g the s a m e s t a i n i n g p r o c e d u r e a s that  involving flow c y t o m e t r y , i m a g e s of cell s a m p l e s a r e t h e n t a k e n u s i n g f l u o r e s c e n c e m i c r o s c o p y rather then s u b m i t t i n g the s a m p l e s to c y t o m e t r i c a n a l y s i s . The  three  fluorochromes  used  in  fluorescence  microscopy  experiments  were  Bcl-2-FITC  ( d e s c r i b e d a b o v e ) , M i t o T r a c k e r R e d to d e t e r m i n e m i t o c h o n d r i a l c o n t e n t in c e l l s , a n d D A P I , a D N A stain u s e d to a s s e s s D N A c l e a v a g e d u r i n g a p o t o s i s . M i t o T r a c k e r R e d is a n o t h e r m i t o c h o n d r i a l d y e that a c c u m u l a t e s in this structure d u e to a thioreactive c h l o r o m e t h y l moiety that k e e p s the d y e a s s o c i a t e d with m i t o c h o n d r i a after fixation. Excitation of 4 7 8 n m a n d a E m i s s i o n of 5 9 9  nm.  D A P I ( 4 ' , 6 - D i a m i d i n o - 2 - p h e n y l i n d o l e ) is a D N A stain that b i n d s to A T rich r e g i o n s . ^excitation of 3 5 9 n m a n d a ^  e m m  i s s i o n of 461  It It h a s a  nm.  It h a s a  78  1.5.3 REFERENCES 1  C i s p l a t i n D r u g M o n o g r a p h . F a u l d i n g ( C a n a d a ) Inc. M o n t r e a l , Q u e b e c , C a n a d a  2  D o x o r u b i c i n D r u g M o n o g r a p h . F a u l d i n g ( C a n a d a ) Inc. M o n t r e a l , Q u e b e c , C a n a d a  T a n n o c k , l a n F., a n d Hill, R i c h a r d P . 1 9 9 8 . The Scientific Basis of Oncology. T o r o n t o : M c G r a w - H i l l , p. 3 8 4 .  3  Taxol™ Drug M o n o g r a p h . M e a d J o h n s o n O n c o l o g y Products. Bristol-Myers S q u i b b C o . Princeton, New Jersey, U.S.A.  4  S e n c h e n k o v A , Litvak D A , C a b o t M C . T a r g e t i n g c e r a m i d e m e t a b o l i s m - a s t r a t e g y for o v e r c o m i n g d r u g r e s i s t a n c e . J Natl Cancer Inst. 2 0 0 1 M a r 7 ; 9 3 ( 5 ) : 3 4 7 - 5 7 . R e v i e w  5  6  W a k e l i n g A E , D u k e s M , B o w l e r J . A potent s p e c i f i c p u r e a n t i e s t r o g e n with c l i n i c a l potential.  Cancer Res. 1991 A u g 1;51(15):3867-73. D a u v o i s S , W h i t e R, P a r k e r M G . T h e a n t i e s t r o g e n ICI 1 8 2 7 8 0 d i s r u p t s e s t r o g e n r e c e p t o r n u c l e o c y t o p l a s m i c shuttling. J Cell Sci. 1 9 9 3 D e c ; 1 0 6 ( Pt 4 ) : 1 3 7 7 - 8 8 .  7  C a i l l e a u R, Y o u n g R, O l i v e M , R e e v e s W J J r . B r e a s t t u m o r cell lines f r o m p l e u r a l e f f u s i o n s . J Natl Cancer Inst. 1 9 7 4 S e p ; 5 3 ( 3 ) : 6 6 1 - 7 4 . 8  S o u l e H D , V a z g u e z J , L o n g A , A l b e r t S , B r e n n a n M . A h u m a n cell line f r o m a p l e u r a l e f f u s i o n d e r i v e d f r o m a b r e a s t c a r c i n o m a . J Natl Cancer Inst. 1 9 7 3 N o v ; 5 1 ( 5 ) : 1 4 0 9 - 1 6 . 9  B i e d e r m a n n K A , S u n J R , G i a c c i a A J , T o s t o L M , B r o w n J M . s c i d m u t a t i o n in m i c e c o n f e r s h y p e r s e n s i t i v i t y to ionizing radiation a n d a d e f i c i e n c y in D N A d o u b l e - s t r a n d b r e a k repair. Proc Natl Acad Sci U S A. 1991 F e b 15;88(4): 1 3 9 4 - 7 . 1 0  S h i n k a i Y , R a t h b u n G , L a m K P , O l t z E M , S t e w a r t V , M e n d e l s o h n M , C h a r r o n J , D a t t a M , Y o u n g F, Stall A M , et a l . R A G - 2 - d e f i c i e n t m i c e lack m a t u r e l y m p h o c y t e s o w i n g to inability to initiate V ( D ) J r e a r r a n g e m e n t . Cell. 1 9 9 2 M a r 6 ; 6 8 ( 5 ) : 8 5 5 - 6 7 . 1 1  C a r m i c h a e l J , DeGraff W G , G a z d a r A F , M i n n a J D , Mitchell J B . Evaluation of a tetrazolium-based s e m i a u t o m a t e d c o l o r i m e t r i c a s s a y : a s s e s s m e n t o f c h e m o s e n s i t i v i t y t e s t i n g . Cancer Res. 1 9 8 7 F e b 15;47(4):936-42. 1 2  C h e n C F , H w a n g J M , W u C H , C h e n C S , C h e n K Y . E v a l u a t i o n o f a rapid t e t r a z o l i u m - b a s e d c o l o r i m e t r i c a s s a y for s e l e c t i n g a n t i c a n c e r d r u g s . Zhonghua Yi Xue Za Zhi (Taipei). 1 9 9 0 Jul;46(1 ):7-16. 1 3  79  1.6 SUMMARY OF HYPOTHESES  T h e following s u m m a r i z e s the two h y p o t h e s i s that f o r m the b a s i s of the r e s e a r c h outlined in this t h e s i s a n d the c o n d i t i o n s by w h i c h t h e s e h y p o t h e s e s w e r e f o r m e d :  Whereas  Condition 1: T h e r e s i s t a n c e of c a n c e r c e l l s to c h e m o t h e r a p y d r u g s is in part d u e to the i m p a i r e d ability of a s u b s e t of c e l l s within in a t u m o r p o p u l a t i o n to u n d e r g o a p o p t o s i s in r e s p o n s e to a s s a u l t s from c y t o t o x i c a g e n t s .  Whereas  Condition 2: T h e conveyance  of  stoichiometric  Bcl-2 f a m i l y of proteins  the ratios  apoptotic of  signal  to  anti-apoptotic  r e g u l a t e c h e m o t h e r a p y - i n d u c e d a p o p t o s i s by downstream  proteins  to  effectors  pro-apoptotic  based  upon  proteins  at  the the  mitochondria.  Whereas  Condition 3:  Bcl-2, a s a significant  a n t i - a p o p t o t i c p r o t e i n , will o p p o s e a p o p t o s i s a n d  p r o m o t e cell s u r v i v a l in c e l l s treated with c h e m o t h e r a p y d r u g s if l e v e l s of this protein a r e e q u a l to, or e x c e e d , the a m o u n t s of the p r o - a p o p t o t i c p r o t e i n s , s p e c i f i c a l l y B a x a n d B a k , within the c e l l .  Then  Hypothesis 1: the u s e of  D o w n - r e g u l a t i o n of  Bcl-2 a n t i s e n s e or  Bcl-2  l e v e l s within the c e l l u l a r e n v i r o n m e n t t h r o u g h  a n t i - e s t r o g e n c o m p o u n d s , s u c h that l e v e l s of this protein  d e c r e a s e in relation to B a x a n d B a k , s h o u l d i n c r e a s e the s u s c e p t i b i l i t y of t h e s e c e l l s to u n d e r g o a p o p t o s i s a n d s e n s i t i z e o t h e r w i s e resistant c e l l s to c h e m o t h e r a p y d r u g s .  Then  Hypothesis 2: T h e inherent level of Bcl-2 in the c e l l , w h e t h e r b y virtue of the ratio of this protein to p r o - a p o p t o t i c h o m o l o g u e s or d u e to the a b s o l u t e l e v e l of Bcl-2 in the c e l l , s h o u l d d e t e r m i n e the vulnerability of this cell to a p o p t o s i s , a n d t h u s its sensitivity to c h e m o t h e r a p y d r u g s ; c e l l s e x p r e s s i n g high ratios of Bcl-2 to B a x a n d B a k s h o u l d b e m o r e resistant to c h e m o t h e r a p y d r u g s t h a n c e l l s with low ratios of Bcl-2 to B a x a n d B a k .  80  1.7 SUMMARY OF RESEARCH OBJECTIVES T h e p u r p o s e of t h e r e s e a r c h that c o m p r i s e s this t h e s i s w a s to test t h e h y p o t h e s e s that Bcl-2 level in n e o p l a s t i c c e l l s is d e t e r m i n a t e in sensitivity of t h e s e c e l l s to c h e m o t h e r a p y d r u g s a n d that m a n i p u l a t i o n of Bcl-2 c o n t e n t is sufficient to s e n s i t i z e resistant c e l l s to c y t o t o x i c a g e n t s . T h e r e s e a r c h o b j e c t i v e s outlined b e l o w a d d r e s s t h e s e h y p o t h e s e s through v a r i o u s m e a n s a n d a t t e m p t to r e s o l v e their validity in the context of the cell lines utilized a n d u n d e r the c o n d i t i o n s e m p l o y e d in t h e s e s t u d i e s .  T h e r e s e a r c h o b j e c t i v e s of e a c h c h a p t e r a r e a s f o l l o w s :  Chapter 2:  T o d o w n - r e g u l a t e t h e l e v e l s of Bcl-2 in two different b r e a s t c a n c e r cell lines e x p r e s s i n g differing l e v e l s of Bcl-2 in vitro u s i n g Bcl-2 a n t i s e n s e in a n a t t e m p t to i n c r e a s e their ability to u n d e r g o a p o p t o s i s , a n d thus, s e n s i t i z e t h e m to a h o s t of c h e m o t h e r a p y a g e n t s  Chapter 3:  T o g e n e r a t e a n d c h a r a c t e r i z e a s e t of h i g h , m e d i u m , a n d l o w e x p r e s s i n g c l o n e s within the s a m e b r e a s t c a n c e r cell line b y w a y of Bcl-2 t r a n s f e c t i o n o f t w o p a r e n t a l cell lines a n d s e l e c t i o n of c l o n a l p o p u l a t i o n s f r o m the t r a n s f e c t e d p o p u l a t i o n .  Chapter 4:  T o d o w n regulate t h e l e v e l s of Bcl-2 in wild-type a n d high a n d l o w Bcl-2 e x p r e s s i n g c l o n a l p o p u l a t i o n s f r o m the same b r e a s t c a n c e r cell lines t h o u g h inhibition of e s t r o g e n in vitro in a n a t t e m p t to s e n s i t i z e t h e m to c h e m o t h e r a p y d r u g s .  Chapter 5:  T o d o w n regulate the l e v e l s of Bcl-2 in high a n d l o w e x p r e s s i n g p o p u l a t i o n s from the same b r e a s t c a n c e r cell lines in vivo with Bcl-2 a n t i s e n s e in a n a t t e m p t to s e n s i t i z e t h e m to c h e m o t h e r a p y d r u g s .  CHAPTER 2 IN  ANALYSIS OF ANTISENSE MODULATION OF BCL-2: EFFECTS O N T H E CHEMOSENSITIVITY AND APOPTOTIC POTENTIAL OF TWO BREAST CANCER C E L L LINES VITRO  82  2.1 INTRODUCTION  In c o n s i d e r a t i o n  of the putative  ability  of B c l - 2 to c o n f e r  r e s i s t a n c e u p o n c e l l s to v a r i o u s  c h e m o t h e r a p y a g e n t s , a n d its a s s o c i a t i o n with a g g r e s s i v e t u m o u r p h e n o t y p e s , w e d e s i g n e d a n u m b e r of  in vitro  e x p e r i m e n t s to quantify t h e i n f l u e n c e of this g e n e o n d r u g r e s i s t a n c e a n d a p o p t o s i s in two b r e a s t  c a r c i n o m a cell l i n e s .  T h e o b j e c t i v e of t h e s e e x p e r i m e n t s is two-fold: 1)  to elucidate what effect the  intrinsic level of Bcl-2 in these cell lines would have on their potential for apoptosis; and, 2) to establish whether these cells can be sensitized to chemotherapeutic agents by down-regulation of the Bcl-2 protein using antisense oligonucleotides. A n u m b e r of s t u d i e s h a v e a n a l y z e d t h e effect that m a n i p u l a t i n g B c l - 2 l e v e l s w o u l d h a v e u p o n t h e g r o w t h of c a n c e r c e l l s a n d their ability to u n d e r g o a p o p t o s i s with a n t i s e n s e o l i g o d e o x y n u c l e o t i d e s  in vitro. D o w n - r e g u l a t i o n  of t h e b c l - 2 protein  h a s b e e n s h o w n to inhibit t h e g r o w t h of p r o s t a t e  1  and non-  H o d g k i n ' s cell l i n e s , a s w e l l a s i n d u c e a p o p t o s i s in g l i o m a s , B - c e l l C h r o n i c L y m p h o c y t i c L e u k e m i a 2  3  c e l l s , a n d a s e r i e s of lung c a n c e r cell l i n e s 4  5 , 6  ' . T h e results of t h e s e s t u d i e s r e v e a l t h e i m p o r t a n c e of the 7  B c l - 2 level in m a i n t a i n i n g cellular integrity, e v e n in t h e a b s e n c e of a p o p t o t i c stimuli, a n d indicate that perturbations in B c l - 2 l e v e l s m a y b e sufficient in itself to i n d u c e w i d e s p r e a d a p o p t o s i s . W h i l e the p r e c e d i n g s t u d i e s e x a m i n e d the effect of B c l - 2 a n t i s e n s e a l o n e , s e v e r a l other  in vitro  s t u d i e s e v a l u a t e d t h e effect of B c l - 2 a n t i s e n s e in c o n j u n c t i o n with c h e m o t h e r a p y a g e n t s in v a r i o u s cell lines with t h e h o p e that d i m i n i s h e d l e v e l s of B c l - 2 w o u l d n e u t r a l i z e r e s i s t a n c e in t h e s e c e l l s to the t h e s e drugs.  A s a result of this r e s e a r c h , a b r o g a t i o n of B c l - 2 w a s s i g n i f i c a n t in s e n s i t i z i n g A c u t e M y e l o i d  L e u k e m i a c e l l s to c y t o s i n e - a r a b i n o s i d e a n d n o n - H o d g k i n ' s l y m p h o m a c e l l s to c y t o s i n e a r a b i n o s i d e a n d 8  methotrexate . 9  F u r t h e r m o r e , B c l - 2 a n t i s e n s e d i s p l a y e d s y n e r g i s t i c cytotoxicity with t h e d r u g s e t o p o s i d e ,  d o x o r u b i c i n , a n d c i s p l a t i n in lung c a n c e r cell l i n e s , with d o x o r u b i c i n in transitional cell c a n c e r c e l l s , a n d 1 0  with d o c e t a x e l in S h i n o g i t u m o u r c e l l s . 1 2  1 1  T h e s e results indicate that m a n i p u l a t i o n of B c l - 2 in b r e a s t  c a n c e r cell lines m a y l i k e w i s e c o n f e r sensitivity to c h e m o t h e r a p y d r u g s . T h e c a p a c i t y to m a n i p u l a t e l e v e l s of protein within a cell m a y b e a c c o m p l i s h e d t h r o u g h s e v e r a l different m e a n s .  H o w e v e r , t h e u s e of a n t i s e n s e o l i g o d e o x y n u c l e o t i d e s is f a v o u r e d in this c i r c u m s t a n c e  b e c a u s e of the e a s e of a d m i n i s t r a t i o n to  in vitro  c u l t u r e s a s w e l l a s t h e t r a n s i e n t n a t u r e of the m o d u l a t i o n  of the protein. T h i s is in c o n t r a s t to transfection with a s t a b l e a n t i s e n s e g e n e w h i c h w o u l d require the  83 s e l e c t i o n of s u r v i v i n g c l o n e s f r o m a p o p u l a t i o n that h a s b e e n t r a n s f e c t e d with a g e n e w h i c h p r o g r a m s for cell d e a t h ; t h u s , t h e s e s u r v i v i n g c l o n e s b y their v e r y nature m a y h a v e a c h i e v e d s u r v i v a l in a m a n n e r i n d e p e n d e n t of B c l - 2 . The  Bcl-2 antisense molecule  applied  in t h e s e  experiments  is G e n a s e n s e ™  (G3139),  an  e i g h t e e n - b a s e - p a i r o l i g o d e o x y n u c l e o t i d e ( O D N ) d i r e c t e d a g a i n s t the first s i x c o d o n s of the B c l - 2 m R N A ; this m o l e c u l e h a s b e e n s h o w n to b e effective in r e d u c i n g both B c l - 2 m R N A a n d protein e x p r e s s i o n in a s e q u e n c e s p e c i f i c m a n n e r . G e n a s e n s e w a s a l s o u s e d in c o n j u n c t i o n with a m i s m a t c h control m o l e c u l e 1 3  ( G 4 1 2 6 ) that is identical to G e n a s e n s e apart f r o m two s i n g l e b a s e - p a i r m i s m a t c h e s at p o s i t i o n s 10 a n d 13 f r o m the 5 ' e n d of t h e m o l e c u l e . T h e two c o m m o n b r e a s t c a n c e r cell lines u s e d in t h e s e s t u d i e s w e r e c h o s e n o n the b a s i s of their intrinsic B c l - 2 l e v e l s , a s well a s their e s t r o g e n r e c e p t o r ( E R ) s t a t u s : M C F - 7 is a n E R - p o s i t i v e cell line with relatively h i g h inherent  B c l - 2 e x p r e s s i o n w h i l e the M D A - 4 3 5 / L C C 6 is a n E R - n e g a t i v e cell line with  relatively l o w inherent B c l - 2 e x p r e s s i o n . M o r e o v e r , t h e s e two cell lines a l s o reflect the c l i n i c a l o b s e r v a t i o n that E R p o s i t i v e b r e a s t t u m o u r s tend to h a v e high B c l - 2 e x p r e s s i o n w h i l e E R - n e g a t i v e b r e a s t t u m o u r s tend to h a v e l o w B c l - 2 e x p r e s s i o n These  two  1 4 , 1 5 , 1 6  cell lines w e r e  .  treated  with G e n a s e n s e a l o n e  or  in c o m b i n a t i o n  with  several  c h e m o t h e r a p y a g e n t s a n d the ability of this a g e n t to i n d u c e a p o p t o s i s a n d s e n s i t i z e c e l l s w a s a s s e s s e d t h o u g h s e v e r a l m o l e c u l a r a n d cell s u r v i v a l a n a l y s e s . T h e results of t h e s e s t u d i e s  r e v e a l u s e f u l insights  into the role of B c l - 2 e x p r e s s i o n in d r u g r e s i s t a n c e in b r e a s t c a n c e r a n d p r o v i d e a b a s i s for further i n v e s t i g a t i o n into this a r e a .  2.2 MATERIALS AND METHODS  Cell Lines M C F - 7 ( E R positive) a n d M D A - 4 3 5 / L C C 6 ( E R n e g a t i v e ) h u m a n b r e a s t c a n c e r cell lines w e r e o b t a i n e d f r o m the N C I ( B e t h e s d a , M D ) a n d Dr. R. C l a r k e ( G e o r g e t o w n U n i v . ) , r e s p e c t i v e l y . C e l l s w e r e m a i n t a i n e d in R P M I or D M E M r e s p e c t i v e l y , with 1 0 % fetal b o v i n e s e r u m at 3 7 ° C a n d 5 % C 0 . 2  84  Antisense Oligodeoxynucleotides (ODNs) Antisense  ODNs  directed  against  the  open  reading  frame  of  bcl-2  mRNA  (5'-  T C T C C C A G C G T G C G C C A T - 3 ' , G 3 1 3 9 , G e n t a I n c o r p o r a t e d , S a n D i e g o , C A ) w e r e i n t r o d u c e d into the b r e a s t c a n c e r c e l l s u s i n g l i p o s o m e s c o m p o s e d of 5 0 % / 5 0 % (by wt.) d i o l e o y l d i m e t h y l a m m o n i u m  chloride  (DODAC)/dioleoyIphosphatidyl-ethanolamine ( D O P E ) purchased from Northern Lipids (Vancouver, B C ) . T o e n s u r e s e q u e n c e specificity, both a s h a m (buffer) t r e a t m e n t (control) a n d a t w o - b a s e m i s m a t c h O D N ( 5 ' - T C T C C C A G C A T G T _ G C C A T - 3 ' , G 4 1 2 6 , G e n t a Incorporated, S a n D i e g o , C A ) were u s e d a s controls. M o n o l a y e r s of c e l l s in e x p o n e n t i a l g r o w t h w e r e i n c u b a t e d with t h e l i p o s o m e - O D N c o m p l e x in s e r u m - f r e e m e d i a for 4 h e v e r y 2 4 h o n t w o c o n s e c u t i v e d a y s .  Western Analysis Bcl-2, and B c l - X available antibodies.  L  protein e x p r e s s i o n w a s e v a l u a t e d b y W e s t e r n a n a l y s i s u s i n g c o m m e r c i a l l y  C e l l l y s a t e s w e r e p r e p a r e d b y w a s h i n g c e l l s o n c e with i c e c o l d p h o s p h a t e buffered  s a l i n e f o l l o w e d b y t h e a d d i t i o n of lysis buffer (50 m M T r i s , 1 5 0 m M N a C l , 0 . 1 % S D S , 0 . 5 % s o d i u m deoxycholate,  1% N P - 4 0 , 0 . 0 2 % s o d i u m azide) containing the C o m p l e t e ™ , Mini protease  inhibitor  c o c k t a i l ( B o e h r i n g e r M a n n h e i m / R o c h e D i a g n o s t i c s , I n d i a n a p o l i s , IN). S a m p l e s w e r e c e n t r i f u g e d a n d the s u p e r n a t a n t s t o r e d at - 7 0 C . L y s a t e s e q u a l i z e d for protein c o n t e n t w e r e s e p a r a t e d in 0 . 1 % S D S , 1 2 . 5 % p o l y a c r y l a m i d e g e l s a n d t r a n s f e r r e d to Immobilon p o l y v i n y l i d e n e difluoride o r n i t r o c e l l u l o s e m e m b r a n e s . M e m b r a n e s w e r e b l o c k e d ( 1 % B S A , 0 . 0 2 % T w e e n - 2 0 , 0 . 0 5 % s o d i u m a z i d e , a n d 1 X P B S for Immobilon m e m b r a n e s ; 5 % milk, 0 . 0 5 % s o d i u m a z i d e , a n d 1 X T B S for n i t r o c e l l u l o s e m e m b r a n e s ) then p r o b e d with a n t i b o d i e s for B c l - 2 ( D A K O , C a r p i n t e r i a , C A ) , B c l - X ( S a n t a C r u z B i o t e c h n o l o g y Inc., S a n t a C r u z , C A ) , B a x ( S a n t a C r u z B i o t e c h n o l o g y Inc., S a n t a C r u z , C A ) a n d Mcl-1 ( S a n t a C r u z B i o t e c h n o l o g y Inc., S a n t a C r u z , C A ) . B l o t s w e r e w a s h e d , i n c u b a t e d with h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d s e c o n d a r y a n t i b o d i e s (DAKO),  then  visualized using a n enhanced chemiluminescence W e s t e r n  ( A m e r s h a m P h a r m a c i a Biotech).  blotting a n a l y s i s  system  85  RNase Protection Assays T o t a l R N A w a s i s o l a t e d u s i n g T r i z o l (Life T e c h n o l o g i e s , R o c k v i l l e , M D ) . A s s a y s w e r e c a r r i e d o u t u s i n g t h e R i b o Q u a n t m u l t i p r o b e ( P h a r m i n g e n , S a n D i e g o , C A ) protection a s s a y s y s t e m with t h e t e m p l a t e set  hApo-2c  as described  phosphoimaging  analysis  in  the m a n u f a c t u r e r ' s  (Phosphoimager  protocol.  and ImageQuant  Quantitation  w a s determined  v 5 . 0 software,  Molecular  using  Dynamics,  Sunnyvale, C A ) .  Viability Assays E q u a l n u m b e r s of c e l l s (2-5 x 1 0 ) w e r e s e e d e d in 9 6 - w e l l microtiter p l a t e s a n d a l l o w e d to a d h e r e 3  overnight.  After  O D N treatments,  48-72  hour  exposures  to v a r y i n g  concentrations  of  cytotoxics  ( d o x o r u b i c i n , p a c l i t a x e l , cisplatin) a n d direct a p o p t o s i s i n d u c i n g a g e n t s ( C 6 c e r a m i d e ) w e r e p e r f o r m e d . Direct c o u n t s w e r e p e r f o r m e d utilizing h e m o c y t o m e t e r a n d t r y p a n b l u e d y e e x c l u s i o n . C e l l viability after O D N a n d / o r c y t o t o x i c t r e a t m e n t w a s a l s o a s s e s s e d for total n u m b e r of v i a b l e c e l l s u s i n g a c o n v e n t i o n a l 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl  tetrazolium  bromide  ( M T T ) d y e reduction  a s s a y a n d optical  d e n s i t y d e t e r m i n e d at 5 7 0 n m . C l o n o g e n i c i t y w a s a s s e s s e d u s i n g plating e f f i c i e n c i e s f o r s e r i a l dilutions of s i n g l e c e l l s u s p e n s i o n s a s d e t e c t e d b y m e t h y l e n e b l u e s t a i n i n g of s i n g l e a d h e r e n t c o l o n i e s . A l l a s s a y s w e r e p e r f o r m e d in triplicate.  Mitochondrial membrane potential measurement and cell cycle analyses A l t e r a t i o n s in m i t o c h o n d r i a l m e m b r a n e potential following O D N t r e a t m e n t w a s m o n i t o r e d u s i n g a c a t i o n i c lipophilic d y e 3 , 3 ' - d i h e x i l o x a d i c a r b o c y a n i n e ( D i O C ) ( 1 3 , 1 4 ) . 6  A t s e l e c t e d t i m e s after O D N  t r e a t m e n t , 5 x 1 0 c e l l s w e r e h a r v e s t e d a n d i n c u b a t e d with D i O C 6 ( 4 0 n M ) f o r 3 0 m i n u t e s at 3 7 ° C . In 5  s o m e e x p e r i m e n t s , s i m u l t a n e o u s cell c y c l e a n a l y s i s w a s e v a l u a t e d u s i n g H o e c h s t s t a i n i n g (20 ug/ml) for 3 0 m i n u t e s at 3 7 ° C .  After i n c u b a t i o n with a p p r o p r i a t e d y e s , c e l l s w e r e w a s h e d t w i c e with c o l d P B S  c o n t a i n i n g 0.1 % B S A ( P B S - B ) , r e - s u s p e n d e d in P B S c o n t a i n i n g 0 . 5 n g / m l P I , a n d w e r e i m m e d i a t e l y a n a l y z e d b y f l o w c y t o m e t r y ( C o u l t e r E p i c s , M i a m i , F L ) . C h a n g e s in t h e m e m b r a n e potential n o r m a l i z e d for t h e c e l l u l a r m i t o c h o n d r i a l m a s s b y s t a i n i n g with 1 0 0 n M n o n y l a c r i d i n e o r a n g e ( N A O ) .  were  86 For  cell c y c l e a n a l y s e s , 1 x 1 0  6  cells were  w a s h e d with  p e r m e a b i l i z e d with 7 0 % e t h a n o l , d r o p w i s e at 4 ° C for 3 0 m i n u t e s .  ice-cold P B S a n d then  slowly  C e l l s w e r e t h e n w a s h e d with P B S - B  a n d R N A a s e - t r e a t e d (1 m g / m l , D N A a s e - f r e e ) for 3 0 m i n . at 3 7 ° C .  F i n a l l y , c e l l s w e r e s t a i n e d with 5 0  u g / m l PI in P B S a n d t h e PI w a s a l l o w e d to equilibrate in c e l l s o n i c e for 3 0 m i n u t e s prior to a n a l y s i s o n flow c y t o m e t e r ( C o u l t e r ) . F i l e s of a m i n i m u m of 1 0 , 0 0 0 u n g a t e d e v e n t s w e r e c o l l e c t e d a n d later a n a l y z e d using E x p o analysis software (Coulter).  Statistical analysis D a t a w e r e a n a l y z e d b y t h e S t u d e n t ' s t-test o r o n e w a y a n a l y s i s of v a r i a n c e ( A N O V A ) . T h e l e v e l s of statistical s i g n i f i c a n c e w e r e s e t at p < 0 . 0 5 ( t w o - s i d e d ) . A l l statistical c a l c u l a t i o n s w e r e p e r f o r m e d u s i n g S P S S for W i n d o w s , v e r s i o n 7.5.1 s o f t w a r e .  2.3 RESULTS  Measurement of Inherent Bcl-2 level of MCF-7 and MDA-435/LCC6 cells and the Effect of Bcl-2 down-regulation on cell growth and survival T o d e t e r m i n e the inherent B c l - 2 protein l e v e l s of the c e l l lines u s e d in this e x p e r i m e n t , W e s t e r n a n a l y s e s w e r e performed on untreated M C F - 7 a n d M D A - 4 3 5 / L C C 6 cells.  R e s u l t s r e v e a l e d that M C F - 7  c e l l s e x h i b i t e d three t i m e s the B c l - 2 level of M D A - 4 3 5 / L C C 6 c e l l s a s d e t e r m i n e d b y d e n s i t o m e t e r a n a l y s i s {Figure 2.1 A).  S u b s e q u e n t to this, t h e s e c e l l lines w e r e treated with t h e B c l - 2 a n t i s e n s e m o l e c u l e  G e n a s e n s e a n d m i s m a t c h control ( G 4 1 2 6 ) for t w o f o u r - h o u r s e s s i o n s o v e r a 4 8 h o u r time c o u r s e c o n s i s t e n t with that u s e d for later c h e m o s e n s i t i z a t i o n a s s a y s .  D O D A C / D O P E l i p o s o m e s w e r e required  for sufficient t r a n s f e c t i o n of the a n t i s e n s e m o l e c u l e s in vitro, a n d s t u d i e s i n d i c a t e d that a lipid to D N A c h a r g e ratio of 1.3 to 1.0 ( l i p i d / O D N w e i g h t ratio of 5) p r o d u c e d t h e m o s t o p t i m a l d o w n - r e g u l a t i o n of B c l - 2 protein in both c e l l l i n e s .  In r e g a r d to c o n c e n t r a t i o n s of D N A u s e d in t h e s e e x p e r i m e n t s , a n O D N  c o n c e n t r a t i o n of 8 0 0 n M p r o v i d e d the g r e a t e s t d e g r e e of B c l - 2 d o w n r e g u l a t i o n with c o n c e n t r a t i o n s b e l o w 6 0 0 n M p r o d u c e d l e s s than 5 0 % r e d u c t i o n s in protein c o m p a r e d to c o n t r o l w h i l e c o n c e n t r a t i o n s g r e a t e r than  1 0 0 0 u M incurred  significant  cytotoxicity  to  the cells  E x p e r i m e n t s e m p l o y i n g B c l - 2 a n t i s e n s e i n d i c a t e d that two four-  (unrelated  to  sequence  specificity).  87  3  > 2  g  ON  ~J  -Actin '-Bcl-2  B M D A - 435/LCC6 AS  MCF-7  MM  C ~ Bcl-2  AS M M -Actin Bcl-2  FIGURE 2.1 Inherent Bcl-2 Level in MDA-435/LCC6 and MCF-7 Cells and Expression of Bcl-2 After Treatment with Bcl-2 Antisense. (A) Western analysis of intrinsic Bcl-2 protein expression in MDA-435/LCC6 and MCF-7 cells. (B) Bcl-2 levels in MDA435/LCC6 and MCF-7 cells 48 hours after treatment with Bcl-2 antisense (AS), mismatch control (MM), or no treatment (C).  88  h o u r p u l s e s o f G e n a s e n s e w e r e c a p a b l e o f d e c r e a s i n g B c l - 2 l e v e l s in both M C F - 7 a n d M D A 4 3 5 / L C C 6 c e l l s in c o m p a r i s o n to u n t r e a t e d c o n t r o l s , w h i l e t h e m i s m a t c h control h a d a n e g l i g i b l e effect in this r e g a r d (Figure 2.1 B).  A n a l y s i s u s i n g d e n s i t o m e t r y r e v e a l e d that B c l - 2 l e v e l s w e r e s u p p r e s s e d  b e t w e e n 8 0 % to 9 5 % b y G e n a s e n s e in c o m p a r i s o n to u n t r e a t e d control w h i l e t h e m i s m a t c h O D N i n c u r r e d l e s s t h a n a 2 0 % r e d u c t i o n in c o m p a r i s o n to this c o n t r o l .  F u r t h e r m o r e , this s e q u e n c e - s p e c i f i c effect w a s  m a i n t a i n e d f r o m 4 8 to 9 6 h o u r s after initial t r e a t m e n t with G e n a s e n s e (Figure 2.2). In a d d i t i o n to protein l e v e l s , r i b o n u c l e a s e protection a s s a y s w e r e u n d e r t a k e n to d e t e r m i n e t h e s t a t u s o f m R N A l e v e l s of a n u m b e r o f B c l - 2 protein f a m i l y m e m b e r s d u r i n g a n t i s e n s e treatment.  (All RNA analyses in both MCF-7  and MDA-435/LCC6 cells were performed by Dr. Anne Wallis). R N A a n a l y s i s in M D A - 4 3 5 / L C C 6 c e l l s , B c l - 2 m R N A l e v e l s in M D A - 4 3 5 / L C C 6 c e l l s w e r e r e d u c e d b y 6 0 - 8 0 % 2 4 h o u r s after initial a n t i s e n s e t r e a t m e n t a n d s o m e r e d u c t i o n w a s still e v i d e n t after 9 6 h o u r s (Figure 2.3). A n a l y s i s o f other m R N A s in the M D A - 4 3 5 / L C C 6 c e l l line r e v e a l e d that B a d m R N A l e v e l s w e r e slightly d e p r e s s e d , a l t h o u g h like t h o s e in t h e M C F - 7 , this c h a n g e w a s not significant. A s f o r the other B c l - 2 f a m i l y m e m b e r s , M c l - 1 , B c l - w , B c l - x , B a k , a n d B a x m R N A l e v e l s r e m a i n e d u n c h a n g e d in t h e c o u r s e o f a n t i s e n s e t r e a t m e n t .  In c o n t r a s t to  altered l e v e l s ' o f m R N A a c h i e v e d u s i n g G e n a s e n s e in both t h e M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l l i n e s , B c l 2 m R N A l e v e l s in m i s m a t c h treated c e l l s w e r e r e d u c e d b y o n l y 2 5 % o n e d a y after initiation of treatment A s with t h e f i n d i n g s in M D A - 4 3 5 / L C C 6 c e l l s , in M C F - 7 c e l l s 2 4 h o u r s after t h e initial G e n a s e n s e t r e a t m e n t r e v e a l e d a r e d u c t i o n in B c l - 2 m R N A w h i c h c o n t i n u e d f o r a n a d d i t i o n a l 4 8 h o u r s after w h i c h time B c l - 2 m R N A l e v e l s returned to a level c o m p a r a b l e with u n t r e a t e d c o n t r o l s .  A n a l y s i s of other m R N A s in  M C F - 7 c e l l s i n d i c a t e d a slight, a l t h o u g h n o t significant, d e c r e a s e in B c l - X a n d B a d .  A i l other m R N A s  e v a l u a t e d - B c l - w , B a k , a n d B a x - r e m a i n e d u n c h a n g e d (data not s h o w n ) . A d d i t i o n a l W e s t e r n a n a l y s e s to d e t e r m i n e if a n y m o d u l a t i o n s o f B c l - X , o c c u r r e d in t h e c o u r s e o f a n t i s e n s e t r e a t m e n t i n d i c a t e d n o L  c h a n g e s in t h e l e v e l s of this protein (Figure 2.3). P r i o r to t r e a t m e n t of c e l l s with c h e m o t h e r a p e u t i c a g e n t s , t h e c y t o t o x i c effect o f G e n a s e n s e a n d control m o l e c u l e s a l o n e w a s e s t a b l i s h e d b y quantifying t h e a m o u n t o f live c e l l s a n d p e r c e n t viability at various times  post-treatment.  Treatment, which  involved  a 4 h o u r e x p o s u r e to t h e a n t i s e n s e - l i p i d  c o m p l e x e s f o l l o w e d b y a n a d d i t i o n a l e x p o s u r e 2 4 h o u r s later, r e s u l t e d in a s i g n i f i c a n t r e d u c t i o n in t h e a m o u n t of v i a b l e c e l l s o v e r 7 2 h o u r s . F o r M D A - 4 3 5 / L C C 6 c e l l s 7 2 h o u r s after t r e a t m e n t with G e n a s e n s e o n l y 5 0 % o f v i a b l e c e l l s r e m a i n e d in c o m p a r i s o n to t h e initial plating n u m b e r o f 0 . 2 X 1 0 c e l l s . 6  In  89  MCF-7  MDA-435/LCC6  g  CL  CQ  CD  ro  40  CC  24  48  72  !  0  Time (hours after ODN exposure) Control  Antisense — — Mismatch A  24  48  72  Time (hours after ODN exposure) •••»  Control — •  Antisense -  • Mismatch  FIGURE 2.2 Relative Bcl-2 Protein Levels in MDA-435/LCC6 and MCF-7 Cell Lines Over 96 Hours After Treatment with Bcl-2 Antisense. Timeline over 96 hours of the level of Bcl-2 protein in MDA-435/LCC6 and MCF-7 cells treated with Bcl-2 antisense or mismatch control in comparison to Bcl-2 levels in untreated controls.  90  MDA-435/LCC6  MCF-7  AS  AS  MM -Actin  Actin  -Bcl-x,  Bcl-x  48h  24h  B .  MM  MM  T  72h +DOX  72h  AS M M A S M M A S M M A S  Bel w Bclx -  Bad< Bik -Bak -Bax  mmm* mmmw  <m*m mwww  teilti wmimw  tommlm  M  t  t Atu^M ^^^^^^— ^^^T^^  tf^M  Bel 2 Mcl  1 «  L32  G A P D H --  *IP*flp  ^WMP  min  FIGURE 2.3 Relative mRNA and Protein Levels in MDA-435/LCC6 and MCF-7 Cell Lines After Treatment with Bcl-2 Antisense. A . Bcl-XL levels in MCF-7-7 and MDA-MB-435/LCC6 cells 48 hours after treatment with Bcl-2 antisense. B . mRNA levels of Bcl-2 family proteins in MDA-MB-435/LCC6 cells 24 hours after treatment with Bcl-2 antisense.  Time (hours) —a- Control — t — Antisense ±— IVlsmatch  2.4 Viability of MDA-435/LCC6 Cells After Bcl-2 Antisense Treatment as Assessed by Trypan Blue Exclusion. Direct counts of viable cell MDA-435/LCC6 cells  FIGURE  utilizing trypan blue dye exclusion after treatment with Bcl-2 antisense, mismatch control, or untreated control.  92 c o m p a r i s o n to c o n t r o l s , t h e a m o u n t  of v i a b l e  c e l l s in t h e group treated with G e n a s e n s e w a s o n l y 1 0 % that  of c e l l s w h i c h w e r e left u n t r e a t e d (Figure 2.4).  In M C F - 7 c e l l s a s i m i l a r pattern e m e r g e d with o n l y 4 %  v i a b l e c e l l s in c o m p a r i s o n to untreated c o n t r o l s (data not s h o w n ) .  In both cell lines t h e m i s m a t c h control  i n d u c e d s o m e cytotoxicity; h o w e v e r , t h e r e d u c t i o n in v i a b l e c e l l s w a s l e s s t h a n 2 0 % in both c a s e s . In a d d i t i o n to e v a l u a t i n g t h e p e r c e n t a g e of v i a b l e c e l l s r e m a i n i n g after a n t i s e n s e treatment, t h e n u m b e r of n o n - v i a b l e c e l l s w a s a l s o a s s e s s e d in o r d e r to indicate w h e t h e r a n t i s e n s e treatment i n d u c e s direct cytotoxicity rather than s i m p l e growth i m p a i r m e n t . c e l l s , control g r o u p s in  U s i n g trypan b l u e a s a n indicator of n o n - v i a b l e  MCF-7 a n d M D A - 4 3 5 / L C C 6 c o n t a i n e d 9% a n d 2 . 5 % d e a d c e l l s , r e s p e c t i v e l y  (Figure 2.5). T h e a m o u n t of n o n - v i a b l e c e l l s in m i s m a t c h treated g r o u p s w a s slightly h i g h e r at 1 1 % f o r M C F - 7 a n d 6 % for M D A - 4 3 5 / L C C 6 cells.  In c o n t r a s t to t h e s e p r e v i o u s g r o u p s h o w e v e r , G e n a s e n s e  treated g r o u p s c o n t a i n e d high n u m b e r s of d e a d c e l l s , with 4 7 % a n d 3 8 % n o n - v i a b l e c e l l s in M C F - 7 a n d M D A - 4 3 5 / L C C 6 , respectively. Finally, p o p u l a t i o n s of c e l l s in e a c h cell line w e r e treated with G e n a s e n s e or m i s m a t c h O D N s , o r not treated at a l l , a n d t h e n re-plated to e s t a b l i s h the r e p r o d u c t i v e potential a n d g r o w t h rates of the v a r i o u s p o p u l a t i o n s . 7 2 h o u r s after re-plating, t h e n u m b e r of live c e l l s in t h e G e n a s e n s e t r e a t e d g r o u p w a s 4 2 % c o m p a r e d to u n t r e a t e d c o n t r o l s w h i l e t h e m i s m a t c h treated g r o u p c o n t a i n e d 7 2 % of v i a b l e c e l l s in c o m p a r i s o n to u n t r e a t e d c o n t r o l s (Figure 2.6).  Analysis of apoptotic mechanisms associated with Genasense-Induced Cytotoxicity In o r d e r to e s t a b l i s h that cytotoxicity  incurred through  treatment  with G e n a s e n s e  involved  a p o p t o s i s , a n u m b e r of e x p e r i m e n t s w e r e c o n d u c t e d to a n a l y z e s p e c i f i c m o r p h o l o g i c a l a n d m o l e c u l a r attributes of this p r o c e s s .  Initial s t u d i e s in both M D A - 4 3 5 / L C C 6 a n d M C F - 7 cell lines e n d e a v o r e d to  c h a r a c t e r i z e t h e c e l l u l a r m o r p h o l o g y of t h e s e c e l l s u p o n treatment with B c l - 2 a n t i s e n s e . treated c e l l s ,  In G e n a s e n s e  DAPI s t a i n i n g r e v e a l e d c e l l s w h o s e m o r p h o l o g y e x h i b i t e d f e a t u r e s c o n s i s t e n t with n u c l e a r  b l e b b i n g w h o s e c h r o m a t i n h a d c o n d e n s e d , both qualities w h i c h a r e i n d i c a t i v e of a p o p t o s i s (Figure 2.7). F u r t h e r m o r e , m a n y c e l l s in this treatment g r o u p c o n t a i n e d v e r y d i m a n d f a d e d D A P I s t a i n i n g c o n s i s t e n t with c e l l s in a d v a n c e d s t a g e s of a p o p t o s i s w h e r e D N A h a s b e e n c l e a v e d a n d t h e n u c l e u s itself d i s s o l v e d . In c o n t r a s t to this treatment g r o u p , c e l l s w h i c h w e r e a d m i n i s t e r e d m i s m a t c h control a n t i s e n s e c o n t a i n e d  93  60 -,  50  H  MDA-435/LCC6  MCF-7  FIGURE 2.5 Percentage of Non-Viable MDA-435/LCC6 and MCF-7 Cells After Bcl-2 Antisense Treatment as Assessed by Inclusion of Trypan Blue. Bcl-2 antisense induces cytotoxic effects on MCF-7 and MDA-435/LCC6 cells. Percentage of non-viable cells utilizing direct cell counts and trypan blue dye exclusion 48 hours after ODN treatment  94  MDA-435/LCC6 0.80.70.6c o LO^  0.5 -  >.  Q "ra .9  0.3-  CL  O  0.20.1 -  ()  1  I  24  1  I  •  48  i  72  Time (hours after replating) — a — Control — • — Antisense — ± — Mismatch  FIGURE 2.6 Long Term Viability of MDA-435/LCC6 Cells After Bcl-2 Antisense Treatment as Assessed by MTT. Total number of viable cells as determined by MTT dye reduction assay after treatment with Bcl-2 antisense, mismatch control, or left untreated (Control).  95  F I G U R E 2.7 Fluorescent Micrographs of Bcl-2 A n t i s e n s e or M i s m a t c h Control Treated M D A - 4 3 5 / L C C 6 C e l l s Stained with the D N A dye DAPI. A. M D A - 4 3 5 / L C C 6 c e l l s treated with m i s m a t c h control B. M D A - 4 3 5 / L C C 6 c e l l s treated with B c l - 2 a n t i s e n s e . A n t i s e n s e treated c e l l s d i s p l a y a p o p t o t i c m o r p h o l o g y ( p u n c t u a t e d f l u o r e s c e n c e with c o n d e n s e d n u c l e i [arrow 1] a n d faint s t a i n i n g of n u c l e a r r e m n a n t s [arrow 2]) in c o m p a r i s o n to m i s m a t c h treated c e l l s .  96 bright a n d e v e n l y - d i s t r i b u t e d  D A P I s t a i n i n g in m o s t c e l l s .  While nuclear morphology a n d c a s p a s e  a c t i v a t i o n r e p r e s e n t relatively late e n d p o i n t s in the a p o p t o t i c p r o c e s s , m i t o c h o n d r i a l m e m b r a n e potential d i s r u p t i o n , a n e a r l y a p o p t o t i c e v e n t , w a s a l s o e v a l u a t e d . (All Flow Cytometry work in this chapter was produced by Dr. Daniel Menezies and Visia Dragowska). Dr. D a n i e l M e n e z i e s a n d V i s i a D r a g o w s k a c o n d u c t e d f l o w c y t o m e t r i c a n a l y s i s u s i n g D i O C to m e a s u r e m i t o c h o n d r i a l m e m b r a n e potential w h i l e 6  p r o p i d i u m i o d i d e (PI) w a s e m p l o y e d to e s t a b l i s h live v s . d e a d c e l l s in M D A - 4 3 5 / L C C 6 c e l l s ( n o n y l a c r i d i n e o r a n g e w a s u s e d to n o r m a l i z e c e l l s for m i t o c h o n d r i a l content). U n t r e a t e d c e l l s a n d c e l l s treated with m i s m a t c h control c o n t a i n e d a l a r g e p e r c e n t a g e of live c e l l s with intact m i t o c h o n d r i a l m e m b r a n e potential - 51 % a n d 4 5 % , r e s p e c t i v e l y (Quadrant A4 in Figure 2.8A and 2.8B) - a n d f e w e r c e l l s with d i s r u p t e d . m e m b r a n e potential - 2 4 % a n d 2 3 % , r e s p e c t i v e l y (Quadrant A3 in Figure 2.8A and 2.8B). In contrast, after 24 h o u r s o n l y 1 9 % of c e l l s treated with G e n a s e n s e c o n t a i n e d c e l l s with intact m i t o c h o n d r i a l m e m b r a n e potential w h i l e in 6 0 % of t h e s e c e l l s it w a s d i s r u p t e d (Quadrants A4 and A3 in Figure 2.8C). T h e a n a l y s i s of c e l l c y c l e v e r s u s viability in r e s p o n s e to t r e a t m e n t with G e n a s e n s e t r e a t m e n t w a s a l s o i n v e s t i g a t e d b y Dr. M e n e z i e s in M D A - 4 3 5 / L C C 6 c e l l s in s e v e r a l a s s a y s .  T h e first a s s a y e m p l o y e d  H o e c h s t 3 3 3 4 2 stain for cell c y c l e a n a l y s i s in c o m b i n a t i o n with PI to e s t a b l i s h c e l l viability (Figure 2.9). In control c e l l s , 6 5 % o f c e l l s d i s p l a y e d a c h a r a c t e r i s t i c live-cell D N A profile (Quadrant A1, Figure 2.9A) with f e w a p o p t o t i c c e l l s (Quadrants A2 and A3, Figure 2.9A). C e l l s treated with m i s m a t c h control d i s p l a y e d a s i m i l a r live-cell D N A profile a s in t h e u n t r e a t e d g r o u p , albeit with f e w e r v i a b l e c e l l s a n d m o r e a p o p t o t i c c e l l s (Figure 2.9B).  F i n a l l y , in G e n a s e n s e treated  cells, t h e number of apoptotic  cells increased  d r a s t i c a l l y to 6 2 % (Quadrants A2 and A3, Figure 2.9C) w h i l e relatively f e w v i a b l e c e l l s - 1 7 % - r e m a i n e d (Quadrant A1, Figure 2 . 9 C ) ; t h e D N A profile o f t h e s e v i a b l e c e l l s , h o w e v e r , w a s s i m i l a r to t h o s e of t h e m i s m a t c h treated a n d u n t r e a t e d c o n t r o l s . A s e c o n d cell c y c l e a s s a y w a s c o n d u c t e d u s i n g p e r m e a b l i z e d c e l l s in c o m b i n a t i o n with PI (Figure 2.10). A n a l y s i s r e v e a l e d s i m i l a r c e l l c y c l e distributions for u n t r e a t e d , m i s m a t c h t r e a t e d , a n d G e n a s e n s e treated c e l l s (Figure 2.1 OA I, II and III); h o w e v e r , a g a i n G e n a s e n s e t r e a t e d c e l l s d i s p l a y e d h i g h e r a m o u n t s o f a p o p t o t i c c e l l s (61%) in c o m p a r i s o n to m i s m a t c h treated o r u n t r e a t e d c e l l s ( 2 5 % a n d 3 0 % , respectively). S i m i l a r r e s u l t s w e r e a c h i e v e d in t h e s e three f l o w c y t o m e t r i c a s s a y s in M C F - 7 c e l l s ; h o w e v e r , t h e i n c r e a s e d fragility of t h e s e c e l l s to p r o c e s s i n g s t e p s d u r i n g p r e p a r a t i o n for a n a l y s i s r e s u l t e d in e l e v a t e d n o n - v i a b l e c e l l s in control  a n d O D N treatment groups, which  p r e c l u d e d quantitative  comparisons.  97  E CCj  5?  *"!  60  .23  .fl  H-H  o  •c  •*-» CD O  C  o  Vi  tp  o  fi  a 4—»  T3  CD  fl  o  -fl  °  a ^  co  CD  T3  co (D  CO  .TH  03  .  00 CD  53 | l II I  I I I  Mill  I  _  I "  PQ  (A  1  C  <  CD  ca  a -4—»  CD  vo CU  O Q  o >  CD  -A  <3  CD  JZ  °  rfl fl"  W nfl  fl  "•fl  CD  3  03  CD  cd  CO  fl 1-  H  co  fl  >>l  W> CD  fl  o  fl  U  OH CD  fl u CO a u 1 2 w VO  IIII I  II  I  Mill  I  I II  I  I  II  I I I  I  I  u o  «?5  in  -fl o ° -rt G ^ CD CO CD  H^ KS  o o *a= o oa  fl CU  o  PH  o is s ©  CD  s a -o .2  I—I  CO  UH  ^H  >HH  ^  fl .a s co  O  cu  <L>  2  -s oDH _ca  a tS -tS  i—i  PH  S 9vo § cu Q .2 ^3 ^Q 5i> , S  LH  "O C  CO  CD O  kH  •S <-> 8 o U  w  § ^  T3  T «s* -< « Q 00  Id  a  ex !S  S  vU  CS  a a CD  CD  VH  CD  O  c^ O  98  •a u  •  V  3  a .2  rl.  C3 CJ  1  t:« S3 C  c3  I  " 3  <u o 3  £ .3 "2 s > o  3? I  •a  I  I  I  I  I  I  J3 <u o C c  I  u u  c  00  a < •B % o «-g  U .9 J .a £ tf  %l "5 S T o 2 «  s cs  c/i  Q *S  O tl  U  . a  a _! W  , < • «N--  PQ  on  O  CJ  tn  'C u  u  '  -a s  -3  I  o TS £ 5 C m ocn j3 « 3 •J3 13 CD  I  PH  «*> O—S  <•> s a> e a. ° 2 c3  3  s3 O  u .22  a j  J3  a  5 3  T3  e  S3  <D  ~H  ©  9  <; iu  ce ^< te se  C3  ^•9 S13a. 2  S^ 2 9 § a  •a"  I CJ  cy c -a a o  • - cu  £ -9 <2 o O «  T! to  ST 1  i  i  i  i  i  i  i  i  2  §  2 "S  iipaoH  < x Of  (U  u  fc. , 60 IU  T3  a o O T3 §• •o a. a ta  99  F I G U R E 2.10 Quantitative Cell Cycle Analysis of Genasense and Doxorubicin Treated Cells Using Propidium Iodide Staining and Flow Cytometry. MDA-435/LCC6 cells were either left untreated (Row I), or treated with mismatch ODN (Row II) or Genasense (Row III) followed by exposure to doxorubicin. Columns refer to cells 24 hours after treatment with: Column A) no doxorubicin exposure; Column B) after a 2 hour exposure to doxorubicin; Column C) after a 24 hour exposure to doxorubicin. All treatments were conducted with a doxorubicin concentration of 0.5 uM.  100 A d d i t i o n a l l y , K 5 6 2 c e l l s , w h i c h c o n t a i n n e g l i g i b l e a m o u n t s of B c l - 2 p r o t e i n , w e r e u s e d a s control c e l l s a h a n a l y z e d with t h e s a m e flow c y t o m e t r i c a n a l y s i s .  In t h e s e c e l l s , G e n a s e n s e h a d n o effect  upon  m i t o c h o n d r i a l m e m b r a n e potential, PI profile, o r H o e c h s t / P I s t a i n i n g .  Treatment Combining Genasense and Chemotherapy  T r e a t m e n t of M C A - 4 3 5 / L C C 6 a n d M C F - 7 c e l l s with G e n a s e n s e , a n d t h e s u b s e q u e n t d o w n regulation  of B c l - 2 protein  that o c c u r s , l e a d s to direct cytotoxicity  r e q u i r e m e n t of additional a p o p t o t i c stimuli.  in t h e s e c e l l lines without t h e  In spite of t h e s e f i n d i n g s , h o w e v e r , a great d e a l of interest  e x i s t s in the ability of G e n a s e n s e to not o n l y i n d u c e a p o p t o s i s in c e l l s , but a l s o to s e n s i t i z e c e l l s to other a p o p t o t i c stimuli, s u c h a s c h e m o t h e r a p y d r u g s . T h e r e f o r e , w e c o n d u c t e d s t u d i e s e v a l u a t i n g t h e effect of d o w n - r e g u l a t i o n of B c l - 2 t h r o u g h G e n a s e n s e  treatment  in c o m b i n a t i o n with t h e c h e m o t h e r a p y d r u g  doxorubicin F l o w c y t o m e t r i c a n a l y s i s u s i n g p e r m e a b l i z e d c e l l s in c o m b i n a t i o n with PI w e r e u n d e r t a k e n to a s s e s s both t h e c e l l c y c l e profile, a n d the d e g r e e of a p o p t o s i s , in c e l l s treated with G e n a s e n s e .  These  s t u d i e s i n d i c a t e d a n i n c r e a s e in a p o p t o t i c c e l l s in u n t r e a t e d a n d m i s m a t c h treated c e l l s w h e n e x p o s e d to doxorubicin.  C e l l s w h i c h w h e r e not treated with O D N p r o g r e s s e d f r o m 3 0 % o f c e l l s in a p o p t o s i s to 5 7 %  a p o p t o t i c c e l l s after 2 4 h o u r s of e x p o s u r e to d o x o r u b i c i n .  L i k e w i s e , m i s m a t c h control treated c e l l s  i n c r e a s e d f r o m 2 5 % a p o p t o t i c c e l l s to 5 5 % c e l l s in a p o p t o s i s after t h e s a m e t i m e p e r i o d (Figure 2.10 Al and Cl versus All and CU); f u r t h e r m o r e , in both of t h e s e t r e a t m e n t g r o u p s , a c e l l c y c l e a r r e s t at G / M w a s 2  o b s e r v e d (Figure 2.10 Cl and CH). A s for t h e c o m b i n e d effect of d o x o r u b i c i n a n d G e n a s e n s e treatment, the n u m b e r of a p o p t o t i c c e l l s i n c r e a s e d in this t r e a t m e n t g r o u p a s w e l l u p o n e x p o s u r e to d o x o r u b i c i n ; f r o m 6 1 % a p o p t o t i c c e l l s to 9 1 % after 2 4 h o u r s of e x p o s u r e to d o x o r u b i c i n (Figure 2.10 Alll, Bill, and CHI). A d d i t i o n a l l y , f l o w c y t o m e t r i c a n a l y s i s d e m o n s t r a t e d i n c r e a s i n g m i t o c h o n d r i a l m e m b r a n e potential d e p o l a r i z a t i o n in t u m o r c e l l s after addition of d o x o r u b i c i n in a m a n n e r qualitatively s i m i l a r to that a c h i e v e d with G 3 1 3 9 t r e a t m e n t (data not s h o w n ) . W h i l e t h e p r e c e d i n g d a t a i n d i c a t e s a g r e a t e r d e g r e e of a p o p t o s i s in c e l l s e x p o s e d to both G e n a s e n s e a n d d o x o r u b i c i n , it d o e s n o t e s t a b l i s h w h e t h e r t h e s e cytotoxicities a r e s i m p l y additive, o r w h e t h e r G e n a s e n s e is c a p a b l e of s e n s i t i z i n g c e l l s to d o x o r u b i c i n in a s y n e r g i s t i c m a n n e r .  Therefore, a  n u m b e r of e x p e r i m e n t s w e r e c o n d u c t e d to a s s e s s t h e c h e m o s e n s i t i v i t y of c e l l s to d o x o r u b i c i n that h a d  101 b e e n p r e v i o u s l y treated with G e n a s e n s e .  M D A - 4 3 / L C C 6 a n d M C F - 7 w e r e t r e a t e d with G e n a s e n s e ,  m i s m a t c h c o n t r o l , o r left u n t r e a t e d a n d s u b s e q u e n t l y e x p o s e d to v a r i o u s c o n c e n t r a t i o n s of d o x o r u b i c i n over 4 8 hours.  In o r d e r to d i s t i n g u i s h b e t w e e n i n c r e a s e d cytotoxicity d u e to true c h e m o s e n s i t i v i t y a n d  cytotoxicity i n c u r r e d b y the G e n a s e n s e treatment r e g i m e n , e a c h t r e a t m e n t g r o u p w a s c o m p a r e d with the n u m b e r of v i a b l e c e l l s f r o m p o p u l a t i o n s treated with the appropriate ODN but not drug. R e s u l t s r e v e a l e d virtual identical I C  5 0  v a l u e s for c e l l s treated with G e n a s e n s e , m i s m a t c h c o n t r o l , o r u n t r e a t e d c e l l s for both  cell lines (Figure 2.11). T h i s s t u d y w a s r e p e a t e d u s i n g the c h e m o t h e r a p y d r u g s p a c l i t a x e l a n d c i s p l a t i n , a s well a s t h e a p o p t o s i s - i n d u c i n g a g e n t C 6 - c e r a m i d e ; a s with c h e m o s e n s i t i z a t i o n s t u d i e s doxorubicin, I C  5 0  involving  v a l u e s for c e l l s treated with G e n a s e n s e , m i s m a t c h c o n t r o l , o r left u n t r e a t e d w e r e similar  in r e s p o n s e to t r e a t m e n t with t h e s e a g e n t s (Figures 2.12 and 2.13). In o r d e r to e n s u r e that the results o b t a i n e d in the c h e m o s e n s i t i z a t i o n a s s a y s w e r e not the result of  culturing,  drug  e x p o s u r e , or cytotoxicity  assay  conditions  utilized, a d d i t i o n a l  experiments  were  c o n d u c t e d u s i n g a variety of e x p e r i m e n t a l d e s i g n s . A s s a y s w e r e r e p e a t e d u s i n g a w i d e d e g r e e of plating d e n s i t i e s ( 1 0 - 8 0 % ) , d r u g e x p o s u r e t i m e s (4-120 h o u r s , c o n t i n u o u s a n d p u l s e d ) , c o n c o m i t a n t O D N a n d d r u g e x p o s u r e , p r e - t r e a t m e n t with d r u g , a s well a s additional m e t h o d s o f a s s e s s i n g cell viability ( M T T , trypan b l u e , a n d c l o n o g e n i c i t y ) .  C y t o t o x i c i t y c u r v e s g e n e r a t e d to d o x o r u b i c i n , p a c l i t a x e l , c i s p l a t i n , a n d  C 6 - c e r a m i d e c o n t a i n e d insignificant d i f f e r e n c e s in I C  5 0  values between G e n a s e n s e treated, mismatch  trreated, a n d u n t r e a t e d c o n t r o l s u n d e r all e x p e r i m e n t a l c o n d i t i o n s .  102  MDA-435/LCC6  MCF-7 120 J  CU  o  CD > CO  > ~  > ra co  40  ro cu  rr  mi]—  10  Doxorubicin (uM)  • Control  • Antisense -  Doxorubicin (uM) • Mismatch  - Control —•— Antisense —*— Mismatch  FIGURE 2.11 Cytotoxic Effect of Doxorubicin Against MCF-7 AND MDA435/LCC6 Cells After Treatment with Bcl-2 Antisense or Mismatch Control. MCF-7 or MDA-435/LCC6 cells were treated with Bcl-2 antisense, mismatch control, or left untreated (control) and exposed to various concentrations of doxorubicin for 48 hours. Relative numbers of viable cells for each concentration time-point were assessed using MTT dye reduction assay and compared to cells treated with ODN but not drug (Y-axis).  103  3II _  r  -  r  .  11  CQ  with  HUH-'—I  •  CO  3  u>, "3 re > -C3 i  11 i  u  posed rio ing M'TT redu  LB  d o  O  x:u d <  CO  i j  re tme  co  ! o  e  es  and ssed  111  An  CU cn B cu an  <u  o  CO CO re  u  d  d s 43  B  cu  u r-  •8  rle ion  CO  treal -poi  H a a o scu To 3  o IS  !• H — —I :  «  DJD -< CO  DX)  Q >. a.  ra  HHi-r  <u  ©  B  CH  cu  JZ  U  US  CD  #  ©  o  o  g  CO  B  ,o C|H  CO  *u  D  CO  <u  cN  o  u  C  00  nip  Cd  JS  o o  anti nse s were tre ated withBi umbers f v iab] -axi: OD no  JZ/  inst  d  CD  .> "-+->  t3 _o  CO  i i  I  CO  u>  °  x o P  Ff feet  l l  *uw  u  a  *  "K o o >, U  >  T3  'o  1o  CO  J—1  X  U JS  o  d S _o o o ricu £ re T3 bd Cre i. OJ s CO  - M  Fig or I  • .s  O  ont ro CF-7 ofd or 48 pare dto eel trei  C M  no  >  cone assa  I  ntr eac h con  U "o  104  T3  ID co  O  tt  " O XII  *P  g  •  #  9OJ  JS  H E-i  00 «  a  I I1  CU  i mi i  I  //  a. CO  0  I M HI  T3 E CS u <U  im  T3  c«  H  C3 <U OJ  V-  CU  '3  OH I  U a vo S U M  ID  (3  o  ca fc C  f"',t^'ii  11  I'TI  I I  8 !  I—I I' f* I a  »  ^1 «S CQ  UJ  : •  '7 r LU  CQ  i B  B  •  i 9  l  ce •J  !> <D  Q  £  "3  & £ M  1  CS V. cy  >*3  © <u  > ^  > o •8 (3  «  ID  o  73 .> a  w "5 Z  u  r i ^  =a ^  n H  1  <  Pac:  fd  R  O o  •S 13  < J-r-rcs  1) o (3  ID 13  o <; 'C P es  co  1-  o  1o X  o  fix  Q  1  a co  1  a co  £ u .a x  •  CN  S 2  •§ ^ , c*-i  X cs 1 0 2 S 'to  ID  1-1  crt  Io" cj  ce  1  •  •*  co  O 33 « 9 .S 8  O  —1— —1— —1— —r  T3 <D  "5 .3 to cS  .a  a  Q  rS ^ 'I 00 > O  m o '-J fN  «U  CJ I-  cu ce  2  5 3  S tS  ox a  1 « (3 c3 M  S  S S3 o .2  fa <  O  <D  105  2.4 DISCUSSION  T h e s t u d i e s d e s c r i b e d h e r e w e r e u n d e r t a k e n in o r d e r to c h a r a c t e r i z e the  in vitro  c y t o t o x i c arid  c h e m o s e n s i t i z i n g effects of B c l - 2 a n t i s e n s e treatment in a p h a r m a c o l o g i c a l c o n t e x t a n d to c o r r e l a t e s u c h o b s e r v a t i o n s with m o l e c u l a r e n d p o i n t s for s p e c i f i c c e l l u l a r p r o c e s s e s a s s o c i a t e d with a p o p t o s i s . In d o i n g s o , w e i n v e s t i g a t e d the effects of B c l - 2 a n t i s e n s e e x p o s u r e u s i n g a w i d e v a r i e t y of culturing c o n d i t i o n s , drug  concentrations  and  exposure times  a s well  as  several  methods  for  assessing  cytotoxicity.  F u r t h e r m o r e , p h a r m a c o l o g i c a l c o r r e l a t i o n s f o c u s e d o n quantitating the a b s o l u t e n u m b e r of v i a b l e c e l l s r e m a i n i n g after t r e a t m e n t s i n c e this is ultimately the p a r a m e t e r that will reflect t h e r a p e u t i c r e s p o n s e . T h e a n t i s e n s e p r o t o c o l u s e d in this e x p e r i m e n t of two f o u r - h o u r p u l s e s , 2 4 h o u r s apart, w a s c r e a t e d in c o n s i d e r a t i o n of the half-life of the B c l - 2 protein ( a p p r o x i m a t e l y 12 to 18 h o u r s  1 7 , 1 8  ).  The  m e t h o d o l o g y e m p l o y e d w a s u s e d to b a l a n c e the ability to sufficiently b e s t d o w n - r e g u l a t e t h e B c l - 2 protein w h i l e limiting toxicity p r o d u c e d b y either the c a t i o n i c lipids u s e d to d e l i v e r t h e O D N or the c h e m i c a l structure of the a n t i s e n s e m o l e c u l e itself. T h e results p r e s e n t e d h e r e i n d i c a t e that G e n a s e n s e is c a p a b l e of p r o d u c i n g a l a r g e a n d s u s t a i n e d d o w n - r e g u l a t i o n of B c l - 2 m R N A a n d protein in both M C F - 7 a n d M D A 4 3 5 / L C C 6 c e l l s . P r o t e i n l e v e l s r e m a i n e d d e p r e s s e d for o v e r 7 2 h o u r s in both c e l l lines a n d this d o w n regulation w a s s e q u e n c e s p e c i f i c a s B c l - 2 l e v e l s w e r e not r e d u c e d in c e l l s t r e a t e d with m i s m a t c h c o n t r o l . F u r t h e r m o r e , G e n a s e n s e t r e a t m e n t w a s s p e c i f i c for B c l - 2 , a s t r e a t m e n t of either c e l l line did not result in significant alterations in the l e v e l s of m R N A or protein of a n y other B c l - 2 f a m i l y m e m b e r s . T h i s r e d u c t i o n of the a m o u n t of B c l - 2 protein in e a c h c e l l line r e s u l t s in results in significantly r e d u c e d n u m b e r s of v i a b l e c e l l s a n d a n i n c r e a s e in c e l l d e a t h b y a p o p t o s i s ; m o r e o v e r , the g r o w t h ability of c e l l s s u r v i v i n g G e n a s e n s e treatment is i m p a i r e d . A g a i n , t h e s e effects u p o n c e l l viability, a m o u n t of c e l l d e a t h , a n d g r o w t h potential w e r e s e q u e n c e s p e c i f i c a s m i s m a t c h treated c e l l s did not i n c u r s i m i l a r r e s u l t s . W h i l e initial a n a l y s e s i n v e s t i g a t e d the g e n e r a l viability of c e l l s , c e l l d e a t h , a n d growth patterns of MDA-435/LCC6  and  MCF-7  cell  in  r e s p o n s e to  G e n a s e n s e treatment,  molecular  assays  m i t o c h o n d r i a l m e m b r a n e potential, cell c y c l e a n a l y s i s , a n d D N A integrity i n d i c a t e d that this directly i n d u c e d a p o p t o s i s in both c e l l l i n e s .  probing treatment  L i k e w i s e , t r e a t m e n t of c e l l s with c h e m o t h e r a p e u t i c a g e n t s ,  s u c h a s d o x o r u b i c i n , i n c u r r e d a p o p t o s i s a s m e a s u r e d by t h e s o m e of the s a m e a s s a y s . W h e n t h e s e two treatment m o d a l i t i e s w e r e c o m b i n e d , i n c r e a s e d cytotoxicity w a s o b s e r v e d a s reflected b y r e d u c e d n u m b e r s of v i a b l e c e l l s . T h i s d e c r e a s e d viability w o u l d s u g g e s t e n h a n c e d sensitivity  106 to c h e m o t h e r a p y i n d u c e d by B c l - 2 r e d u c t i o n ; h o w e v e r , from a p h a r m a c o l o g i c a l s t a n d p o i n t , this implied synergy should be separated from cytotoxic agents.  i n c r e a s e d cytotoxicity  T h u s , the q u e s t i o n r e m a i n s :  incurred by two independent and  Is the i n c r e a s e d cytotoxicity  additive  induced by combining  G e n a s e n s e with c h e m o t h e r a p y d r u g s s i m p l y the effect of two i n d e p e n d e n t c y t o t o x i c a g e n t s ?  O r is  G e n a s e n s e s e n s i t i z i n g s u r v i v i n g c e l l s to c h e m o t h e r a p y d r u g s to w h i c h t h e y w e r e o n c e r e s i s t a n t ?  The  results p r e s e n t e d h e r e i n d i c a t e d that it is the f o r m e r . W h e n c e l l s f r o m both cell lines w e r e treated with B c l - 2 a n t i s e n s e a n d s u b s e q u e n t l y e x p o s e d to v a r i o u s c o n c e n t r a t i o n s of c h e m o t h e r a p y a g e n t s , there w a s n o d e v i a t i o n in t h e I C  5 0  of a n y of the three  t r e a t m e n t g r o u p s a n a l y z e d , a n d t h u s , n o c h e m o s e n s i t i z a t i o n . It is important to note that e a c h g r o u p w a s c o m p a r e d to the n u m b e r of v i a b l e c e l l s in p o p u l a t i o n s of c e l l s that w e r e t r e a t e d with ODN but not drug ( Y a x i s ) : G e n a s e n s e treated  c e l l s e x p o s e d to d o x o r u b i c i n w e r e  c o m p a r e d to c e l l s treated  with  only  G e n a s e n s e ; m i s m a t c h treated c e l l s e x p o s e d to d o x o r u b i c i n w e r e c o m p a r e d to c e l l s treated o n l y with mismatch;  a n d , untreated  oligonucleotide.  c e l l s e x p o s e d to  doxorubicin  were  compared  to  c e l l s treated  with  no  In this w a y , p o p u l a t i o n s of c e l l s m a y b e s e p a r a t e d f r o m t h o s e that w o u l d h a v e d i e d a s  the result of a n t i s e n s e treatment f r o m t h o s e that p e r i s h d u e to i n c r e a s e d sensitivity to t h e c h e m o t h e r a p y drug. W e w e r e u n a b l e to s h o w c h e m o s e n s i t i v i t y of c e l l s treated with G e n a s e n s e in s p i t e of u s i n g a w i d e spectrum  of  conditions  and  assays  to  assess  cell  survival.  Genasense  did  not  result  in  c h e m o s e n s i t i z a t i o n in the c e l l lines M D A - 4 3 5 / L C C 6 a n d M C F - 7 to d o x o r u b i c i n , t a x o l , c i s p l a t i n , or C 6 c e r a m i d e , all of w h i c h i n d u c e a p o p t o s i s t h r o u g h differing m e c h a n i s m s . O n e m a y a r g u e that the l a c k of sensitivity of G e n a s e n s e treated c e l l s lies in fact that c e l l s s u r v i v i n g G e n a s e n s e d o not d i s p l a y d o w n r e g u l a t e d B c l - 2 , a n d that t h o s e that c o n t a i n e d d e c r e a s e d l e v e l s of this protein h a d a l r e a d y s u c c u m b e d to apoptosis.  In spite of this c o n t e n t i o n , c e l l s r e c o v e r e d for B c l - 2 protein d e t e r m i n a t i o n w e r e h a r v e s t e d at  the s a m e time a s t h o s e for the cytotoxicity s t u d i e s ; this, t h e r e f o r e , p r e c l u d e s a n a r g u m e n t a g a i n s t c e l l p o p u l a t i o n h e t e r o g e n e i t y a s it a p p l i e s to B c l - 2 level a s a r e a s o n for l a c k of sensitivity. W h i l e the results p r e s e n t e d h e r e m a y c o n t r a d i c t the f i n d i n g s of o t h e r s t u d i e s w h i c h  purport  c h e m o s e n s i t i z i n g effects of B c l - 2 a n t i s e n s e , in m a n y of t h e s e s t u d i e s the cytotoxicity i n d u c e d by the a n t i s e n s e m o l e c u l e itself w a s  not t a k e n  into a c c o u n t in c h e m o s e n s i t i z a t i o n a s s a y s  1 9 , 2 0  .  Although  c o m b i n e d t r e a t m e n t s of c h e m o t h e r a p y d r u g s a n d B c l - 2 a n t i s e n s e i n c r e a s e d cytotoxicity, n o r m a l i z a t i o n of  107 the d a t a for t h e s u r v i v a l probability of the individual c y t o t o x i c a g e n t s g e n e r a t e s cytotoxicity c u r v e s v e r y c o m p a r a b l e to t h o s e o b t a i n e d h e r e . In t h o s e s t u d i e s w h e r e i n the c y t o t o x i c effect of the B c l - 2 a n t i s e n s e m o l e c u l e w a s integrated into s e n s i t i z a t i o n d a t a or s t u d i e s w h e r e n o a n t i s e n s e - a s s o c i a t e d cytotoxicity w a s o b s e r v e d , the results of our experiments  m a y still a p p e a r c o n t r a d i c t o r y  2 1 , 2 2 , 2 3  .  H o w e v e r , the  inability  of G e n a s e n s e to  produce  c h e m o s e n s i t i z a t i o n t o w a r d s the c h e m o t h e r a p y d r u g s u s e d m a y b e d u e t h e f a c t o r s s p e c i f i c to the cell or tissue type.  A t p r e s e n t , n o s t u d y h a s r e p o r t e d the ability of the d o w n - r e g u l a t i o n of B c l - 2 to i n d u c e  c h e m o s e n s i t i z a t i o n to a n y c h e m o t h e r a p y drug in either of the cell lines u s e d in o u r e x p e r i m e n t s , or i n d e e d , a n y b r e a s t c a n c e r cell line. W h i l e this h a s b e e n a c h i e v e d in o t h e r c a n c e r s , s u c h a s l y m p h o m a s a n d m e l a n o m a s , the l a c k of c h e m o s e n s i t i z a t i o n in other cell t y p e s r e v e a l s the v a r i a b l e i n f l u e n c e of this protein d e p e n d i n g u p o n t i s s u e or cell t y p e . In a d d i t i o n to the a n a l y s i s of the ability of G e n a s e n s e to i n d u c e c h e m o s e n s i t i z a t i o n in M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l s , the i m p o r t a n c e of the intrinsic level of B c l - 2 in t h e s e two cell lines o n s u s c e p t i b i l i t y to a p o p t o s i s w a s a l s o i n v e s t i g a t e d .  their  M a n y p r e v i o u s s t u d i e s in a v a r i e t y of t i s s u e t y p e s h a v e  positively c o r r e l a t e d the inherent B c l - 2 e x p r e s s i o n level with r e s i s t a n c e to a p o p t o s i s i n d u c e d b y a n u m b e r of s o u r c e s i n c l u d i n g c h e m o t h e r a p y d r u g s a n d h o r m o n e w i t h d r a w a l  2 4 , 2 5  .  Likewise, additional  studies  i n v e s t i g a t e d t h e r e s p o n s e of c e l l s c o n t a i n i n g v a r y i n g l e v e l s of B c l - 2 to B c l - 2 a n t i s e n s e a n d f o u n d t h o s e with h i g h e r l e v e l s of B c l - 2 to e n d u r e l e s s c y t o t o x i c i t y  26,27  .  In the c a s e of o u r p a r t i c u l a r cell l i n e s , a l t h o u g h  M C F - 7 c e l l s c o n t a i n e d o n a v e r a g e a l m o s t three t i m e s the level of B c l - 2 protein a s M D A - 4 3 5 / L C C 6 , both cell lines d i s p l a y e d s i m i l a r r e s p o n s e s to G e n a s e n s e in r e g a r d to cell viability, c e l l d e a t h , a n d long-term viability.  Furthermore,  both cell lines d i s p l a y e d a l m o s t  identical  IC  5 0  values upon  t r e a t m e n t with  c h e m o t h e r a p y a g e n t s s u c h a s d o x o r u b i c i n a n d neither cell line w a s s e n s i t i z e d b y t r e a t m e n t of G e n a s e n s e in c o m b i n a t i o n with c h e m o t h e r a p e u t i c a g e n t s . T h e s e results i n d i c a t e the i m p o r t a n c e of the relative d o w n regulation of B c l - 2 a n d its b a s e l i n e ratio to other anti-apoptotic p r o t e i n s , rather t h a n the a b s o l u t e a m o u n t of B c l - 2 , in dictating r e s p o n s e s to B c l - 2 a n t i s e n s e a n d c o m b i n e d a n t i s e n s e - d r u g cytotoxicity in t h e s e cell lines in vitro . The  results  presented  here  indicate the  peril  in o v e r s i m p l i f y i n g  c o m p o n e n t s of the a p o p t o t i c p a t h w a y s for t h e r a p e u t i c benefit.  the  targeting  of  individual  C l e a r l y , d e c r e a s i n g the l e v e l of B c l - 2  protein in the cell t h r o u g h intervention with G e n a s e n s e c a n i n d u c e a p o p t o s i s a n d w h e n c o m b i n e d with c h e m o t h e r a p y , a n d d e c r e a s e cell viability to a g r e a t e r e x t e n d than is a c c o m p l i s h e d with e a c h a g e n t  108  in vivo,  alone.  T h i s in itself is significant a n d h o l d s p r o m i s e for further e x p e r i m e n t s  o r e v e n t u a l l y , in the  clinic.  H o w e v e r , the ability of G e n a s e n s e to o v e r c o m e d r u g r e s i s t a n c e is t e n u o u s at b e s t .  O n l y with  further s t u d i e s i n v o l v i n g e x p a n d e d o b j e c t i v e s , s i m i l a r to the o n e s o u t l i n e d in t h e s e s t u d i e s , c a n w e clarify the e f f i c a c y a n d u s e f u l n e s s of this important t h e r a p e u t i c tool.  109  2.5 REFERENCES C a m p b e l l M J , D a w s o n M , K o e f f l e r H P . G r o w t h inhibition o f D U - 1 4 5 p r o s t a t e c a n c e r c e l l s b y a B c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e is e n h a n c e d b y N - ( 2 - h y d r o x y p h e n y l ) a l l - t r a n s r e t i n a m i d e . BrJ Cancer. 1 9 9 8 M a r ; 7 7 ( 5 ) : 7 3 9 - 4 4 . 1  S m i t h M R , A b u b a k r Y , M o h a m m a d R, X i e T , H a m d a n M , a l - K a t i b A . A n t i s e n s e o l i g o d e b x y r i b o n u c l e o t i d e d o w n - r e g u l a t i o n o f b c l - 2 g e n e e x p r e s s i o n inhibits g r o w t h of the l o w - g r a d e n o n - H o d g k i n ' s l y m p h o m a cell line W S U - F S C C L . Cancer Gene Ther. 1 9 9 5 S e p ; 2 ( 3 ) : 2 0 7 - 1 2 .  2  J u l i e n T , F r a n k e l B , L o n g o S, K y l e M , G i b s o n S , Shillitoe E , R y k e n T . A n t i s e n s e - m e d i a t e d inhibition of the b c l - 2 g e n e i n d u c e s a p o p t o s i s in h u m a n m a l i g n a n t g l i o m a . Surg Neurol. 2 0 0 0 A p r ; 5 3 ( 4 ) : 3 6 0 - 8 . 3  P e p p e r C , T h o m a s A , H o y T , C o t t e r F, B e n t l e y P . A n t i s e n s e - m e d i a t e d s u p p r e s s i o n o f B c l - 2 highlights its pivotal role in failed a p o p t o s i s in B - c e l l c h r o n i c l y m p h o c y t i c l e u k a e m i a . Br J Haematol. 1 9 9 9 Dec;107(3):611-5.  4  Z a n g e m e i s t e r - W i t t k e U , L e e c h S H , O l i e R A , S i m o e s - W u s t A P , G a u t s c h i O , L u e d k e G H , Natt F, H a n e r R, M a r t i n P , H a l l J , N a l i n C M , S t a h e l R A . A n o v e l b i s p e c i f i c a n t i s e n s e o l i g o n u c l e o t i d e inhibiting both bcl-2 a n d b c l - x L e x p r e s s i o n efficiently i n d u c e s a p o p t o s i s in t u m o r c e l l s . Clin Cancer Res. 2 0 0 0 J u n ; 6 ( 6 ) : 2 5 4 7 55.  5  K o t y P P , Z h a n g H , Levitt M L . A n t i s e n s e b c l - 2 t r e a t m e n t i n c r e a s e s p r o g r a m m e d cell d e a t h in n o n - s m a l l cell lung c a n c e r cell l i n e s . Lung Cancer. 1 9 9 9 F e b ; 2 3 ( 2 ) : 1 1 5 - 2 7 . 6  Z i e g l e r A , L u e d k e G H , F a b b r o D, A l t m a n n K H , S t a h e l R A , Z a n g e m e i s t e r - W i t t k e U . Induction o f a p o p t o s i s in s m a l l - c e l l l u n g c a n c e r c e l l s b y a n a n t i s e n s e o l i g o d e o x y n u c l e o t i d e t a r g e t i n g the B c l - 2 c o d i n g s e q u e n c e . J Natl Cancer Inst. 1 9 9 7 J u l 1 6 ; 8 9 ( 1 4 ) : 1 0 2 7 - 3 6 . 7  K o n o p l e v a M , T a r i A M , E s t r o v Z , H a r r i s D, X i e Z , Z h a o S , L o p e z - B e r e s t e i n G , A n d r e e f f M . L i p o s o m a l B c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e s e n h a n c e proliferation, s e n s i t i z e a c u t e m y e l o i d l e u k e m i a to c y t o s i n e a r a b i n o s i d e , a n d i n d u c e a p o p t o s i s i n d e p e n d e n t o f other a n t i a p o p t o t i c p r o t e i n s . Blood. 2 0 0 0 J u n 15;95(12):3929-38.  8  K i t a d a S , T a k a y a m a S , D e R i e l K, T a n a k a S , R e e d J C . R e v e r s a l of c h e m o r e s i s t a n c e o f l y m p h o m a c e l l s by a n t i s e n s e - m e d i a t e d r e d u c t i o n of b c l - 2 g e n e e x p r e s s i o n . Antisense Res Dev. 1 9 9 4 S u m m e r ; 4 ( 2 ) : 7 1 - 9 .  9  Z a n g e m e i s t e r - W i t t k e U , S c h e n k e r T , L u e d k e G H , S t a h e l R A . S y n e r g i s t i c cytotoxicity o f b c l - 2 a n t i s e n s e o l i g o d e o x y n u c l e o t i d e s a n d e t o p o s i d e , d o x o r u b i c i n a n d c i s p l a t i n o n s m a l l - c e l l l u n g a n c e r cell l i n e s . BrJ Cancer. 1 9 9 8 O c t ; 7 8 ( 8 ) : 1 0 3 5 - 4 2 . 1 0  Bilim V , K a s a h a r a T , N o b o r u H , T a k a h a s h i K, T o m i t a Y . C a s p a s e i n v o l v e d s y n e r g i s t i c cytotoxicity of b c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e s a n d a d r i a m y c i n o n transitional cell c a n c e r c e l l s . Cancer Lett. 2 0 0 0 J u l 3 1 ; 1 5 5 ( 2 ) : 1 9 1 - 8 . 1 1  G l e a v e M E , M i a y a k e H , G o l d i e J , N e l s o n C , T o l c h e r A . T a r g e t i n g b c l - 2 g e n e to d e l a y a n d r o g e n i n d e p e n d e n t p r o g r e s s i o n a n d e n h a n c e c h e m o s e n s i t i v i t y in prostate c a n c e r u s i n g a n t i s e n s e b c l - 2 o l i g o d e o x y n u c l e o t i d e s . Urology. 1 9 9 9 D e c ; 5 4 ( 6 A S u p p l ) : 3 6 - 4 6 1 2  K i t a d a S , M i y a s h i t a T , T a n a k a S , R e e d J C . Investigations o f a n t i s e n s e o l i g o n u c l e o t i d e s t a r g e t e d a g a i n s t b c l - 2 R N A s . Antisense Res Dev. 1 9 9 3 S u m m e r ; 3 ( 2 ) : 1 5 7 - 6 9 . 1 3  H e l l e m a n s P , v a n D a m P A , W e y l e r J , v a n O o s t e r o m A T , B u y t a e r t P , V a n M a r c k E . P r o g n o s t i c v a l u e of bcl-2 e x p r e s s i o n in i n v a s i v e b r e a s t c a n c e r . BrJ Cancer. 1 9 9 5 A u g ; 7 2 ( 2 ) : 3 5 4 - 6 0 . 1 4  S i l v e s t r i n i R, B e n i n i E , V e n e r o n i S , D a i d o n e M G , T o m a s i c G , S q u i c c i a r i n i P , S a l v a d o r i B . p 5 3 a n d bcl-2 e x p r e s s i o n c o r r e l a t e s with c l i n i c a l o u t c o m e in a s e r i e s o f n o d e - p o s i t i v e b r e a s t c a n c e r patients. J Clin 1 5  110  Oncol. 1 9 9 6 M a y ; 14(5): 1 6 0 4 - 1 0 . E g u c h i H , S u g a K, S a j i H , T o i M , N a k a c h i K, H a y a s h i S I . Different e x p r e s s i o n patterns o f B c l - 2 f a m i l y g e n e s in b r e a s t c a n c e r by e s t r o g e n r e c e p t o r s t a t u s with s p e c i a l r e f e r e n c e to p r o - a p o p t o t i c B a k g e n e . Cell Death Differ. 2 0 0 0 M a y ; 7 ( 5 ) : 4 3 9 - 4 6 . 1 6  R e e d J C . A d a y in the life o f the B c l - 2 protein: d o e s the t u r n o v e r rate o f B c l - 2 s e r v e a s a b i o l o g i c a l c l o c k for c e l l u l a r l i f e s p a n r e g u l a t i o n ? L e u k R e s . 1 9 9 6 F e b ; 2 0 ( 2 ) : 1 0 9 - 1 1 . 1 7  K i t a d a S , T a k a y a m a S , D e R i e l K, T a n a k a S , R e e d J C . R e v e r s a l o f c h e m o r e s i s t a n c e o f l y m p h o m a c e l l s b y a n t i s e n s e - m e d i a t e d r e d u c t i o n o f b c l - 2 g e n e e x p r e s s i o n . Antisense Res Dev. 1 9 9 4 S u m m e r ; 4 ( 2 ) : 7 1 - 9 . 1 8  Bilim V , K a s a h a r a T , N o b o r u H , T a k a h a s h i K, T o m i t a Y . C a s p a s e i n v o l v e d s y n e r g i s t i c cytotoxicity o f b c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e s a n d a d r i a m y c i n o n transitional c e l l c a n c e r c e l l s . Cancer Lett. 2 0 0 0 J u l 31 ;155(2): 1 9 1 - 8 . 1 9  K o n o p l e v a M , T a r i A M , E s t r o v Z , H a r r i s D, X i e Z , Z h a o S , L o p e z - B e r e s t e i n G , A n d r e e f f M . L i p o s o m a l B c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e s e n h a n c e proliferation, s e n s i t i z e a c u t e m y e l o i d l e u k e m i a to c y t o s i n e a r a b i n o s i d e , a n d i n d u c e a p o p t o s i s i n d e p e n d e n t of other a n t i a p o p t o t i c p r o t e i n s . Blood. 2 0 0 0 J u n 15;95(12):3929-38.  2 0  Z a n g e m e i s t e r - W i t t k e U , S c h e n k e r T, L u e d k e G H , S t a h e l R A . S y n e r g i s t i c cytotoxicity o f b c l - 2 a n t i s e n s e o l i g o d e o x y n u c l e o t i d e s a n d e t o p o s i d e , d o x o r u b i c i n a n d c i s p l a t i n o n s m a l l - c e l l l u n g c a n c e r c e l l l i n e s . BrJ Cancer. 1 9 9 8 O c t ; 7 8 ( 8 ) : 1 0 3 5 - 4 2 . 2 1  K i t a d a S , T a k a y a m a S , D e R i e l K, T a n a k a S , R e e d J C . R e v e r s a l o f c h e m o r e s i s t a n c e o f l y m p h o m a c e l l s by a n t i s e n s e - m e d i a t e d r e d u c t i o n o f b c l - 2 g e n e e x p r e s s i o n . Antisense Res Dev. 1 9 9 4 S u m m e r ; 4 ( 2 ) : 7 1 - 9 .  2 2  G l e a v e M E , M i a y a k e H , G o l d i e J , N e l s o n C , T o l c h e r A . T a r g e t i n g b c l - 2 g e n e to d e l a y a n d r o g e n i n d e p e n d e n t p r o g r e s s i o n a n d e n h a n c e c h e m o s e n s i t i v i t y in prostate c a n c e r u s i n g a n t i s e n s e b c l - 2 o l i g o d e o x y n u c l e o t i d e s . Urology. 1 9 9 9 D e c ; 5 4 ( 6 A S u p p l ) : 3 6 - 4 6 .  2 3  G a z i t t Y , R o t h e n b e r g M L , H i l s e n b e c k S G , F e y V , T h o m a s C , M o n t e g o m r e y W . B c l - 2 o v e r e x p r e s s i o n is a s s o c i a t e d with r e s i s t a n c e to p a c l i t a x e l , but not g e m c i t a b i n e , in multiple m y e l o m a c e l l s . IntJ Oncol. 1 9 9 8 Oct;13(4):839-48.  2 4  L a s o r e l l a A , l a v a r o n e A , Israel M A . Differentiation of n e u r o b l a s t o m a e n h a n c e s B c l - 2 e x p r e s s i o n a n d i n d u c e s alterations o f a p o p t o s i s a n d d r u g r e s i s t a n c e . Cancer Res. 1 9 9 5 O c t 1 5 ; 5 5 ( 2 0 ) : 4 7 1 1 - 6 .  2 5  Z a n g e m e i s t e r - W i t t k e U , S c h e n k e r T , L u e d k e G H , S t a h e l R A . S y n e r g i s t i c cytotoxicity o f b c l - 2 a n t i s e n s e o l i g o d e o x y n u c l e o t i d e s a n d e t o p o s i d e , d o x o r u b i c i n a n d c i s p l a t i n o n s m a l l - c e l l l u n g a n c e r cell l i n e s . BrJ Cancer. 1 9 9 8 O c t ; 7 8 ( 8 ) : 1 0 3 5 - 4 2 2 6  K i t a d a S , T a k a y a m a S , D e R i e l K, T a n a k a S , R e e d J C . R e v e r s a l o f c h e m o r e s i s t a n c e o f l y m p h o m a c e l l s by a n t i s e n s e - m e d i a t e d r e d u c t i o n o f b c l - 2 g e n e e x p r e s s i o n . Antisense Res Dev. 1 9 9 4 S u m m e r ; 4 ( 2 ) : 7 1 - 9 .  2 7  111  CHAPTER 3 GENERATION AND CHARACTERIZATION OF CLONES EXPRESSING DIFFERING LEVELS OF BCL-2  112  3.1 INTRODUCTION  In the p r e v i o u s c h a p t e r , our findings i n d i c a t e d that differing B c l - 2 l e v e l s b e t w e e n the two different cell lines u s e d a p p e a r e d to m a k e little d i f f e r e n c e in their sensitivity to c h e m o t h e r a p y d r u g s o r in their ability to u n d e r g o a p o p t o s i s .  In light of t h e s e r e s u l t s , w e e n d e a v o r e d to i n v e s t i g a t e this a r e a further b y  creating s t a b l e c l o n e s e x p r e s s i n g a r a n g e of B c l - 2 l e v e l s a n d then s u b s e q u e n t l y a n a l y z e t h e s e c l o n e s f o r their drug sensitivity a n d a p o p t o t i c potential.  A significant d i f f e r e n c e b e t w e e n t h e s e c l o n e s a n d t h e cell  lines u s e d p r e v i o u s l y is that this a l l o w e d c o m p a r i s o n s b e t w e e n c e l l s of the s a m e cell line. T h i s effectively a l l o w s for a m o r e a c c u r a t e a s s e s s m e n t of the effects of B c l - 2 l e v e l o n the c e l l u l a r r e s p o n s e a n d c a p a c i t y for a p o p t o s i s s i n c e c o n s i d e r a t i o n s involving c e l l - l i n e - s p e c i f i c effects h a v e e s s e n t i a l l y b e e n a b o l i s h e d .  Thus, the objectives of the experiments outlined in this chapter were to generate and characterize a s e t of high, medium, and low expressing clones from Bcl-2 transfected populations of parent MCF-7 and MDA-435/LCC6 cells. W i t h t h e c r e a t i o n of t h e s e c e l l s , further e x p e r i m e n t s involving t h e d o w n - r e g u l a t i o n of B c l - 2 both in vitro a n d in vivo c o u l d b e c o n d u c t e d to m o r e a c c u r a t e l y a s s e s s the effect of B c l - 2 o v e r - e x p r e s s i o n u p o n the induction of a p o p t o s i s a n d sensitivity to c h e m o t h e r a p y d r u g s . W h i l e m a n y s t u d i e s investigating t h e effect of B c l - 2 level in t h e c e l l u p o n t h e regulation of a p o p t o s i s h a v e u s e d c e l l s lines w h i c h typically o v e r - e x p r e s s B c l - 2 , m a n y o t h e r s h a v e i n d u c e d Bcl-2 o v e r e x p r e s s i o n b y t r a n s f e c t i n g a particular cell line with a c o n s t r u c t c o n t a i n i n g the bcl-2 g e n e . In this m a n n e r , the effect of B c l - 2 o v e r - e x p r e s s i o n m a y b e s t u d i e d in a n y d e s i r e d cell line, a n d not limited to t h o s e that naturally o v e r - e x p r e s s this protein.  F u r t h e r m o r e , c o n d i t i o n s m a y b e m o r e strictly c o n t r o l l e d , a n d t h e  i n f l u e n c e of B c l - 2 m o r e e x a c t l y d e l i n e a t e d , b y c o m p a r i n g p o p u l a t i o n s of c e l l s f r o m t h e s a m e cell line that differ o n l y in w h e t h e r t h e y h a v e or h a v e not b e e n t r a n s f e c t e d with a c o n s t r u c t c o n t a i n i n g the bcl-2 g e n e . In vitro a n a l y s e s u s i n g Bcl-2 t r a n s f e c t e d  c e l l s h a v e r e v e a l e d s i m i l a r results to t h o s e  different cell lines w h i c h naturally c o n t a i n v a r y i n g a m o u n t s of B c l - 2 .  using  In t h e h u m a n prostate L N C a P cell  line, B c l - 2 o v e r - e x p r e s s i o n t h r o u g h transfection resulted in p r o s t a t e c e l l s m o r e resistant to a p o p t o s i s i n d u c e d b y nutrient d e p r i v a t i o n , a n d r o g e n withdrawal  a n d apoptotic agents s u c h a s phorbol e s t e r s  1 , 2  .  S i m i l a r s t u d i e s in b l a d d e r c e l l s f o u n d that o v e r - e x p r e s s i o n of B c l - 2 i n c u r r e d r e s i s t a n c e to cisplatin a n d t r a n s f e c t e d p 5 3 , w h i l e transfection of Bcl-2 into lung c a n c e r c e l l s r e s u l t e d in i n c r e a s e d r e s i s t a n c e to doxorubicin and C i s p l a t i n  3 , 4 , 5  .  L i k e w i s e , a h e p a t o c e l l u l a r c a r c i n o m a cell line t r a n s f e c t e d with t h e bcl-2  g e n e w a s m o r e resistant to paclitaxel a n d d o x o r u b i c i n than o n e t r a n s f e c t e d with a v e c t o r . 6  113 W h i l e the a b o v e s t u d i e s f o u n d s i m i l a r results to t h o s e u s i n g c e l l l i n e s with intrinsically high B c l - 2 l e v e l s , a f e w s t u d i e s f o u n d that o v e r - e x p r e s s i o n of B c l - 2 did not affect the ability of a cell line to u n d e r g o a p o p t o s i s or r e s p o n d to c h e m o t h e r a p y d r u g s ; in s o m e c a s e s , o v e r - e x p r e s s i o n a c t u a l l y s e n s i t i z e d c e l l s to apoptotic a g e n t s  7 , 8  .  Although these divergent results m a y be explained b a s e d upon tissue- a n d cell-  s p e c i f i c d i f f e r e n c e s , t h e y a l s o highlight the difficulties in utilizing c e l l s that c o n t a i n protein l e v e l s that h a v e b e e n artificially a l t e r e d d u e to t r a n s f e c t i o n .  E l e v a t i o n s of a protein of interest within the c e l l m a y  s u b s e q u e n t l y alter the e x p r e s s i o n l e v e l s a n d s t a t u s of other g e n e s i n s i d e the c e l l , g e n e r a t i n g c h a n g e s that m a y o b s c u r e a n d c o m p l i c a t e r e s u l t s . A d d i t i o n a l l y , s i m i l a r p r o b l e m s m a y a r i s e w h e n insertion of the c o n s t r u c t into the g e n o m e alters or s i l e n c e s g e n e s a n d i n d u c e s c h a n g e s in g e n e e x p r e s s i o n . In addition to t h e s e c o n s i d e r a t i o n s , a n d a l s o quite significant, is the fact that t r a n s f e c t i o n of a p o p u l a t i o n with c e l l s i n v o l v e s a c q u i r i n g c e l l s c o n t a i n i n g the c o n s t r u c t t h r o u g h s e l e c t i o n p r o c e d u r e s e m p l o y i n g antibiotics.  By  its v e r y n a t u r e , this s e l e c t i o n of c e l l s w h i c h c o n t a i n c o n s t r u c t s f r o m a n o v e r a l l p o p u l a t i o n p r o d u c e s a s u b p o p u l a t i o n that m a y exhibit s u b t l e d i f f e r e n c e s f r o m the p a r e n t a l p o p u l a t i o n f r o m w h i c h it w a s s e l e c t e d differences  which  may  affect  the  way  these  cells  behave  and  function.  However,  with  these  c o n s i d e r a t i o n s in m i n d , the u s e of t r a n s f e c t e d c e l l lines is n o n e t h e l e s s a p o w e r f u l tool in i n v e s t i g a t i n g the functions  of particular  g e n e s under varying  circumstances.  W h i l e the  elimination  of  complicating  v a r i a b l e s is a l w a y s the g o a l of a n y r e s e a r c h e r , the ability of e l i m i n a t i n g all f a c t o r s is e x t r e m e l y difficult.  In  this c a s e , the g e n e r a t i o n of c l o n e s within the s a m e c e l l line that exhibit v a r y i n g l e v e l s of B c l - 2 will h o p e f u l l y p r o v e u s e f u l in clarifying a n d highlighting the effects of this g e n e u p o n the p r o c e s s of a p o p t o s i s a n d sensitivity to different t r e a t m e n t r e g i m e n s .  3.2 MATERIALS AND METHODS  Cell Lines  M D A 4 3 5 / L C C 6 a n d M C F - 7 h u m a n b r e a s t c a r c i n o m a cell lines w e r e o b t a i n e d f r o m the N C I t u m o r r e p o s i t o r y ( B e t h e s d a , M D ) a n d Dr. R. C l a r k e ( G e o r g e t o w n U n i v . ) , r e s p e c t i v e l y . M D A 4 3 5 / L C C 6 a n d M C F 7 B c l - 2 t r a n s f e c t e d c e l l lines a n d c l o n e s w e r e g e n e r a t e d in o u r l a b o r a t o r y b y e l e c t r o p o r a t i n g the c e l l s in the p r e s e n c e of a p l a s m i d c o n t a i n i n g c D N A for the full length c o d i n g r e g i o n of  bcl-2  driven by a C M V  p r o m o t e r a n d the g e n e for n e o m y c i n r e s i s t a n c e to a l l o w s e l e c t i o n of t r a n s f e c t e d c e l l s . H L - 6 0 , K 5 6 2 a n d  114 J u r k a t cell lines w e r e o b t a i n e d f r o m t h e A T C C ( R o c k v i l l e , M D ) a n d t h e D O H H 2 c e l l line w a s a gift f r o m G e n t a Inc. ( S a n D i e g o , C A ) . C e l l s w e r e m a i n t a i n e d in D M E M m e d i a with 1 0 % fetal b o v i n e s e r u m . P B L w e r e o b t a i n e d f r o m p e r i p h e r a l b l o o d of h e a l t h y v o l u n t e e r s b y c e n t r i f u g a t i o n  over a density  gradient  (Histopaque -1077, Sigma, St. Louis, M O ) R  Western Blot Analysis C e l l s w e r e c e n t r i f u g e d a n d treated with i c e - c o l d lysis buffer ( 1 5 0 m M N a C l , 1 % N P - 4 0 , 0 . 5 % sodium deoxycholate, 0 . 1 % S D S , 2.5 m M E D T A , 0 . 1 % sodium azide), containing protease (Complete-Mini  R  inhibitors  p r o t e a s e inhibitor tablets, B o e h r i n g e r M a n n h e i m G m B H , G e r m a n y ) . A f t e r i n c u b a t i o n for  3 0 m i n o n i c e , s a m p l e s w e r e c e n t r i f u g e d at 1 4 , 0 0 0 r p m for 15 m i n , a n d s t o r e d at - 7 0 ° C .  P r o t e i n content  in the l y s e d e x t r a c t s w a s d e t e r m i n e d u s i n g a d e t e r g e n t - c o m p a t i b l e B i o R a d a s s a y ( B i o R a d D C , B i o R a d L a b s , H e r c u l e s , C A ) . E q u a l a m o u n t s of protein ( 2 0 | i g / l a n e ) w e r e s u b j e c t e d to 1 2 . 5 % S D S - P A G E ( B i o R a d ) . G e l s w e r e t r a n s f e r r e d to n i t r o c e l l u l o s e m e m b r a n e s , a n d m e m b r a n e s w e r e b l o c k e d o v e r n i g h t at 4 ° C with 5 % s k i m milk, 0 . 0 5 % s o d i u m a z i d e in T B S ( 2 0 m M T r i s p H 8 . 2 , 1 3 7 m M s o d i u m c h l o r i d e ) . M o u s e m A b to h u m a n B c l - 2 (clone 1 2 4 , D A K O , C a r p i n t e r i a , C A ) 1:3,000 a n d m o u s e a n t i - h u m a n (3-actin m A b ( S i g m a , S t . L o u i s , M O ) 1:10,000 w e r e u s e d diluted in 1 % s k i m milk in T B S , c o n t a i n i n g 0 . 0 5 % Tween-20 and 0 . 0 5 % sodium azide.  T h e m e m b r a n e s were then  i n c u b a t e d for 1 h with  1:3,000  horseradish peroxidase-conjugated anti-mouse IgG ( P r o m e g a C o . , M a d i s o n , WI). Proteins were detected by u s i n g a n e n h a n c e d c h e m i l u m i n e s c e n c e ( E C L , A m e r s h a m P h a r m a c i a B i o t e c h , B u c k i n g h a m s h i r e , E n g l a n d ) m e t h o d , a n d v i s u a l i z e d after e x p o s u r e to K o d a k film. T h e m e m b r a n e s w e r e e x p o s e d at v a r i o u s t i m e s , a n d the s c a n s of shorter e x p o s u r e s w e r e u s e d for t h e o p t i c a l d e n s i t y c a l c u l a t i o n s in o r d e r to s t a y within t h e linear r a n g e .  S c a n n i n g d e n s i t o m e t r y ( M o l e c u l a r D y n a m i c s , S u n n y v a l e , C A ) w a s p e r f o r m e d to  quantify b a n d intensities b y v o l u m e / a r e a integration. T h e a m o u n t of B c l - 2 protein in c e l l s w a s n o r m a l i z e d to their p-actin l e v e l s .  Fixation and Anti-Bcl-2 Staining C e l l s w e r e h a r v e s t e d a n d w a s h e d 2 t i m e s in p h o s p h a t e - b u f f e r e d s a l i n e ( P B S ) p l u s 0 . 1 % b o v i n e s e r u m albumin B S A ( P B S B ) , proteases a n d D N a s e s free ( C a l B i o C h e m , S a n D i e g o , C A ) . Adherent  115 M D A 4 3 5 / L C C 6 and M C F 7 cells were harvested by vigorous pipetting and with T r y p s i n / E D T A (Gibco B R L , Grand Island, NY).  3x10  6  cells per cell type were placed in 1.5 ml V W R  Eppendorf tubes.  After  centrifugation, cell pellets were resuspended slowly in 300 ul freshly made 2% formaldehyde (FA, 16% ultrapure formaldehyde methanol free, Polysciences, Warrington, PA) in P B S . Cells were fixed for 30 min in R T . In order to permeabilize cells final 1% Tween-20 (Sigma, St. Louis, MO) was added for the last 15 min of fixation.  Fixed cells were spun and pellets were resuspended in 150 ul 10% B S A in P B S followed  by 2 washes with 1 ml P B S B resuspended  in  300  ul  containing 0.5%  blocking  reagent  Tween-20  containing  (PBSBT).  PBSBT  with  Subsequently pellets  100  ug/ml  human  IgG  were (ICN  ImmunoBiologicals, Lisle, IL), 100 ug/ml mouse IgG (Cappell, West Chester, PA), 10% normal human serum and 500 ug/ml R N a s e A (boiled) (Sigma, St. Louis, MO).  Samples were incubated for 15 min in  3 7 ° C water bath and placed overnight in the fridge to allow blocking. distributed over three Eppendorf tubes. second aliquot was  Next day each sample was  The first aliquot of cells was used as autofluorescence control,  stained with mouse isotype control-FITC  antibody  (IgGi  F/P  = 4.33,  DAKO,  Carpinteria, C A ) at a final concentration of 4ug/ml, and the third aliquot of cells was stained with mouse anti-human Bcl-2-FITC antibody (IgG! isotype, clone 124, F/P=4.33 D A K O , Carpinteria, C A ) at a final concentration of 4 ug/ml. Cells were incubated for 60 min at R T in dark.  Cells were washed 2 x in 1ml  P B S B T and 1 x with P B S B allowing 5-10 min incubation in a wash buffer before a second and third spin. All centrifugation  steps were performed using a microcentrifuge (Biofuge  pico,  Heraeus, Osterode,  Germany) set at 7,000 rpm/20 sec for live cells and 10,000 rpm/25 sec for fixed cells. Finally, cells were resuspended in 300 ul P B S B or P B S B containing 0.05ug/ml DAPI (Molecular Probes, Eugene, Oregon) if cell cycle analysis was required, and transferred to 352063 Falcon tubes for flow cytometry analysis. Samples were analyzed immediately or stored in the fridge and analyzed 1 day later in which case final 0.05% sodium azide was added to all samples. stored samples.  Flow cytometry  No changes in fluorescence intensity was observed in  116 Samples were analyzed using an EPICS  R  Elite ESP flow cytometer (Beckman-Coulter, Miami,  FL) equipped with Enterprise 621 laser (Coherent, Inc., Santa Clara, CA). In order to obtain MESF values, Quantum™ 24 or 25 FITC premixed beads (Flow Cytometry Standards Corporation, San Juan, PR), were used to calibrate the flow cytometer scale. A median relative fluorescence intensity (MRFI) channel value for each bead populations was calculated using ExpoMFA software.  A calibration curve  was constructed and subsequent data from analyzed samples obtained from MRFI was converted to MESF. All data were corrected for non-specific fluorescence of isotype controls quantified in the same way.  Determining effective fluorescence/protein ratio (F/P ff) and antibody-binding capacity (ABC) e  In order to quantify antibody-binding sites, Simply Cellular beads (Flow Cytometry Standards R  Corporation, San Juan, PR) were used as a standard to determine fluorescent intensity per antibody. The F/Peff ratio was obtained by dividing MESF of Simply Cellular standard labelled to saturation with the R  FITC conjugated antibody by the number of calibrated antibody binding sites on the standard. The ABC was calculated by dividing MESF value obtained from cells stained with a saturated concentration of FITC conjugated antibody by the F/P  eff  ratio of the antibody. To validate the effectiveness of the reagents used  for quantitation of antibody binding sites, ABC for T lymphocytes from normal donors using anti-CD4-FITC antibody (Pharmingen, San Diego, CA) was evaluated.  Mitochondria staining  In order to visualize mitochondria, 3x10 live cells were stained in 600 ul DMEM 10% FBS with 6  400 nM MitoTracker Red CMXRos (MTR) (Molecular Probes, Eugene, Oregon ). After 10 min incubation at 37°C waterbath, cells were washed 3 x with PBSB and put through fixation, permeabilization and staining procedures using the Bcl-2 staining protocol described above.  117  Fluorescence microscopy A l i q u o t s of B c l - 2 - F I T C a n d M T R s u s p e n s i o n s t a i n e d c e l l s w e r e u s e d to p r e p a r e c y t o s p i n s l i d e s . C y t o s p u n c e l l s w e r e m o u n t e d in 10 ul of 2.5% D A B C O anti-fade ( S i g m a , S t , L o u i s , M O ) in 9 0 % g l y c e r o l a p p l i e d to a s e m i - d r i e d cell p r e p a r a t i o n .  I m a g e s of c e l l s w e r e a c q u i r e d with a S e n s i C a m C C D c a m e r a  ( P C O , K e l h e i m , G e r m a n y ) a t t a c h e d to A x i o p l a h 2 m i c r o s c o p e ( Z e i s s , J e n a , G e r m a n y ) u s i n g N o r t h e r n E c l i p s e S o f t w a r e ( E m p i x Imaging Inc., M i s s i s s a u g a , O n t a r i o , C a n a d a ) . I m a g e s for both F I T C a n d M T R stained cells were taken from the s a m e focal plane.  3.3 RESULTS  Production of Bcl-2 Transfected MCF-7 and MDA-435/LCC6 Cells T h e p a r e n t a l Bcl-2 t r a n s f e c t e d cell lines w e r e c r e a t e d b y D r s . A n n e W a l l i s a n d C h o w H w e e L e e u s i n g a p l a s m i d p r o v i d e d b y t h e l a b of Dr. Bill J i a (Figure 3.1). T h i s c o n s t r u c t ( p R c / C M V ) c o n t a i n s a bcl-2 c D N A c o r r e s p o n d i n g to t h e 3 . 5 K b transcript from the bcl-2 g e n e , o n e o f t h r e e k n o w n s p l i c e v a r i a n t s , a n d c o d e s for t h e Bcl-2B i s o t y p e of t h e Bcl-2 p r o t e i n . E x p r e s s i o n of t h e Bcl-2 c o d i n g r e g i o n is controlled b y a 9  Cytomegalovirus  promoter  polyadenylation region.  a n d t h e bcl-2  transcript  is t e r m i n a t e d  by a  bovine  growth  hormone  In addition to the bcl-2 c o d i n g r e g i o n , this c o n s t r u c t a l s o c o n t a i n s a n A m p i c i l l i n  r e s i s t a n c e g e n e in o r d e r to s e l e c t for p r o d u c t i o n of t h e c o n s t r u c t in b a c t e r i a a n d a C o l E 1 r e g i o n for e x t r a c h r o m o s o m a l replication in b a c t e r i a . A s w e l l , t h e p l a s m i d c o n t a i n s a N e o m y c i n r e s i s t a n c e g e n e for s e l e c t i o n in e u k a r y o t e s , w h i c h is u n d e r t h e control of a S V 4 0 viral p r o m o t o r a n d t e r m i n a t e d b y a S V 4 0 polyadenylation region.  L a s t l y , this c o n s t r u c t c o n t a i n s t w o s u p e r f l u o u s e l e m e n t s , a h e r p e s r e p l i c o n o n l y  a c t i v a t e d in h e r p e s - v i r u s infected c e l l s , a n d a n F1 origin u s e d in m a k i n g s i n g l e s t r a n d e d D N A . Briefly, t h e Bcl-2 c D N A w a s inserted at t h e polylinker u s i n g t h e t w o u n i q u e restriction sites H i n d III a n d X b a I f o l l o w e d b y amplification of the p l a s m i d s in b a c t e r i a a n d c o n f i r m a t i o n of t h e p r e s e n c e a n d orientation of the Bcl-2 c D N A through restriction m a p p i n g . M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l s w e r e then t r a n s f e c t e d with t h e c o n s t r u c t v i a e l e c t r o p o r a t i o n a n d s e l e c t e d u s i n g G 4 1 8 .  P CMV: Bcl-2: BGH: Fl ori: SV40: NEO: SpA: ColE 1: AMP: HRP:  Cytomegalvirus Promotor Bcl-2 8.5 kb cDNA Bovine Growth Hormone Polyadenylation Site Fl Origin SV40 Viral Promotor Neomycin Resistance G e n e SV40 Virus Polyadenylation Site Bacterial Extrachromosomal Replication Site Ampicillin Resistance G e n e Herpes Replicon  FIGURE 3.1 Diagram of Plasmid Construct Bearing a Bcl-2 cDNA Transfected into MDA-435/LCC6 and MCF-7 Cells.  119  Co-localization of Bcl-2 with Mitochondria T o d e m o n s t r a t e that B c l - 2 protein w a s l o c a l i z e d in t h e m i t o c h o n d r i a , w e a c q u i r e d f l u o r e s c e n t m i c r o s c o p i c i m a g e s of c e l l s s t a i n e d with F I T C - l a b e l e d a n t i - B c l - 2 a n t i b o d i e s a n d m i t o c h o n d r i a l - s p e c i f i c M i t o T r a c k e r R e d ( M T R ) d y e (Figure 3.2).  M D A - 4 3 5 / L C C 6 a n d M C F - 7 cells which h a d b e e n transfected  with B c l - 2 w e r e c o m p a r e d to D O H H 2 (high inherent B c l - 2 e x p r e s s i n g ) a n d H L - 6 0 ( l o w inherent B c l - 2 e x p r e s s i n g ) c e l l s a s c o n t r o l s . T h e left p a n e l of F i g u r e 3.2 i n d i c a t e s B c l - 2 - F I T C s t a i n i n g , w h i c h is l o c a l i z e d to s m a l l s p h e r i c a l b o d i e s in t h e c y t o p l a s m . T h e pattern of s t a i n i n g e v i d e n t in t h e s e p a n e l s c o r r e s p o n d s to the pattern of s t a i n i n g of t h e M T R d y e in the s a m e c e l l s (right p a n e l ) i n d i c a t i n g t h e c o - l o c a l i z a t i o n of the B c l - 2 - F I T C a n d M T R d y e s in the m i t o c h o n d r i a .  N o significant b a c k g r o u n d s t a i n i n g w a s e v i d e n t u s i n g  I g G ^ F I T C control a n t i b o d y (data not s h o w n ) .  Characterization of Bcl-2 in MCF-7 and MDA-435/LCC6 Bcl-2 Transfected Cells In o r d e r to quantify t h e l e v e l s of B c l - 2 in B c l - 2 t r a n s f e c t e d M D A - 4 3 5 / L C C 6 a n d M C F - 7 c e l l s , flow c y t o m e t r i c a n a l y s i s w a s p e r f o r m e d o n p e r m e a b l i z e d c e l l s s t a i n e d with B c l - 2 - F I T C .  In a d d i t i o n to M C F - 7  a n d M D A - 4 3 5 / L C C 6 c e l l s , H L - 6 0 a n d K 5 6 2 c e l l s (low B c l - 2 e x p r e s s o r s ) a n d D O H H 2 c e l l s (high B c l - 2 expressor) were u s e d a s controls.  A l l s a m p l e s a n a l y z e d w e r e c o m p a r e d with u n s t a i n e d c e l l s , a n d c e l l s  s t a i n e d with I g d - F I T C a n t i b o d y a s b a c k g r o u n d c o n t r o l s .  F o r e a c h c e l l t y p e , t h e i n c r e m e n t of m e d i a n  relative f l u o r e s c e n c e intensity ( A M R F I ) w a s c a l c u l a t e d b y s u b t r a c t i n g t h e M R F I of I g G ^ F I T C s t a i n e d s a m p l e s f r o m B c l - 2 - F I T C l a b e l e d s a m p l e s ; this p r o v i d e d the f l u o r e s c e n c e l e v e l , a n d h e n c e t h e B c l - 2 l e v e l , with the b a c k g r o u n d l e v e l s of a n t i b o d y b i n d i n g a c c o u n t e d for. F i g u r e 3 . 3 s h o w s h i s t o g r a m s of the M R F I f r o m B c l - 2 - F I T C l a b e l e d M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l s t r a n s f e c t e d with B c l - 2 .  Fluorescence  intensities for the M D A - 4 3 5 / L C C 6 c e l l lines v a r i e d f r o m a relative f l u o r e s c e n c e of 1 0 to o v e r 1 0 (arbitrary 1  units), indicating a p o p u l a t i o n of c e l l s with highly v a r i a b l e B c l - 2 l e v e l s .  3  P e a k fluorescence w a s found  b e t w e e n 1 0 a n d 1 0 indicating that t h e majority of c e l l s in this p o p u l a t i o n c o n t a i n e d B c l - 2 l e v e l s within 1  this r a n g e .  2  L i k e w i s e , M C F - 7 c e l l s t r a n s f e c t e d with B c l - 2 d i s p l a y e d a w i d e r a n g e of f l u o r e s c e n c e , with  f l u o r e s c e n c e intensities r a n g i n g a l s o r a n g i n g f r o m 1 0 to 1 0 . In this c a s e , p e a k intensity w a s s o m e w h a t 1  higher, falling b e t w e e n 1 0 a n d 1 0 (Figure 3.3). 2  3  3  F I G U R E 3.2 Fluorescence Microscopy Images of Cells Stained with cc-Bcl-2 F I T C and M T R M i t o c h o n d r i a l Stain. Bcl-2 localization in the mitochrondria as indicated b y Bcl-2-FITC staining of MDA-MB-435/LCC6 and MCF-7 Bcl-2 transfected, HL-60, and DoHH2 cell lines in comparison to the mitochrondrial-specific dye Mitotracker Red  F I G U R E 3.3 Transfected  Profile of Bcl-2 Content in M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 P a r e n t a l c e l l l i n e s . A. MDA-MB-435/LCC6 B. MCF-7  122  Generation of Clones Expressing Discreet Levels of Bcl-2 W i t h t h e o b s e r v a t i o n that B c l - 2 t r a n s f e c t e d M D A - 4 3 5 / L C C 6 a n d M C F - 7 w e r e c o m p r i s e d of c e l l s with a v a r y i n g l e v e l s of B c l - 2 , w e i s o l a t e d c l o n e s f r o m t h e s e p a r e n t a l p o p u l a t i o n s in o r d e r to g e n e r a t e c e l l lines falling into o n e of three c a t e g o r i e s :  high, m e d i u m or low Bcl-2 e x p r e s s i n g .  T h e s e clones were  g e n e r a t e d u s i n g stringent c o n d i t i o n s s u c h that e a c h c l o n a l p o p u l a t i o n c o n t a i n e d a s n a r r o w a n d f a s t i d i o u s a r a n g e of B c l - 2 e x p r e s s i o n a s p o s s i b l e a n d that t h e stability of this e x p r e s s i o n w a s m a i n t a i n e d o v e r time. A s with t h e p a r e n t a l cell l i n e s , f l o w c y t o m e t r y u s i n g B c l - 2 - F I T C w a s e m p l o y e d to a s s e s s B c l - 2 content.  In a d d i t i o n to t h e a n a l y s i s of c l o n a l p o p u l a t i o n s , t h e p a r e n t a l c e l l l i n e s , a n d H L - 6 0 a n d K 5 6 2  c e l l s (low B c l - 2 e x p r e s s o r s ) a n d D O H H 2 c e l l s (high B c l - 2 e x p r e s s o r ) w e r e i n c l u d e d a s c o n t r o l s . O f t h o s e c l o n e s d e r i v e d f r o m B c l - 2 t r a n s f e c t e d M D A - 4 3 5 / L C C 6 c e l l s , a total of n i n e p r o d u c e d p o p u l a t i o n s that w e r e v i a b l e e n o u g h for a n a l y s i s  {Figure 3.4).  O f these nine cell lines, four e x p r e s s e d low  l e v e l s o f B c l - 2 ( C l o n e s 3 , 4 , 1 1 , a n d 12); four e x p r e s s e d m e d i u m l e v e l s of B c l - 2 ( C l o n e s 1, 2 , 5 , a n d 9); a n d o n e e x p r e s s e d high l e v e l s of B c l - 2 ( C l o n e 7). B a s e d u p o n t h e profile o f t h e p a r e n t a l c e l l line f r o m the c l o n e s w e r e d e r i v e d , l o w B c l - 2 e x p r e s s i o n is d e f i n e d a s h a v i n g a m e a n f l u o r e s c e n c e of l e s s than 1 0 0 ; m e d i u m B c l - 2 e x p r e s s i o n c o n s i s t s of a m e a n f l u o r e s c e n c e g r e a t e r t h a n 1 0 0 but l e s s t h a n 4 0 0 ; a n d , high B c l - 2 e x p r e s s i o n is a m e a n f l u o r e s c e n c e g r e a t e r than 4 0 0 . In t h e c a s e of the B c l - 2 t r a n s f e c t e d M C F - 7 c e l l s , a total of s e v e n c l o n e s w e r e a n a l y z e d for B c l - 2 c o n t e n t ( F / g u r e 3.5). O f t h e s e s e v e n p o p u l a t i o n s , t w o e x p r e s s e d l o w l e v e l s of B c l - 2 ( C l o n e s 2 a n d 9) a n d five e x p r e s s e d m e d i u m l e v e l s of B c l - 2 ( C l o n e s 2 , 4 , 5, 7 , a n d 11); n o c l o n e s w e r e a n a l y z e d w h i c h e x p r e s s e d h i g h l e v e l s of B c l - 2 in c o m p a r i s o n with the p a r e n t a l p o p u l a t i o n .  C r i t e r i a for c l a s s i f i c a t i o n of  c l o n e s into h i g h , l o w or m e d i u m e x p r e s s i n g g r o u p s w a s b a s e d u p o n t h e B c l - 2 profile of the p a r e n t a l B c l - 2 transfected M C F - 7 population which s h o w e d a similar range a s the Bcl-2 transfected M D A - 4 3 5 / L C C 6 ; t h e r e f o r e , t h e s a m e criteria w a s u s e d in both cell l i n e s .  Characterization of Selected Clones by Western Analysis and Flow Cytometry After s e t s of c l o n a l p o p u l a t i o n s w e r e g e n e r a t e d a n d a n a l y z e d f r o m both M C F - 7 a n d M D A 4 3 5 / L C C 6 cell lines, c l o n e s representing high, m e d i u m , a n d low B c l - 2 e x p r e s s i n g populations w e r e s e l e c t e d for further a n a l y s i s a n d e x p e r i m e n t s .  C l o n e s w e r e s e l e c t e d b a s e d u p o n t h e B c l - 2 l e v e l s of the  MDA4357LCC6 Parental Population:  MDA435/LCC6 Clone *1:  MDA4351CC6 Clone #2:  MDA435/LCC6 Clone 4:  MDA435/LCC6 Clone #5:  MDA435/LC06 Clone *3:  MDA436VLCC6: Clone «7  mM*  MDA435/LCC6 Clone »11:  MDA4357LCC6 Clone *12:  FIGURE 3.4 Flow Cytometry Histograms of Clonal Populations Isolated from Bcl-2 T ransfected MDA-435/LCC6 Cells. Clones were isolated from Bcl-2 transfected MDA-  435/LCC6 cells (upper left histogram) and analyzed for Bcl-2 content. Of nine clones analyzed, four were low Bcl-2 expressing, four were medium Bcl-2 expressing, and one was high Bcl-2 expressing.  MCF-7 Parental Population:  MCF-7 Clone M:  MCF-7 Clone #4:  MCF-7 Clone #i:  MCF-7 Clone #9:  MCF-7 Clone *11:  MCF-7 Clone *7:  FIGURE 3.5 Flow Cytometry Histograms of Clonal Populations Isolated from Bcl-2 Transfected MCF-7 Cells. Clones were isolated from Bcl-2 transfected MCF-7 cells (upper left histogram) and analyzed for Bcl-2 content. Of seven clones analyzed two were low Bcl-2 expressing and four were medium Bcl-2 expressing.  125 cells, but also for factors such as viability in culture and growth properties.  From the M C F - 7 cell line,  Clone 2 (low Bci-2), Clone 9 (medium Bcl-2), and Clone 7 (medium Bcl-2) were selected while from the M D A - 4 3 5 / L C C 6 cell line, Clone 11 (low Bcl-2), Clone 2 (medium Bcl-2), Clone 5 (medium Bcl-2), and Clone 7 (high Bcl-2) were chosen. These selected clones were then analyzed by flow cytometry and the results subsequently compared to findings from Western analyses to more accurately determine Bcl-2 levels in each population. In analyzing the clonal populations using flow cytometry, we used slightly altered procedures in an attempt to more precisely assess the levels of Bcl-2 in each population.  T h e s e procedures involved  relating the fluorescence measured by the flow cytometer to the actual amount of Bcl-2 protein in the cell by adjusting for the number of fluorochrome molecules attached to each Bcl-2-FITC antibody.  If the  number of fluorochrome molecules per Bcl-2-FITC antibody is known, then fluorescence measured by the flow cytometer can be related to. the number of fluorochrome molecules present, the number of antibodies bound to Bcl-2, and hence, the amount of Bcl-2 protein in the cell  (Figure 3.6). In  order to determine the  amount of fluorochrome bound to each Bcl-2-FITC antibody, standards (Simply Cellular Beads) were employed which contained a known amount of antibody binding sites, or antibody binding capacity (ABC). T h e s e standards were saturated with the Bcl-2-FITC antibody and the molecules of equivalent soluble fluorochrome  (MESF)  of the saturated  standard determined  by comparing it to a standard  curve  generated from bead populations containing known levels of fluorochrome. A ratio, called the F/P f, that ef  indicates the number of molecules of fluorochrome per antibody was then calculated by dividing the M E S F of the standard by the A B C of the standard.  For example, if 20,000 molecules of equivalent  soluble fluorochrome was measured on a standard containing 10,000 known binding sites (ABC), then 20,000 + 10,000 = 2, or 2 molecules of fluorochrome per Bcl-2-FITC antibody.  The amount of Bcl-2  protein in a the cells of a given population can then be determined by measuring the M E S F per cell in a population of cells (using the same standard curve mentioned above), and dividing it by the F / P . This eff  yields the number of antibodies bound per cell, and hence the amount of Bcl-2 protein. Table 1.6 illustrates Bcl-2 levels in different cell types expressed as mean M E S F and A B C values ± standard deviation obtained from a minimum of three different measurements of the Bcl-2 transfected clones and control cells using quantitative fluorescent cytometry ( Q F C M )  measurements.  In addition to  analyzing the clonal populations D O H H 2 , HL-60, K562, Jurkat, M D F - 7 and M D A - 4 3 5 / L C C 6 Wild-Type, and peripheral blood lymphocytes (PBL) were used as controls. From this analysis, a comparison of the  126  M o u s e  a-Bcl-2 Ab  n  Bcl-2-FITC Antibody  Bcl-2 Bound to Standard Step 1: Mix Standards of know dntibody binding capacity with anti-Bcl-2 FITC conjugated antibodies  Fluorochrome Molecule Goat a M o u s e Ab  Standard (10,000  b i n d i n g sites)  '-mmmmmvt0^^k%  Step 2: Ensure sdturation of standards with Bcl-2-FITC  MESFstandard F/PEFF  —  ABCstandard  7,000 5000  S 10000  1 5000  F/PEFF  20000  = 10,000  Molecules of Equivalent Soluble Fluorochrome (MESF) per Cell  Step 3: Determine the molecules of equivdlent soluble fluorochrome (MESF) of the saturated standard using d standard curve generated from beads with known amounts of fluorochrome  - 0.7  Step 4 : Calculate F/Peff by dividing the MESF of the standard by the number of known binding sites.  Antibody Binding =  MESFcells * / EFF F  P  Capacityll  Antibodies  ABCstandard  "TvTESFsalls.  Binding  A  1vTE5f5t««dard  Capacity  (ABCceii) Antibodies 20000  40000  w 60000  52,000 =  Binding  80000  Capacity Molecules of Equivalent Soluble Fluorochrome (MESF) per Cell  Step 5: Calculate MESF of cell sample using the same standdrd curve used above.  74, 300  0.7  (ABCceii) Step 6: Calculate the number of antibodies bound per cell by dividing the MESF of the cells by F/Peff.  FIGURE 3.6 Calculation of P ff indicating the Antibody Binding Capacity of the Cell Calculation of the antibody binding capacity of the cell provides a more accurate level of Bcl-2 e  content per cell than M E S F as it is corrected for the amount of fluorochrome bound per antibody.  127 c l o n e s s e l e c t e d f r o m the M D A - 4 3 5 / L C C 6 cell line r e v e a l s that t h e h i g h e x p r e s s i n g c l o n e ( C l o n e 7) c o n t a i n s o v e r 15 t i m e s the B c l - 2 level of the low e x p r e s s i n g c l o n e ( C l o n e 11) a n d 2 . 3 a n d 3.3 t i m e s the B c l - 2 level of the t w o m e d i u m e x p r e s s i n g c l o n e s ( C l o n e 5 a n d C l o n e 2, r e s p e c t i v e l y ) .  In the c a s e of the  M C F - 7 , t h e h i g h e s t e x p r e s s i n g c l o n e ( C l o n e 7) c o n t a i n i n g 2.7 t i m e s the level of B c l - 2 a s the  lowest  e x p r e s s i n g c l o n e ( C l o n e 2) a n d 1.7 t i m e s the B c l - 2 level of the m e d i u m e x p r e s s i n g c l o n e ( C l o n e 9). It s h o u l d b e n o t e d that the n u m b e r of a n t i b o d i e s b o u n d by a cell is not n e c e s s a r i l y e q u i v a l e n t to the n u m b e r of a n t i g e n m o l e c u l e s e x p r e s s e d by that c e l l , but rather r e p r e s e n t s a n t i b o d y - a c c e s s i b l e b i n d i n g s i t e s only. T h e v a r i a t i o n s in M E S F a n d A B C v a l u e s for e a c h cell t y p e in individual e x p e r i m e n t s c o u l d b e d u e to slight m o d u l a t i o n s of B c l - 2 d u e to culture c o n d i t i o n s , p h a s e of the cell c y c l e , a n d c l o n a l s e l e c t i o n within the cell c u l t u r e .  T h e m o s t r e p r o d u c i b l e results w e r e o b t a i n e d with p r i m a r y h u m a n l y m p h o c y t e s , w h e r e  culture c o n d i t i o n s did not a p p l y .  Variability in B c l - 2 s t a i n i n g m a y a l s o a r i s e f r o m cell l o s s d u r i n g fixation  a n d s t a i n i n g p r o c e d u r e , w h i c h c r e a t e s a different ratio of c e l l s p e r a n t i b o d y c o n c e n t r a t i o n s .  Therefore,  particular c a r e w a s t a k e n in o u r p r o t o c o l to a c h i e v e a high cell r e c o v e r y b y u s i n g s m a l l r e a g e n t v o l u m e s , s m a l l t u b e s , a n d s t a b l e cell n u m b e r s p e r s a m p l e in a d d i t i o n to u s i n g f r e s h l y p r e p a r e d a n d s t a n d a r d i z e d r e a g e n t s . C e l l r e c o v e r y in t h e s e e x p e r i m e n t s w a s in the r a n g e of 6 0 - 9 0 % .  Table 1.6  Flow Cytometric Quantitation of Bcl-2 Level in MCF-7 and MDA-435/LCC6 Clonal Populations  Cell Type  M E S F (1 x 1 0 )  A B C (1 x 1 0 )  M e a n ± Standard Deviation  M e a n ± Standard Deviation  HL-60  8.8 + 4 . 6 23 ± 12.6  12.6 ± 6 . 5 32.9 ± 1 8 200 ± 5 8 . 3 65.1 ± 4.6 139.1 ± 3 3 101.1 ± 7 . 3  K652  a  DOHH2  140 ± 4 0 . 8  Jurkat  45.6 ± 3.2 97.4 ± 70.8 ± 5 . 1  M C F - 7 Wild Type MCF-7 Clone 2 MCF-7 Clone 9 MCF-7 Clone 7  108.7  ±52.4  a  155.3  ±74.9  270.7 ± 84.4 75.6 ± 10.4  MDA-435/LCC6 Clone 2  189.5 + 59.1 59.2 ± 7.3 75.1 ± 18.7 343.8 ± 5 9  MDA-435/LCC6 Clone 5  495.4 ± 2 0 6 . 2  707.7 ± 2 9 0  MDA-435/LCC6 Clone 7  1,145.3 ± 4 4 4 . 3 38.5 ± 1.7  55 ±  M D A - 4 3 5 / L C C 6 Wild Type M D A - 4 3 5 / L C C 6 C l o n e 11  PBL  107.3  ±26.7  491.1  ±84.3  1,636  ±635 2.4  In a d d i t i o n a l to a n a l y z i n g M C F - 7 a n d M D A - 4 3 5 / L C C 6 u s i n g f l o w c y t o m e t r y , W e s t e r n a n a l y s i s w a s a l s o e m p l o y e d to d e t e r m i n e the B c l - 2 level in t h e s e c e l l s a n d the r e s u l t s f r o m the W e s t e r n a n a l y s i s  128 s u b s e q u e n t l y c o r r e l a t e d t o t h e r e s u l t s o b t a i n e d f r o m flow c y t o m e t r i c a n a l y s i s .  Figures 3.7 and 3.8  indicate the r e s u l t s of the W e s t e r n a n a l y s i s of M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 c l o n e s a n d a c o m p a r i s o n of t h e s e r e s u l t s to v a l u e s o b t a i n e d t h r o u g h flow c y t o m e t r y . C o r r e l a t i o n o f d a t a f r o m W e s t e r n blots with flow c y t o m e t r y v a l u e s w a s h i g h , with a c o r r e l a t i o n coefficient of R = 0 . 8 7 8 8 w h e n t h e o p t i c a l d e n s i t y ( a s 2  d e t e r m i n e d b y d e n s i t o m e t r y ) of W e s t e r n blots w a s c o m p a r e d with A B C v a l u e s o b t a i n e d from cytometry.  flow  A l t h o u g h B c l - 2 l e v e l s in c e l l s m e a s u r e d with both flow c y t o m e t r y a n d W e s t e r n a n a l y s i s  e x h i b i t e d t h e s a m e quantitative  trend in B c l - 2 c o n t e n t , t h e quantitation  a p p e a r e d to h a v e h i g h e r r e s o l u t i o n .  r a n g e u s i n g flow  cytometry  A s a c o n s e q u e n c e , d i f f e r e n c e s b e t w e e n l o w a n d high B c l - 2  e x p r e s s o r s in both c e l l lines w e r e m o r e p r o n o u n c e d .  Western Analysis of Levels of the Pro-Apoptotic Proteins Bax and Bak T h e s t a t u s of t h e p r o - a p o p t o t i c proteins B a x a n d B a k w a s d e t e r m i n e d in e a c h of t h e c l o n a l p o p u l a t i o n s a s w e l l a s M C F - 7 a n d M D A - 4 3 5 / L C C 6 W i l d T y p e c e l l s (Figure 3.9). S u b s e q u e n t to this, t h e l e v e l s o f t h e s e p r o t e i n s w e r e t h e n c o m p a r e d to t h e B c l - 2 level of e a c h c e l l line (Figure 3.10).  129  A  C  g-,  LB: LW: LTot: L2:  L B L W L l l L 2 L 5 L7  MDA-435/LCC6 MDA-43S/LCC6 MDA-43S/LCC6 MDA-435/LCC6  Background Wild-Type Bcl-2 Transfected Parental Bcl-2 Transfected Clone 2  L5: L7: Lll:  MDA-435/LCC6 Bcl-2 Transfected Clone 5 MDA-43S/LCC6 Bcl-2 Transfected Clone 7 MDA-435/LCC6 Bcl-2 Transfected Clone 11  FIGURE 3.7 Flow Cytometry and Western Analysis of Selected MDA-MB435/LCC6 Clonal Populations. A. Flow cytometric histograms of clones. B. Results of Western analysis of clones. C. Bcl-2 content of clonal population indicated in Antibody Binding Capacity (ABC). D. Densitometry analysis of Western Blots.  130  OJ  ABC > i  w J8,'  t  t  LU  '1 1  1.  ^  1  *  10'  1'0  2  10°  1IT  MW MTot M2 M9  M7  MW MTot M2 M9  M7  PMT2 log Bcl-2-FITC  D  B  -Actin 46 kDa -Bcl-2 26 kDa MW MW: MTot: M2:  MTot  M2  M9 M7  MCF-7 Wild-Type M7: MCF-7 Bcl-2 Transfected Parental M9: MCF-7 Bcl-2 Transfected Clone 2  MCF-7 Bcl-2 Transfected Clone 7 MCF-7 Bcl-2 Transfected Clone 9  FIGURE 3.8 Flow Cytometry and Western Analysis of Selected MCF-7 Clonal Populations. A. Flow cytometric histograms of clones. B. Results of Western analysis of clones. C. Bcl-2 content of clonal population indicated in Antibody Binding Capacity (ABC). D. Densitometry analysis of Western Blots.  131  In the M D A - 4 3 5 / L C C 6 c e l l line, l e v e l s of B a x protein v a r i e d f r o m 8 to 15 t i m e s h i g h e r in C l o n e 11 a n d C l o n e 7 t h a n the l e v e l s of B a x protein in C l o n e 5, C l o n e 2 or t h e W i l d - T y p e c e l l s . T h e o p p o s i t e w a s true in the l e v e l s of B a k protein, w h e r e C l o n e 5 a n d the W i l d - T y p e c e l l s d i s p l a y e d 1 to 3 t i m e s h i g h e r l e v e l s of this protein t h a n C l o n e 7 or C l o n e 1 1 . G e n e r a l l y , a c r o s s all of the M D A - 4 3 5 / L C C 6 c l o n e s a n d c o n t r o l , B a k l e v e l s w e r e h i g h e r than B a x l e v e l s , but not n e a r l y a s e l e v a t e d a s B c l - 2 l e v e l s . c a s e , the W i l d - T y p e c e l l s , w a s there m o r e B a k protein than B c l - 2 .  In o n l y o n e  Interestingly, w h i l e B a x a n d B a k l e v e l s  v a r i e d b e t w e e n the c l o n e s a n d control cell line, there w a s no c o r r e l a t i o n b e t w e e n the l e v e l s of t h e s e p r o t e i n s a n d the level of B c l - 2 .  T h e ratios of B c l - 2 to B a x a n d B a k in t h e M D A - 4 3 5 / L C C 6 C l o n e s a r e  o u t l i n e d in T a b l e 1.7. In the M C F - 7 c e l l line, B a x a n d B a k l e v e l s w e r e m u c h m o r e s i m i l a r b e t w e e n c l o n e s , with virtually identical l e v e l s of B a x a n d B a k protein for the three c l o n e s a n d t h e W i l d - t y p e c e l l s a n a l y z e d  (Figure 3.10).  A g a i n , a s with the M D A - 4 3 5 / L C C 6 c e l l s , B a x a n d B a k l e v e l s did not c o r r e l a t e with B c l - 2 l e v e l s in the M C F - 7 c l o n a l or W i l d - T y p e p o p u l a t i o n s . T h e ratios of B c l - 2 to B a x a n d B a k in the M C F - 7 C l o n e s a r e d i s p l a y e d in T a b l e 1.7.  Table 1.7 Ratio of Bcl-2 to Bax and Bak Individually or Combined in Clonal and Wild-type Cells* to Bak to Combined Ratio of Bcl-2 to Bax MDA-435/LCC6 Clone 11 MDA-435/LCC6 Clone 7 MDA-435/LCC6 Clone 5 MDA-435/LCC6 Clone 2 MDA-435/LCC6 Wild-Type MCF-7 Clone 2 MCF-7 Clone 5 MCF-7 Clone 7 MCF-7 Wild-Type  5 31 161 5 60 3 6 7 7  1 9 2 5 1 2 3 5 4  2 12 2 75 1 5 7 13 9  L a s t l y , the e x p r e s s i o n l e v e l s i n d i c a t e d h e r e w e r e m a i n t a i n e d in e a c h p o p u l a t i o n o v e r t i m e .  Bcl-2  levels in both M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l s , a s a s s e s s e d by b o t h f l o w c y t o m e t r y a n d W e s t e r n analysis,  were  unchanged  in  cells  after  over  * A l l ratios r o u n d e d to the n e a r e s t w h o l e n u m b e r  20  passages  in  cell  culture  (data  not  shown).  132  Bax rl  Actin Bax  Bak r-;  <N  a,  ^  ^  s s § § 3 5 2 Actin Bak  FIGURE 3.9 Western Analysis of Bax and Bak Protein Levels in MDA-MB435/LCC6 and MCF-7 Clones.  133  4.356  c  CD  Q 2.500^ 1.724  Q. 2.0CXH  o 1.113 0.694  Bcl-2 • Bax • Bak L2: L7: LW:  M D A - 4 3 5 / L C C 6 Bcl-2 Transfected Clone 2 M D A - 4 3 5 / L C C 6 Bcl-2 Transfected Clone 7 M D A - 4 3 5 / L C C 6 Wild-Type  L5: L l 1:  M D A - 4 3 S / L C C 6 Bcl-2 Transfected Clone 5 M D A - 4 3 5 / L C C 6 Bcl-2 Transfected Clone 11  2.223  1.587  1.240  0.212  M7  •0.227.  MW  M9  • Bcl-2 • Bax • Bak MW: M2:  M C F - 7 Wild-Type M7: M C F - 7 Bcl-2 Transfected Clone 2 M 9 :  M C F - 7 B c l - 2 Transfected Clone 7 M C F - 7 B c l - 2 Transfected Clone 9  FIGURE 3.10 Relative Bcl-2, Bax and Bak Levels of MDA-435/LCC6 and MCF-7 Clonal Populations as Determined by Western Analysis.  134  3.4 DISCUSSION  T r a n s f e c t i o n of M C F - 7 a n d M D A - 4 3 5 / L C C 6 c e l l s with a p l a s m i d c o n t a i n i n g the B c l - 2 c o d i n g r e g i o n p r o d u c e d a p o p u l a t i o n of c e l l s with a w i d e - r a n g e of B c l - 2 e x p r e s s i o n . B c l - 2 l e v e l s in individually t r a n s f e c t e d c e l l s a r e d e p e n d e n t u p o n the strength of the p r o m o t o r u s e d in t h e c o n s t r u c t , the n u m b e r of c o p i e s of t h e c o n s t r u c t integrated into the g e n o m e , a n d the l o c a t i o n of t h e s e i n t e g r a t i o n s .  Predictably,  the B c l - 2 profile of t h e s e c e l l lines d i s p l a y e d a distribution w h e r e i n the majority of the c e l l in the p o p u l a t i o n c o n t a i n e d m e d i u m l e v e l s of B c l - 2 , a s d e f i n e d b y the m e d i a n relative f l u o r e s c e n t intensity of the p o p u l a t i o n ( M R F I ) . W h i l e m o s t of the c e l l s within the t r a n s f e c t e d M C F - 7 a n d M D A - 4 3 5 / L C C 6 p o p u l a t i o n s c o n t a i n e d m e d i u m l e v e l s of B c l - 2 , a s m a l l n u m b e r c o n t a i n e d h i g h or low l e v e l s of this p r o t e i n . In light of the o b s e r v e d distribution of B c l - 2 l e v e l s in t h e s e t r a n s f e c t e d p o p u l a t i o n s , w e a t t e m p t e d to d e r i v e individual c l o n a l p o p u l a t i o n s e x p r e s s i n g either h i g h , m e d i u m or l o w l e v e l s of B c l - 2 with a n a r r o w r a n g e of e x p r e s s i o n a n d the m a i n t e n a n c e of this e x p r e s s i o n o v e r t i m e .  O f nine clonal populations  a n a l y z e d in the t r a n s f e c t e d M D A - 4 3 5 / L C C 6 cell line, four w e r e c h o s e n for further a n a l y s i s a n d u s e in future e x p e r i m e n t s . T h e s e w e r e c h o s e n o n the b a s i s of a n u m b e r of f a c t o r s , i n c l u d i n g the d e s i r e d B c l - 2 l e v e l , t h e r a n g e of B c l - 2 e x p r e s s i o n in the p o p u l a t i o n , a n d g r o w t h a n d c e l l c u l t u r e attributes.  In the c a s e  of the latter r e q u i r e m e n t , s o m e p o p u l a t i o n s w e r e e x c l u d e d b e c a u s e of the p r e s e n c e of l a r g e n u m b e r s of d e a d c e l l s in c u l t u r e or e x t r e m e l y s l o w g r o w t h rates in c o m p a r i s o n to W i l d - T y p e c e l l s . O u r a t t e m p t s to p r o d u c e c l o n a l p o p u l a t i o n s with v a r y i n g l e v e l s of B c l - 2 w a s m o r e s u c c e s s f u l in the M D A - 4 3 5 / L C C 6 c e l l line than the M C F - 7 cell line, a s B c l - 2 ratios b e t w e e n h i g h , m e d i u m , a n d low e x p r e s s o r s w a s 1 5 : 3 : 2 : 1 . In r e g a r d to t r a n s f e c t e d M C F - 7 c e l l s , s e v e n c l o n a l p o p u l a t i o n s w e r e a n a l y z e d for B c l - 2 c o n t e n t a n d t h r e e s e l e c t e d for further a n a l y s i s a n d e x p e r i m e n t s .  O n e notable  distinction  b e t w e e n the M C F - 7 c l o n e s a n d t h o s e s e l e c t e d f r o m the t r a n s f e c t e d M D A - 4 3 5 / L C C 6 c e l l s w a s that n o n e of t h e s e p o p u l a t i o n s c o n t a i n e d h i g h e r o v e r a l l B c l - 2 l e v e l s t h a n the p a r e n t a l p o p u l a t i o n s f r o m w h i c h they w e r e d e r i v e d . H o w e v e r , the three c l o n e s s e l e c t e d did c o n t a i n h i g h , m e d i u m a n d low l e v e l s of B c l - 2 w h e n c o m p a r e d to o n e a n o t h e r , a n d a r e f e a s i b l e for s t u d i e s investigation utilizing c e l l s of v a r y i n g B c l - 2 content. A f t e r the c l o n e s w e r e s e l e c t e d , the B c l - 2 l e v e l s in e a c h p o p u l a t i o n w a s m o r e p r e c i s e l y a s s e s s e d t h r o u g h f l o w c y t o m e t r y a n d W e s t e r n a n a l y s i s . W h i l e W e s t e r n a n a l y s i s is t h e m o r e c o m m o n t e c h n i q u e of d e t e r m i n i n g protein l e v e l s within a s a m p l e of c e l l s , both rely u p o n s i m i l a r m e c h a n i s m s to d e t e r m i n e protein c o n t e n t a n d c o n t a i n s i m i l a r r e s o l u t i o n in this r e g a r d .  B o t h e m p l o y p r i m a r y a n t i b o d i e s to B c l - 2 in  135 c o n j u n c t i o n with m a r k e r s u s e d to e v a l u a t e the a m o u n t of p r i m a r y a n t i b o d y b o u n d - f l u o r o c h r o m e in the c a s e of flow c y t o m e t r y a n d h o r s e r a d i s h p e r o x i d a s e b o u n d to s e c o n d a r y a n t i b o d i e s in W e s t e r n a n a l y s i s . F a c t o r s affecting the a c c u r a c y of e a c h t e c h n i q u e i n c l u d e the a m o u n t of a n t i b o d y u s e d , the a m o u n t of m a r k e r p e r a n t i b o d y , a n d the n u m b e r of p o s s i b l e b i n d i n g s i t e s in the s a m p l e a s s a y e d .  D e s p i t e the  potential variability of t h e s e e l e m e n t s , results f r o m a n a l y s i s of c l o n a l p o p u l a t i o n s u s i n g t h e s e  two  p r o c e d u r e s c o r r e l a t e d w e l l . W h i l e both flow c y t o m e t r y a n d W e s t e r n a n a l y s i s indirectly m e a s u r e the Bcl-2 protein c o n t e n t in the c e l l , the similarity in the results of t h e s e two a s s a y s s u g g e s t s a n a c c u r a t e d e t e r m i n a t i o n of the l e v e l of this protein in the s a m p l e s a n a l y z e d . T h e final c o m p o n e n t in the c h a r a c t e r i z a t i o n of t h e s e cell lines w a s the d e t e r m i n a t i o n of the proa p o p t o t i c proteins B a x a n d B a k . T h e m e a s u r e m e n t of the l e v e l s of t h e s e p r o t e i n s is e s p e c i a l l y significant a s the sensitivity of c l o n e s to cytotoxic a g e n t s or their ability to u n d e r g o a p o p t o s i s is not s o m u c h a function of their Bcl-2 content, but rather the ratio of Bcl-2 to p r o - a p o p t o t i c p r o t e i n s  1 0  .  T h e r e f o r e , the  l e v e l s of B a x a n d B a k w e r e e s t a b l i s h e d a n d ratios of t h e s e t w o p r o t e i n s to Bcl-2 g e n e r a t e d .  Because  l e v e l s of e a c h of t h e s e proteins w a s low relative to Bcl-2 in the majority of the c l o n a l p o p u l a t i o n s , the o v e r a l l effect of high Bcl-2 c o n t e n t w a s p r e s e r v e d .  I n d e e d , e v e n with the l e v e l s of t h e s e two proteins  a c c o u n t e d for, the c l o n e s r e t a i n e d relative Bcl-2 l e v e l s (i.e. h i g h , m e d i u m , or low) in relation to o n e a n o t h e r , albeit not to a s great a d e g r e e a s b e f o r e . W h e n c o n s i d e r i n g of the a m o u n t s of B a k a n d B a x in the c l o n a l p o p u l a t i o n s , o n e m a y a r g u e that a s s e s s i n g the l e v e l s of t h e s e two p r o t e i n s r e p r e s e n t s o n l y a limited s e l e c t i o n of the Bcl-2 f a m i l y of p r o t e i n s , o t h e r s of w h i c h m a y a l s o affect the a p o p t o t i c potential of these cells.  I n d e e d , the status of other p r o - a p o p t o t i c p r o t e i n s , a s w e l l a s a n t i - a p o p t o t i c p r o t e i n s , m a y b e  significant in this r e g a r d .  W h i l e t h e s e c o n s i d e r a t i o n s a r e c e r t a i n l y v a l i d , the s h e e r n u m b e r of apoptotic  family m e m b e r s p r e c l u d e s the a s s e s s m e n t of the protein l e v e l s of e a c h c o m p o n e n t in the c l o n a l populations generated.  F o r the s a k e of practicability, o n l y the l e v e l s of B a x a n d B a k protein —  c o m m o n a n d well c h a r a c t e r i z e d p r o - a p o p t o t i c proteins — in e a c h cell line w a s a s s e s s e d W i t h the g e n e r a t i o n of r e p r e s e n t a t i v e c l o n a l p o p u l a t i o n s f r o m t r a n s f e c t e d M C F - 7 a n d M D A -  435/LCC6 cell lines c o n t a i n i n g h i g h , m e d i u m a n d low e x p r e s s i o n l e v e l s of Bcl-2, s t u d i e s w e r e then u n d e r t a k e n to d e t e r m i n e the effect of Bcl-2 level o n sensitivity to c h e m o t h e r a p y a g e n t s . T h e following two c h a p t e r s d i s c u s s this t o p i c .  two  136  3.5 REFERENCES R a f f o A J , P e r l m a n H , C h e n M W , D a y M L , S t r e i t m a n J S , B u t t y a n R. O v e r e x p r e s s i o n of b c l - 2 p r o t e c t s p r o s t a t e c a n c e r c e l l s f r o m a p o p t o s i s in vitro a n d c o n f e r s r e s i s t a n c e to a n d r o g e n d e p l e t i o n in v i v o . Cancer Res. 1 9 9 5 O c t 1 ; 5 5 ( 1 9 ) : 4 4 3 8 - 4 5 . 1  K a j i w a r a T , T a k e u c h i T , U e k i T , M o r i y a m a N , U e k i K, K a k i z o e T , K a w a b e K. Effect of B c l - 2 o v e r e x p r e s s i o n in h u m a n p r o s t a t e c a n c e r c e l l s in vitro a n d in v i v o . Int J Urol. 1 9 9 9 O c t ; 6 ( 1 0 ) : 5 2 0 - 5 . 2  M i y a k e H , H a n a d a N , N a k a m u r a H , K a g a w a S , F u j i w a r a T, H a r a I, E t o H , G o h j i K, A r a k a w a S , K a m i d o n o S , S a y a H . O v e r e x p r e s s i o n of B c l - 2 in b l a d d e r c a n c e r c e l l s inhibits a p o p t o s i s i n d u c e d b y cisplatin a n d a d e n o v i r a l - m e d i a t e d p 5 3 g e n e transfer. Oncogene. 1 9 9 8 F e b 1 9 ; 1 6 ( 7 ) : 9 3 3 - 4 3 .  3  M i y a k e H , H a r a I, Y a m a n a k a K, A r a k a w a S , K a m i d o n o S . S y n e r g i s t i c e n h a n c e m e n t of r e s i s t a n c e to cisplatin in h u m a n b l a d d e r c a n c e r c e l l s b y o v e r e x p r e s s i o n of m u t a n t - t y p e p 5 3 a n d B c l - 2 . J Urol. 1 9 9 9 Dec;162(6):2176-81. 4  O h m o r i T, P o d a c k E R , N i s h i o K, T a k a h a s h i M , M i y a h a r a Y , T a k e d a Y , K u b o t a N , F u n a y a m a Y , O g a s a w a r a H , O h i r a T , et a l . A p o p t o s i s of lung c a n c e r c e l l s c a u s e d by s o m e a n t i - c a n c e r a g e n t s ( M M C , C P T - 1 1 , A D M ) is inhibited b y b c l - 2 . Biochem Biophys Res Commun. 1 9 9 3 A p r 1 5 ; 1 9 2 ( 1 ) : 3 0 - 6 .  5  L u o D, C h e n g S C , X i e H , X i e Y . C h e m o s e n s i t i v i t y of h u m a n h e p a t o c e l l u l a r c a r c i n o m a cell line Q G Y 7 7 0 3 is r e l a t e d to b c l - 2 protein l e v e l s . Tumour Biol. 1 9 9 9 N o v - D e c ; 2 0 ( 6 ) : 3 3 1 - 4 0 .  6  A r r i o l a E L , R o d r i g u e z - L o p e z A M , H i c k m a n J A , C h r e s t a C M . B c l - 2 o v e r e x p r e s s i o n results in r e c i p r o c a l d o w n r e g u l a t i o n of B c l - X ( L ) a n d s e n s i t i z e s h u m a n testicular g e r m cell t u m o u r s to c h e m o t h e r a p y - i n d u c e d a p o p t o s i s . Oncogene. 1 9 9 9 F e b 18; 18(7): 1 4 5 7 - 6 4 . 7  B e a l e P J , R o g e r s P , B o x a l l F, S h a r p S Y , K e l l a n d L R . B C L - 2 f a m i l y protein e x p r e s s i o n a n d p l a t i n u m d r u g r e s i s t a n c e in o v a r i a n c a r c i n o m a . BrJ Cancer. 2 0 0 0 J a n ; 8 2 ( 2 ) : 4 3 6 - 4 0 .  8  T s u j i m o t o Y , C r o c e C M . A n a l y s i s of the structure, transcripts, a n d protein p r o d u c t s of b c l - 2 , the g e n e i n v o l v e d in h u m a n follicular l y m p h o m a . Proc Natl Acad Sci U S A. 1 9 8 6 J u l ; 8 3 ( 1 4 ) : 5 2 1 4 - 8 .  9  Oltvai Z N , K o r s m e y e r S J . C h e c k p o i n t s of d u e l i n g d i m e r s foil d e a t h w i s h e s . 21 ; 7 9 ( 2 ) : 1 8 9 - 9 2 . 1 0  Cell.  1994 Oct  1  CHAPTER 4 BCL-2 MODULATION IN MDA-MB-435/LCC6 AND MCF-7 CELL LINES VIA ESTROGEN INHIBITION: EFFECTS ON CELL GROWTH AND CHEMOSENSITIVITY  138  INTRODUCTION  O u r initial i n v e s t i g a t i o n s c o n c e r n i n g B c l - 2 a n d its effect o n c h e m o s e n s i t i v i t y in vitro utilized B c l - 2 a n t i s e n s e a s a m e a n s to d o w n - r e g u l a t e B c l - 2 .  In o r d e r to v a l i d a t e a n d c o r r o b o r a t e t h e f i n d i n g s of t h e s e  e x p e r i m e n t s , w e utilized e s t r o g e n inhibition a s a n o t h e r m e t h o d of d i m i n i s h i n g t h e a m o u n t of B c l - 2 in t h e cell a n d s u b s e q u e n t l y a n a l y z e r e s p o n s e s to c h e m o t h e r a p y .  W h i l e w e i n c l u d e d both o f t h e original w i l d -  type M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 c e l l s u s e d in p r e v i o u s e x p e r i m e n t s , w e a l s o i n c l u d e d t h e c l o n a l p o p u l a t i o n s w e h a d g e n e r a t e d w h i c h e x p r e s s e d variant l e v e l s of B c l - 2 . T h u s , t h e o b j e c t i v e of t h e s e experiments  is t w o - f o l d :  1) to elucidate what effect the variant level of Bcl-2 in the clonal  populations would have on their chemosensitivity; and, 2) to establish whether these cells can be sensitized to chemotherapeutic agents by down-regulation of the Bcl-2 protein through estrogen inhibition. E s t r o g e n h a s p r o v e n to b e intimately i n v o l v e d in t h e r e g u l a t i o n of c e l l g r o w t h a n d a p o p t o s i s in estrogen-receptor positive cell lines. T h e m e c h a n i s m s by which estrogen m a y influence these cell types m a y , in part, b e d u e to its r e g u l a t i o n of B c l - 2 a n d o t h e r h o m o l o g o u s p r o t e i n s .  T h e bcl-2 g e n e contains  two e s t r o g e n r e s p o n s e e l e m e n t s in its c o d i n g r e g i o n s , a n d it is n o t s u r p r i s i n g that l e v e l s of this protein a r e m o d u l a t e d in a d o s e d e p e n d e n t m a n n e r b y 1 7 p - e s t r a d i o l , o n e of t h e m o s t c o m m o n e s t r o g e n s . S t u d i e s 1  h a v e s h o w n that e s t r o g e n w i t h d r a w a l results in a m a r k e d d e c r e a s e in B c l - 2 m R N A a n d protein l e v e l s , w h i l e l e v e l s of B a x r e m a i n c o n s t a n t  2,3  .  T h e s e d o w n - r e g u l a t i o n s of B c l - 2 protein in t h e f a c e of static B a x  l e v e l s l e a d to d r a s t i c d e c r e a s e s in t h e B c l - 2 to B a x ratio, a n d m a y i n d u c e i n c r e a s e d a p o p t o s i s . A l t h o u g h 4  f e w e r s t u d i e s h a v e b e e n c o n d u c t e d , t h e c o m b i n a t i o n of c h e m o t h e r a p y with a g e n t s that b l o c k t h e action of e s t r o g e n h a s i n d i c a t e d that s o m e s y n e r g y m a y exist b e t w e e n t h e two. T a m o x i f e n h a s s h o w n t h e ability to i n c r e a s e cytotoxicity o r inhibit g r o w t h w h e n c o m b i n e d with d o x o r u b i c i n o r p a c l i t a x e l in a n u m b e r of cell i-  lines  5,6  . W h i l e m o s t s t u d i e s h a v e i n v e s t i g a t e d t h e effects of e s t r o g e n inhibition in vitro, t h e results of in  vivo s t u d i e s p r e s e n t s i m i l a r f i n d i n g s .  It h a s l o n g b e e n r e c o g n i z e d that e s t r o g e n w i t h d r a w a l will result in  the s h r i n k a g e of h o r m o n e - d e p e n d e n t t u m o r s , a n d B c l - 2 s t a t u s is m o s t likely i n v o l v e d in this p r o c e s s . O n e s t u d y utilizing x e n o t r a n p l a n t e d  M C F - 7 c e l l s in n u d e m i c e i n d i c a t e d that t u m o r s  c o m p r i s e d of c e l l s  139 t r a n s f e c t e d with B c l - 2 w e r e t w i c e the s i z e of t u m o r s c o n s i s t i n g of u n t r a n s f e c t e d c e l l s after e s t r o g e n withdrawal.  F u r t h e r m o r e , this s t u d y i n d i c a t e d that the l e v e l s of other B c l - 2 f a m i l y m e m b e r s r e m a i n e d  u n c h a n g e d , r e g a r d l e s s of B c l - 2 level or e s t r o g e n s t a t u s . 7  T h e e s t r o g e n - i n h i b i t i n g c o m p o u n d u s e d in t h e s e s t u d i e s w a s the p u r e a n t i - e s t r o g e n c o m p o u n d F a s l o d e x (ICI 1 8 2 7 8 0 ) , w h i c h l a c k s a n y a g o n i s t i c tropic e s t r o g e n - l i k e e f f e c t s f o u n d with c o m p o u n d s s u c h a s T a m o x i f e n , a n d thus s p e c i f i c a l l y b l o c k s all stimulatory a c t i o n s of e s t r o g e n s .  F u r t h e r m o r e , in direct  c o m p a r i s o n s with T a m o x i f e n , F a s l o d e x p r o v e d to b e better at d i m i n i s h i n g B c l - 2 protein l e v e l s , inhibiting cell g r o w t h , a n d i n d u c i n g a p o p t o s i s . 8  W h i l e the a p o p t o t i c potential of F a s l o d e x m a y b e related to  r e g u l a t i o n of B c l - 2 p r o t e i n s , the m a n n e r in w h i c h it inhibits c e l l g r o w t h is l e s s c l e a r .  S o m e studies have  i n d i c a t e d that F a s l o d e x m a y b e c a p a b l e of r e d u c i n g g r o w t h factor e x p r e s s i o n — s u c h a s insulin g r o w t h factor ( I G F ) — in b r e a s t c a n c e r c e l l s , a l t h o u g h the extent to w h i c h this o c c u r s still r e m a i n s u n c l e a r . 9  T h e cell l i n e s utilized in t h e s e s t u d i e s w e r e the wild-type M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 c e l l l i n e s , the f o r m e r b e i n g e s t r o g e n - r e c e p t o r n e g a t i v e w h i l e the later is e s t r o g e n - r e c e p t o r p o s i t i v e ; thus all M D A - M B - 4 3 5 / L C C 6 l i n e s s e r v e a s c o n t r o l s in e x p e r i m e n t s i n v o l v i n g F a s l o d e x . A d d i t i o n a l l y , the h i g h e s t a n d l o w e s t B c l - 2 e x p r e s s i n g c l o n a l p o p u l a t i o n s g e n e r a t e d f r o m t h e s e two c e l l l i n e s w e r e a l s o e m p l o y e d , not o n l y to a n a l y z e their r e s p o n s e to c h e m o t h e r a p y after t r e a t m e n t with e s t r o g e n inhibitors, but a l s o to s i m p l y a s s e s s a n y inherent d i f f e r e n c e s in c h e m o s e n s i t i v i t y b e t w e e n t h e c e l l lines  (chemosensitivity  b e t w e e n c l o n e s in the a b s e n c e of e s t r o g e n inhibitors).  4.2 MATERIALS AND METHODS  Cell lines  T h e h u m a n b r e a s t c a n c e r c e l l line M D A - M B - 4 3 5 / L C C 6  w a s o b t a i n e d f r o m Dr. R. C l a r k e ,  G e o r g e t o w n U n i v e r s i t y a n d t r a n s f e c t e d with either a v e c t o r or p l a s m i d c o n t a i n i n g t h e b c l - 2 c o d i n g r e g i o n . C l o n e s w e r e c r e a t e d f r o m t r a n s f e c t e d p o p u l a t i o n s a n d c h a r a c t e r i z e d a s d e s c r i b e d in c h a p t e r 2.  Cells  w e r e g r o w n a s a d h e r e n t c u l t u r e s a n d m a i n t a i n e d in D M E M s u p p l e m e n t e d with 1 0 % F B S at 3 7 ° C a n d 5 % C 0 . C e l l s w e r e u s e d in e x p o n e n t i a l g r o w t h p h a s e , u p to a m a x i m u m of 2 5 p a s s a g e s . 2  140  Western Blot analysis of Bcl-2 expression.  C e l l lines w e r e g r o w n in t h e a b s e n c e o r p r e s e n c e of 1 X 1 0 M F a s l o d e x o v e r five d a y s with 7  lysates taken every 24 hours.  C e l l l y s a t e s in i c e - c o l d l y s i s buffer ( 1 5 0 m M N a C l , 1 % N P - 4 0 , 0 . 5 %  s o d i u m d e o x y c h o l a t e , 0.1 % S D S , 2 . 5 m M E D T A , 0.1 % s o d i u m a z i d e ) , c o n t a i n i n g p r o t e a s e inhibitors (Complete-Mini  R  p r o t e a s e inhibitor t a b l e t s , B o e h r i n g e r M a n n h e i m G m B H , G e r m a n y ) .  c e n t r i f u g e d at 1 4 , 0 0 0 r p m f o r 1 5 m i n , a n d s t o r e d at - 7 0 ° C .  S a m p l e s were  P r o t e i n c o n t e n t in t h e l y s e d e x t r a c t s w a s  d e t e r m i n e d u s i n g a d e t e r g e n t - c o m p a t i b l e B i o R a d a s s a y ( B i o R a d L a b s , H e r c u l e s , C A ) . E q u a l a m o u n t s of protein (20 p g / l a n e ) w e r e s u b j e c t e d to 12.5 % S D S - P A G E ( B i o r a d ) f o r 4 5 m i n at 150 V in a g l y c i n e buffer (1.92 M g l y c i n e , 2 5 m M T r i s p H 8.3) c o n t a i n i n g 1 % S D S .  G e l s w e r e t r a n s f e r r e d to n i t r o c e l l u l o s e  m e m b r a n e s in a g l y c i n e t r a n s f e r buffer with 2 0 % m e t h a n o l f o r 1 h at 7 0 V . M e m b r a n e s w e r e b l o c k e d o v e r n i g h t at 4 ° C with 5 % s k i m milk, 0 . 0 5 % s o d i u m a z i d e in P B S ( 2 0 m M T r i s p H 8 . 2 , 1 3 7 m M s o d i u m chloride).  M o u s e m A b to h u m a n B c l - 2 ( D A K O , D e n m a r k ) , 1:3,000, h u m a n B a x ( U p s t a t e B i o t e c h ) 1:1000,  or B a k 1:1000 ( U p s t a t e , B i o t e c h ) a n d m o u s e a n t i - h u m a n (3-actin m A b ( S i g m a , S t . L o u i s , M O ) , 1:10,000 w e r e u s e d diluted in 1 % s k i m milk in T B S , c o n t a i n i n g 0 . 0 5 % T w e e n - 2 0 a n d 0 . 0 5 % s o d i u m a z i d e . T h e m e m b r a n e s w e r e t h e n i n c u b a t e d f o r 1 h with 1:3,000 h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d a n t i - m o u s e I g G (Promega, M a d i s o n , WI). Proteins were detected by using a n e n h a n c e d chemiluminescence ( E C L , A m e r s h a m P h a r m a c i a B i o t e c h , B u c k i n g h a m s h i r e , E n g l a n d ) m e t h o d , a n d v i s u a l i z e d after e x p o s u r e to K o d a k film.  S c a n n i n g d e n s i t o m e t r y ( M o l e c u l a r D y n a m i c s , S u n n y v a l e , C A ) w a s p e r f o r m e d to quantify  b a n d intensities b y v o l u m e / a r e a integration. T h e a m o u n t o f B c l - 2 protein in c e l l s / t u m o r w a s n o r m a l i z e d to their (3-actin l e v e l s .  Cytotoxicity and Growth Assays  F o r cytotoxicity a s s a y s , c e l l s w e r e p l a c e in m e d i a with o r without 1 X 1 0 M F a s l o d e x a n d e q u a l 7  n u m b e r s of c e l l s (2-5 x 1 0 ) w e r e s e e d e d in 9 6 - w e l l microtiter p l a t e s a n d a l l o w e d to a d h e r e overnight. 3  D r u g dilutions of d o x o r u b i c i n a n d c i s p l a t i n w e r e a d d e d to t h e p l a t e s after 2 4 h o u r s a n d c e l l survivability w a s a s s e s s e d after 4 8 h o u r s f o r total n u m b e r of v i a b l e c e l l s u s i n g a c o n v e n t i o n a l 3 - ( 4 , 5 - D i m e t h y l t h i a z o l 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m b r o m i d e ( M T T ) d y e r e d u c t i o n a s s a y a n d o p t i c a l d e n s i t y d e t e r m i n e d at 5 7 0 nm.  141 F o r g r o w t h a s s a y s 1 X 1 0 c e l l s w e r e plated in s i x - w e l l p l a t e s with o r without 1 X 1 0 M F a s l o d e x 5  7  a n d cell viability a s s e s s e d e v e r y 2 4 h o u r s u s i n g 3 - ( 4 , 5 - D i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l  tetrazolium  b r o m i d e ( M T T ) d y e r e d u c t i o n a s s a y a n d optical d e n s i t y d e t e r m i n e d a t 5 7 0 n m .  RESULTS  Western Analysis of MDA-MB-435/LCC6 and MCF-7 Cell Lines Treated with Faslodex  A d m i n i s t r a t i o n of F a s l o d e x (1 X 1 0  7  M ) c a u s e d down-regulation  of B c l - 2 e x p r e s s i o n within 2 4  h o u r s of t r e a t m e n t w h e r e B c l - 2 protein l e v e l s w e r e r e d u c e d in M C F - 7 W i l d - T y p e , M C F - 7 C l o n e 2 , a n d M C F - 7 C l o n e 7 to a p p r o x i m a t e l y 3 5 % , 2 5 % a n d 6 5 % of pre-treatment c o n t r o l s , r e s p e c t i v e l y (Figure 4.1) F u r t h e r m o r e , B c l - 2 l e v e l s in t h e s e c e l l s r e m a i n e d s u p p r e s s e d up f o r the entire d u r a t i o n of treatment. B c l 2 d o w n - r e g u l a t i o n in t h e M C F - 7 cell lines a p p e a r e d to c o r r e l a t e with initial B c l - 2 c o n t e n t , a s t h e l o w e s t e x p r e s s i n g c l o n e ( C l o n e 2) d i s p l a y e d t h e g r e a t e s t d e g r e e of d o w n r e g u l a t i o n , f o l l o w e d b y M C F - 7 W i l d T y p e , a n d finally M C F - 7 C l o n e 7 w h i c h c o n t a i n e d t h e h i g h e s t level of B c l - 2 .  In r e g a r d t o the e s t r o g e n -  r e c e p t o r n e g a t i v e M D A - M B - 4 3 5 / L C C 6 cell l i n e s , n o c h a n g e in B c l - 2 w a s o b s e r v e d o v e r t h e c o u r s e of F a s l o d e x t r e a t m e n t (Figure 4.1). A d d i t i o n a l l y , B a x a n d B a k l e v e l s w e r e a n a l y z e d in t h e M C F - 7 a n d M D A - M B - 4 3 5 / L C C 6 cell lines w h e n treated with F a s l o d e x o v e r the s a m e t i m e s c o u r s e . O u r f i n d i n g s i n d i c a t e n o c h a n g e s in t h e l e v e l s of B a x or B a k in either cell line u p o n e x p o s u r e to F a s l o d e x (Figure 4.2).  Growth Curves of MDA-MB-435/LCC6 and MCF-7 Cell Lines Treated with Faslodex  M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 cell lines w e r e g r o w n o v e r the c o u r s e of s i x d a y s in the p r e s e n c e or a b s e n c e of F a s l o d e x (1 X 1 0 M ) to c h a r a c t e r i z e the g r o w t h p r o p e r t i e s of t h e s e c e l l s u n d e r t r e a t m e n t 7  c o n d i t i o n s . A l l M C F - 7 cell lines d i s p l a y e d i m p a i r e d g r o w t h rates in the p r e s e n c e of F a s l o d e x in  142  A.  MCF-7 (ER Positive)  MDA-435/LCC6 (ER Negative) Day 2  Day 1  « o  £ i  Q o o a  •a  S  U o  _7~  O  S  H -  P  Day 3  I i  O  ct CD  I  Q o  3  O  3  a » —i i-—  QQ o" 5" 3  3  ~  ft  ro  ro  3  ro  H —  e o o £• Si 2  0? S ro  s  Q Q  st a  3  ro  3  ro ro K>  Day 4  Day 3  Day 4  oQ  S A  S  a  9~4  Day 2  Day 1  e= o o  3 3 ro ro to ~J  3  ro  3  ro  i  - M C F - 7 Wild-Type M C F - 7 Clone 2 M C F - 7 Clone 7  5  M D A - M B - 4 3 5 / L C C 6 Wild-Type  0-  M D A - M B - 4 3 5 / L C C 6 Clone 7 M D A - M B - 4 3 5 / L C C 6 C l o n e 11  1  2  3  E x p o s u r e to F a s l o d e x ( D a y s )  E x p o s u r e to F a s l o d e x ( D a y s )  FIGURE 4.1 Western Analysis of Bcl-2 Levels in MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell lines Exposed to Faslodex over 96 Hours. A. Western analysis of MDA-MB-435/LCC6; Bcl-2 levels compared to pre-treatment controls B. Western analysis of MCF-7 Cells; Bcl-2 levels compared to pre-treatment controls.  Dayl  5  Day  2  Day  3  Day  4  r-  Actin Bak  B. Day  Day 2  1  Day  3  Day  4  H s  •3  Actin Bak  c.  Dayl  Day  a .-  2  Day  3  Day  4  r-  Actin  Bax  D. Dayl  0  Day  H 2 u  2  Day  -  3  Day  4  fi a  5 Actin Bax  FIGURE 4.2 Western Analysis of Bax and Bak Levels in Bcl-2-Transfected MDA-435/LCC6 and MCF-7 Cell Exposed to Fasoldex over 4 Days. A. B a k levels in Bcl-2-Transfected M D A - M B - 4 3 5 / L C C 6 C e l l s B. B a k levels in Bcl-2-Transfected M C F - 7 C e l l s C . B a x levels in B c l - 2 - T r a n s f e c t e d M D A - M B - 4 3 5 / L C C 6 C e l l s . D. B a k levels in B c l - 2 Transfected M C F - 7 C e l l s  144 c o m p a r i s o n to the s a m e c e l l lines g r o w n in n o r m a l m e d i a  (Figure 4.3).  Growth suppression occurred 48  h o u r s after initial e x p o s u r e to F a s l o d e x a n d c o n t i n u e d for the length of the treatment.  In contrast, all  M D A - M B - 4 3 5 / L C C 6 c e l l lines g r e w at c o m p a r a b l e rates r e g a r d l e s s of w h e t h e r t h e y w e r e treated with F a s l o d e x or not  (Figure 4.3).  Chemosensitivity of MDA-MB-435/LCC6 and MCF-7 Cell Lines Treated with Faslodex  M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 cell lines w e r e treated with F a s l o d e x or left untreated  and  e x p o s e d to v a r y i n g c o n c e n t r a t i o n s of d o x o r u b i c i n a n d c i s p l a t i n for 4 8 h o u r s in o r d e r to a s s e s s the ability of F a s l o d e x to c h e m o s e n s i t i z e t h e s e cell lines.  W i t h r e g a r d to d o x o r u b i c i n , both the M C F - 7 a n d M D A -  M B - 4 3 5 / L C C 6 c e l l lines d i s p l a y e d identical I C  v a l u e s r e g a r d l e s s of t r e a t m e n t with F a s l o d e x .  5 0  Notably,  n o variation in c h e m o s e n s i t i v i t y w a s d e t e c t e d b e t w e e n the e s t r o g e n - r e c e p t o r p o s t i v e M C F - 7 c e l l lines treated with F a s l o d e x a n d t h o s e that w e r e not treated with F a s l o d e x  (Figure 5.4).  W h i l e there w a s n o  a p p a r e n t c h a n g e in d r u g sensitivity b e t w e e n cell treated with F a s l o d e x a n d t h o s e left u n t r e a t e d , there w a s a l s o no variation in the I C  5 0  value between high-Bcl-2 expressing, low-Bcl-2 expressing, and Wild Type  cell lines in both the M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 l i n e s . W h e n the M D A - M B - 4 3 5 / L C C 6 a n d M C F - 7 c e l l lines w e r e e x p o s e d to c i s p l a t i n , F a s l o d e x did not i n d u c e a n y d r u g sensitivity; in fact, treatment of e s t r o g e n - r e c e p t o r p o s i t i v e M C F - 7 lines with F a s l o d e x m a d e t h e s e lines slightly m o r e resistant ( a p p r o x i m a t e l y 1.0 log d i f f e r e n c e in I C , 5 0  Figure 5.5).  A s with the  results i n d i c a t e d u p o n d o x o r u b i c i n e x p o s u r e , there w a s n o v a r i a t i o n b e t w e e n sensitivity of h i g h - B c l - 2 e x p r e s s i n g , l o w - B c l - 2 e x p r e s s i n g , a n d wild type c e l l s to c i s p l a t i n in either the M D A - M B - 4 3 5 / L C C 6 or M C F - 7 cell l i n e s .  -Hi— Wild-Type * Clone 2 (Low Bcl-2 Expressor) - T — Clone 7 (High Bcl-2 Expressor) Wild-Type in Faslodex Clone 2 (Low Bcl-2 Expressor) in Faslodex • Clone 7 (High Bcl-2 Expressor) in Faslodex  A.  CO  a  tu  Q  "3 o  -4—I  PH  o  Exposure to Faslodex (days)  B. 6.56.05.55.0-  - Wild-type Clone 7 (High Bcl-2) -Clone 11 (LowBd-2) Wild-type in Faslodex Clone 7 (High Bcl-2) in Faslodex Clone 11 (Low Bcl-2) in Faslodex  4.5to c  Q "ci  4.03.5-  o  3.0-  o  2.5-  a  2.01.51.02  3  4  Exposure to Faslodex (days)  FIGURE 4.3 Growth Rates of MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell lines Exposed to Faslodex over 120 Hours. A . MCF-7 Cells B . MDA-MB-435/LCC6 Cells  A. 140  MDA-436/LCC6 Wild-Type in Faslodex MDA-435/LCC6 Clone 7 (High Bcl-2) in Faslodex MDA-435/LCC6 Clone 11 (Low Bcl-2) in Faslodex MDA-435/LCC6 Wild-Type MDA-43S/LCC6 Clone 7 (High Bcl-2) MDA-435/LCC6 Clone 11 (Low Bcl-2) n  120 4 100-^  c o O 4MI  c  CD  80 H 60 H  o  1—  CD LL  40 4 20-  1E-4  -i—i—i i 11 • 1E-3  0.01  i—i—i i i 1111 0.1  i i i i i 1111 1  ri  1:0  Concentration of Doxorubicin (uM)  B.  -•— • *• •-•  120  •«  MCF-7 Wild-Type in Faslodex MCF-7 Clone 7 (High Bcl-2) in Faslodex MCF-7 Clone 2 (Low Bcl-2) in Faslodex MCF-7 Wild-Type MCF-7 Clone 2 (Low Bcl-2) MCF-7 Clone 7 (High Bcl-2)  100  80 4  o O  60  0) o 0  40  CL  20  I  I  1 T I I  111  1E-3  -l—r  i i i ii]  0.01  T 111J  0.1  -I—I  I M i l ]  1  10  C o n c e n t r a t i o n of D o x o r u b i c i n ( u M )  FIGURE 4.4 Cytotoxicity of MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell Lines Exposed to Doxorobucin over 48 Hours. A . MDA-MB-435/LCC6 B. MCF-7  147  A. 140  - Wild Type Clone 7 (High Bcl-2) - Clone 11 (Low Bcl-2) Wild Type in Faslodex Clone 7 (High Bcl-2) in Faslodex Clone 11 (Low Bcl-2) in Faslodex  n  0.01  0.1  1  10  Concentration of Cisplatin (uM)  B.  -A-  ••->-••  140  •  Wild Type Clone 2 (Low Bcl-2) Clone 7 (High Bcl-2) Wild-Type in Faslodex Clone 2 (Low Bcl-2) in Faslodex Clone 7 (High Bcl-2) in Faslodex  c o o c  o  i_ CD CL  1E-3  0.01  0.1  1  10  100  Concentration of Cisplatin (uM)  FIGURE 4.5 Cytotoxicity of MDA-MB-435/LCC6 (ER-) and MCF-7 (ER+) Cell Lines Exposed to Cisplatin over 48 Hours. A. MDA-MB-435/LCC6 B. MCF-7  148  DISCUSSION  E s t r o g e n s a r e a k n o w n risk factor in the d e v e l o p m e n t of b r e a s t c a n c e r a n d it is well e s t a b l i s h e d t h a t e s t r o g e n w i t h d r a w a l i n d u c e s t u m o r r e g r e s s i o n . F u r t h e r m o r e , s t u d i e s i n v o l v i n g the effect of e s t r o g e n s o n m a m m a r y c a r c i n o m a c e l l s both  in vitro  and  in vivo  i n d i c a t e the p o w e r f u l proliferative effect of t h e s e  a g e n t s , . W h i l e the i n c r e a s e d growth properties of c e l l s e x p o s e d to e s t r o g e n s m a y b e in part attributed 1 0  1 1  to alteration in c e l l u l a r p a t h w a y s controlling cell d i v i s i o n a n d g r o w t h , i n c r e a s e s in the o v e r a l l proliferative rate m a y a l s o b e d u e to d e c r e a s e d a p o p t o s i s through m a n i p u l a t i o n of g e n e s controlling this p r o c e s s . O u r initial s t u d i e s with Bcl-2 a n t i s e n s e i n d i c a t e d that s u p p r e s s i o n of the Bcl-2 protein  was  sufficient to c a u s e a p o p t o s i s in b r e a s t c a n c e r c e l l s but not s e n s i t i z e t h e s e c e l l s to c h e m o t h e r a p y d r u g s ; therefore, in o r d e r to i n v e s t i g a t e t h e s e findings further, w e s o u g h t o t h e r m e a n s to m a n i p u l a t e the Bcl-2 level in cell p o p u l a t i o n s .  E s t r o g e n inhibition, s p e c i f i c a l l y by u s i n g the a n t i - e s t r o g e n c o m p o u n d F a s l o d e x ,  p r o v e d useful in this r e g a r d . T h e results p r e s e n t e d h e r e indicate that treatment of e s t r o g e n s e n s i t i v e M C F - 7 cell lines with F a s l o d e x r e s u l t e d in the d o w n - r e g u l a t i o n of Bcl-2 l e v e l s in the cell with n o a p p a r e n t c h a n g e s in B a x or B a k l e v e l s a n d that n o c h a n g e s o c c u r r e d in the l e v e l s a n y of t h e s e p r o t e i n s in the e s t r o g e n - r e c e p t o r negative MDA-MB-435/LCC6.  T h e s e results indicate the specificity of F a s l o d e x a n d d e m o n s t r a t e s that  the results o b s e r v e d in M C F - 7 c e l l s w e r e the result of inhibition of e s t r o g e n i c e f f e c t s .  A d d i t i o n a l l y , the  g r o w t h c h a r a c t e r i s t i c s of cell lines e x p o s e d to F a s l o d e x w e r e c o m p a r e d with t h o s e g r o w n in n o r m a l m e d i a a n d the results i n d i c a t e d a n i m p a i r e d growth ability in M C F - 7 cell l i n e s t r e a t e d with F a s l o d e x ; results c o r r e l a t e d to findings f r o m other s t u d i e s  1 2 , 1 3  .  these  A s no g r o w t h i m p a i r m e n t o c c u r r e d in e s t r o g e n -  r e c e p t o r n e g a t i v e M D A - M B - 4 3 5 / L C C 6 l i n e s , the d e c r e a s e d proliferation of M C F - 7 treated with F a s l o d e x m a y be attributed to e s t r o g e n inhibition. W h i l e e s t r o g e n - s p e c i f i c effects w e r e o b s e r v e d in the cell l i n e s u s e d in t h e s e  experiments,  e x p o s u r e of F a s l o d e x treated cell lines c o n f e r r e d no i n c r e a s e d sensitivity to d o x o r u b i c i n or c i s p l a t i n . T h e i n c r e a s e in c i s p l a t i n r e s i s t a n c e o b s e r v e d in M C F - 7 c e l l s treated with F a s l o d e x m a y b e d u e to the i m p a i r e d growth rate of t h e s e c e l l s . C i s p l a t i n c r o s s - l i n k s to D N A , f o r m i n g intra- a n d inter-strand a d d u c t s , w h i c h b e n d a n d u n w i n d the d u p l e x a n d inhibit D N A repair. T h e resulting D N A d a m a g e triggers c e l l - c y c l e arrest a n d a p o p t o s i s , a n d a l t h o u g h cisplatin is a c t i v e d u r i n g all p h a s e s of the cell c y c l e , c e l l s a r e e s p e c i a l l y  149 s u s c e p t i b l e d u r i n g S p h a s e w h e n D N A replication is o c c u r r i n g .  G r o w t h inhibition of c e l l p o p u l a t i o n s by  F a s l o d e x w o u l d result in d e c r e a s e d c e l l c y c l i n g a n d l e s s sensitivity to c i s p l a t i n . T h e s e f i n d i n g s c o r r e l a t e with other s t u d i e s c o m b i n i n g F a s l o d e x with c i s p l a t i n t r e a t m e n t that i n d i c a t e d that F a s l o d e x w a s c a p a b l e i  of inhibiting c e l l g r o w t h , but did not perturb the cell c y c l e or affect the i n d u c t i o n of a p o p t o s i s . 1 4  • O n e difficulty in e m p l o y i n g e s t r o g e n inhibitors to s t u d y t h e effects of B c l - 2 d o w n - r e g u l a t i o n o n r e s p o n s e to c h e m o t h e r a p y is the n u m b e r of cellular t a r g e t s in the c e l l w h i c h m a y l i k e w i s e b e a f f e c t e d b y e s t r o g e n inhibition. function  of other  U n d e n i a b l y , inhibition of e s t r o g e n u s i n g F a s l o d e x m a y alter the l e v e l s a n d affect the proteins  in the c e l l , a n d this m a y affect the o u t c o m e of e x p e r i m e n t a l a n a l y s e s .  F u r t h e r m o r e , the feasibility of a s s e s s i n g the role of e s t r o g e n inhibition of the r e g u l a t i o n of a p o p t o s i s , a n d e s p e c i a l l y in r e s p o n s e to c h e m o t h e r a p y d r u g s , is c o m p l i c a t e d b y t h e fact the e x a c t m e c h a n i s m s a n d p a t h w a y b y w h i c h e s t r o g e n r e g u l a t e s cell growth a n d a p o p t o s i s a r e not w e l l u n d e r s t o o d . the r e s u l t s in r e g a r d to c h e m o s e n s i t i v i t y p r e s e n t e d h e r e o n l y s e r v e to u n d e r s c o r e the initial generated using Bcl-2 antisense.  If nothing e l s e ,  in vitro  results  W h i l e it is p o s s i b l e that the alteration of o t h e r c e l l u l a r targets c o u l d  p o s s i b l y c o u n t e r the a p o p t o t i c effects of d e c r e a s e d B c l - 2 l e v e l , this r e m a i n s to b e s e e n .  O u r results  i n d i c a t e d no c h a n g e s in the l e v e l s of B a x a n d B a k , a n d this c o r r e l a t e s with d a t a f r o m other s t u d i e s A l t h o u g h in theory, a d e c l i n e in the B c l - 2 to B a x ratio s h o u l d s e n s i t i z e c e l l s to c h e m o t h e r a p y , d e c r e a s e d B c l - 2 to B a x ratio i n c u r r e d with the treatment  of c e l l s with F a s l o d e x w a s not  h o w e v e r , to c h e m o s e n s i t i z e t h e s e c e l l s to d o x o r u b i c i n or c i s p l a t i n . showed  1 5 , 1 6  .  the  sufficient,  T h e fact that the wild type c e l l s  no d i f f e r e n c e s in c h e m o s e n s i t i v i t y in c o m p a r i s o n to t r a n s f e c t e d  c e l l s further s u p p o r t s  the  c o n c l u s i o n that B c l - 2 is not sufficient in p r o v i d i n g c h e m o s e n s i t i v i t y . T h e s e f i n d i n g s relate the difficulty of d e t e r m i n i n g the r e l a t i o n s h i p b e t w e e n e s t r o g e n inhibitors a n d c h e m o t h e r a p y in r e g a r d to regulation of a p o p t o s i s . I n d e e d , c l i n i c a l s t u d i e s investigating the c o m b i n a t i o n of e s t r o g e n inhibition v i a T a m o x i f e n with c h e m o t h e r a p y h a v e h i g h l i g h t e d s e e m i n g l a c k of s y n e r g y of t h e s e t r e a t m e n t s .  While Tamoxifen and  c h e m o t h e r a p y a r e effective a s individual adjuvant t r e a t m e n t s , c o m b i n a t i o n of the two h a s not b e e n p r o v e n to  p r o v i d e a d d e d b e n e f i t s , e s p e c i a l l y in c o n s i d e r a t i o n of the  a d d e d toxicity  generated  by  c o m b i n i n g t h e s e a g e n t s . T h i s is m o s t likely d u e to the c y t o s t a t i c n a t u r e of e s t r o g e n inhibitors a n d their 1 7  ability to b l o c k c e l l s at the Gi p h a s e of the c e l l c y c l e ; the resulting g r o w t h inhibition r e n d e r s c h e m o t h e r a p y treatment l e s s effective, a s w a s i n d i c a t e d in the results p r e s e n t e d h e r e . E v e n though s o m e studies have indicated  in vitro  that e s t r o g e n inhibition is c a p a b l e of i n d u c i n g  a p o p t o s i s t h r o u g h a m e c h a n i s m involving B c l - 2 d o w n - r e g u l a t i o n , a n d that this effect m a y s y n e r g i s t i c with  150 drugs  such  as  paclitaxel,  the  chemotherapy remains unclear  1 8 , 1 9  consequence .  of  modulating  estrogen  levels  in  combination  with  T h e f i n d i n g s h e r e a n d in other s t u d i e s i n d i c a t e that the d e c l i n e of  Bcl-2 l e v e l s in t h e c e l l , a l t h o u g h p o s s i b l y i n c r e a s i n g the potential for a p o p t o s i s , m a y not b e sufficient o n its o w n to i n d u c e this p r o c e s s or s e n s i t i z e cell the c y t o t o x i c a g e n t s .  F u r t h e r c o n d i t i o n s in the cellular  e n v i r o n m e n t , s p e c i f i c a l l y in r e f e r e n c e to proliferation rates a n d r e g u l a t i o n of the c e l l c y c l e , m a y require m o d i f i c a t i o n for c h e m o r e s p o n s i v e n e s s to i m p r o v e .  151  4.5 REFERENCES  P e r i l l o B , S a s s o A , A b b o n d a n z a C , P a l u m b o G . 1 7 b e t a - e s t r a d i o l inhibits a p o p t o s i s in M C F - 7 c e l l s , i n d u c i n g b c l - 2 e x p r e s s i o n v i a t w o e s t r o g e n - r e s p o n s i v e e l e m e n t s p r e s e n t in t h e c o d i n g s e q u e n c e . Mol Cell Biol. 2 0 0 0 A p r ; 2 0 ( 8 ) : 2 8 9 0 - 9 0 1 . 1  W a n g T T , P h a n g J M . E f f e c t s o f e s t r o g e n o n a p o p t o t i c p a t h w a y s in h u m a n b r e a s t c a n c e r cell line M C F 7. Cancer Res. 1 9 9 5 J u n 1 5 ; 5 5 ( 1 2 ) : 2 4 8 7 - 9 .  2  H u a n g Y , R a y S, R e e d J C , Ibrado A M , T a n g C , N a w a b i A , B h a l l a K. E s t r o g e n i n c r e a s e s intracellular p 2 6 B c l - 2 to p21 B a x ratios a n d inhibits t a x o l - i n d u c e d a p o p t o s i s o f h u m a n b r e a s t c a n c e r M C F - 7 c e l l s .  3  Breast Cancer Res Treat. 1 9 9 7 J a n ; 4 2 ( 1 ) : 7 3 - 8 1 . D e t r e S , S a l t e r J , B a r n e s D M , R i d d l e r S , Hills M , J o h n s t o n S R , Gillett C , A ' H e r n R , D o w s e t t M . T i m e related effects o f e s t r o g e n w i t h d r a w a l o n proliferation- a n d cell d e a t h - r e l a t e d e v e n t s in M C F - 7 x e n o g r a f t s . Int J Cancer. 1 9 9 9 A p r 12;81 ( 2 ) : 3 0 9 - 1 3 .  4  P o m m e r e n k e E , M a t t e r n J , V o l m M . M o d u l a t i o n o f d o x o r u b i c i n - t o x i c i t y b y t a m o x i f e n in m u l t i d r u g resistant t u m o r c e l l s in vitro a n d in v i v o . J Cancer Res Clin Oncol. 1 9 9 4 ; 1 2 0 ( 7 ) : 4 2 2 - 6 .  5  G u W Z , C h e n Z , T a h i r S K , R o s e n b e r g S H , N g S C . S y n e r g i s t i c effect o f p a c l i t a x e l a n d 4 h y d r o x y t a m o x i f e n o n e s t r o g e n r e c e p t o r - n e g a t i v e c o l o n c a n c e r a n d l u n g c a n c e r cell l i n e s . Anticancer Drugs. 1 9 9 9 N o v ; 1 0 ( 1 0 ) : 8 9 5 - 9 0 1 . 6  Pratt M A , K r a j e w s k i S , M e n a r d M , K r a j e w s k a M , M a c l e o d H , R e e d J C . E s t r o g e n w i t h d r a w a l - i n d u c e d h u m a n b r e a s t c a n c e r t u m o u r r e g r e s s i o n in n u d e m i c e is p r e v e n t e d b y B c l - 2 . FEBS Lett. 1 9 9 8 D e c 4;440(3):403-8. 7  D i e l P , S m o l n i k a r K, M i c h n a H . T h e pure a n t i e s t r o g e n ICI 1 8 2 7 8 0 is m o r e effective in t h e induction o f a p o p t o s i s a n d d o w n regulation o f B C L - 2 t h a n t a m o x i f e n in M C F - 7 c e l l s . Breast Cancer Res Treat. 1 9 9 9 Nov;58(2):87-97.  8  H u y n h H , N i c k e r s o n T , P o l l a k M , Y a n g X . R e g u l a t i o n of insulin-like g r o w t h factor I r e c e p t o r e x p r e s s i o n by the p u r e a n t i e s t r o g e n ICI 1 8 2 7 8 0 . Clin Cancer Res. 1 9 9 6 D e c ; 2 ( 1 2 ) : 2 0 3 7 - 4 2 .  9  A n d e r s o n E , C l a r k e R B , Howell A . Estrogen r e s p o n s i v e n e s s and control of normal h u m a n breast proliferation. J Mammary Gland Biol Neoplasia. 1 9 9 8 J a n ; 3 ( 1 ):23-35. 1 0  v a n d e r B u r g B , d e G r o o t R P , I s b r u c k e r L, Kruijer W , d e L a a t S W . D i r e c t s t i m u l a t i o n b y e s t r o g e n o f g r o w t h factor s i g n a l t r a n s d u c t i o n p a t h w a y s in h u m a n b r e a s t c a n c e r c e l l s . J Steroid Biochem Mol Biol. 1992 Sep;43(1-3):111-5. 1 1  D e F r i e n d D J , A n d e r s o n E , B e l l J , W i l k s D P , W e s t C M , M a n s e l R E , H o w e l l A . E f f e c t s of 4 h y d r o x y t a m o x i f e n a n d a n o v e l p u r e a n t i o e s t r o g e n (ICI 1 8 2 7 8 0 ) o n t h e c l o n o g e n i c g r o w t h o f h u m a n b r e a s t c a n c e r c e l l s in vitro. BrJ Cancer. 1 9 9 4 A u g ; 7 0 ( 2 ) : 2 0 4 - 1 1 . 1 2  D i e l P , S m o l n i k a r K, M i c h n a H . T h e p u r e a n t i e s t r o g e n ICI 1 8 2 7 8 0 is m o r e effective in t h e induction o f a p o p t o s i s a n d d o w n regulation o f B C L - 2 t h a n t a m o x i f e n in M C F - 7 c e l l s . Breast Cancer Res Treat. 1 9 9 9 Nov;58(2):87-97. 1 3  Ercoli A , Battaglia A , R a s p a g l i o G , Fattorossi A , Alimonti A , Petrucci F, Caroli S , M a n c u s o S , S c a m b i a G . Activity o f c i s p l a t i n a n d ICI 1 8 2 , 7 8 0 o n e s t r o g e n r e c e p t o r n e g a t i v e o v a r i a n c a n c e r c e l l s : cell c y c l e a n d cell replication rate p e r t u r b a t i o n , c h r o m a t i n texture alteration a n d a p o p t o s i s i n d u c t i o n . Int J Cancer. 2 0 0 0 J a n 1 ;85(1 ) : 9 8 - 1 0 3 . 1 4  Z h a n g G J , K i m i j i m a I, O n d a M , K a n n o M , S a t o H , W a t a n a b e T , T s u c h i y a A , A b e R , T a k e n o s h i t a S . T a m o x i f e n - i n d u c e d a p o p t o s i s in b r e a s t c a n c e r c e l l s r e l a t e s to d o w n - r e g u l a t i o n o f b c l - 2 , but not b a x a n d 1 5  152  b c l - X ( L ) , without alteration o f p 5 3 protein l e v e l s . Clin Cancer Res. 1 9 9 9 O c t ; 5 ( 1 0 ) : 2 9 7 1 - 7 . W a n g T T , P h a n g J M . E f f e c t s of e s t r o g e n o n a p o p t o t i c p a t h w a y s in h u m a n b r e a s t c a n c e r cell line M C F 7. Cancer Res. 1 9 9 5 J u n 1 5 ; 5 5 ( 1 2 ) : 2 4 8 7 - 9 . 1 6  T o l c h e r A W . C u r r e n t s t a t u s of p o s t - c h e m o t h e r a p y t a m o x i f e n in e a r l y b r e a s t c a n c e r . Oncology (Huntington). 1 9 9 4 N o v ; 8 ( 1 1 ) : 3 9 - 4 3 ; d i s c u s s i o n 4 7 - 9 . R e v i e w .  1 7  H u a n g Y , R a y S , R e e d J C , Ibrado A M , T a n g C , N a w a b i A , B h a l l a K. E s t r o g e n i n c r e a s e s intracellular p 2 6 B c i - 2 to p21 B a x ratios a n d inhibits t a x o l - i n d u c e d a p o p t o s i s o f h u m a n b r e a s t c a n c e r M C F - 7 c e l l s . B r e a s r Cancer Res Treat. 1 9 9 7 J a n ; 4 2 ( 1 ) : 7 3 - 8 1 . 1 8  G u W Z , C h e n Z , T a h i r S K , R o s e n b e r g S H , N g S C . S y n e r g i s t i c effect o f p a c l i t a x e l a n d 4 h y d r o x y t a m o x i f e n o n e s t r o g e n r e c e p t o r - n e g a t i v e c o l o n c a n c e r a n d l u n g c a n c e r cell l i n e s . Anticancer Drugs. 1 9 9 9 N o v ; 1 0 ( 1 0 ) : 8 9 5 - 9 0 1 . 1 9  CHAPTER 5 IN  ANALYSIS OF ANTISENSE MODULATION OF BCL-2: EFFECTS ON T H E CHEMOSENSITIVITY OF HIGH AND LOW BCL-2-EXPRESSING BREAST CANCER CELL LINES VIVO  154  5.1  INTRODUCTION  E x p e r i m e n t s involving  Bcl-2 antisense  in vitro  i n d i c a t e d that this m o l e c u l e w a s c a p a b l e of  i n d u c i n g a p o p t o s i s u n d e r a v a r i e t y of c o n d i t i o n s . A l t h o u g h o u r  in vitro  d a t a s h o w e d that B c l - 2 a n t i s e n s e  u s e d in c o n j u n c t i o n with c h e m o t h e r a p y d r u g s i n c u r r e d a n a d d i t i v e effect, n o i n c r e a s e d sensitivity w a s apparent.  In spite of this, w e c o n d u c t e d additional  in vivo  experiments  with B c l - 2 a n t i s e n s e  and  c h e m o t h e r a p y d r u g s to a s s e s s the a p p a r e n t c o n c u r r e n t e f f e c t s of t h e s e a g e n t s in a n i m a l m o d e l s . F u r t h e r m o r e , the t r e a t m e n t of two c l o n a l cell lines — high a n d l o w B c l - 2 e x p r e s s i n g — a l l o w e d for the a s s e s s m e n t of B c l - 2 a s a n m e d i a t o r of d r u g r e s i s t a n c e experiments were:  in vivo.  T h e r e f o r e , the o b j e c t i v e s of t h e s e  1) to analyze the of Bcl-2 antisense and doxorubicin alone or in combination to  inhibit tumor growth in xenotransplanted mice; and, 2) to elucidate whether high and low Bcl-2 e x p r e s s i n g tumor would respond in varying degrees to the antisense and chemotherapy treatment regimen. , T h e c y t o t o x i c p r o p e r t i e s of B c l - 2 a n t i s e n s e h a v e b e e n d o c u m e n t e d  in vivo  have been revealed chemotherapy  drugs  8 , 9 , 1 0  '  1 1 , 1 2  .  as well  1 , 2  '  3 , 4  Inevitably,  '  5 , 6 , 7  .  in vitro,  a n d s i m i l a r results  M o r e o v e r , this effect is i n c r e a s e d w h e n c o m b i n e d with  a s will b e d i s c u s s e d later,  the  cytotoxic  effect  of  Bcl-2  c o m p l i c a t e s t h e d e t e r m i n a t i o n of w h e t h e r this a g e n t s i m p l y a d d s to t h e c y t o t o x i c effect of c h e m o t h e r a p y d r u g s pr a u g m e n t s it t h r o u g h s e n s i t i z i n g t u m o r s to t h e s e c o m p o u n d s .  Ultimately, this point m a y b e  s u p e r f l u o u s w h e n o n e c o n s i d e r s the c l i n i c a l i m p l i c a t i o n s of the i n c r e a s e d r e s p o n s e of c o m b i n i n g B c l - 2 a n t i s e n s e with c h e m o t h e r a p y . T h e B c l - 2 a n t i s e n s e m o l e c u l e u s e d in t h e s e e x p e r i m e n t s w a s G e n a s e n s e ( G 3 1 3 9 ) , this t i m e in c o n j u n c t i o n with a r e v e r s e polarity control s e q u e n c e . T h e s e o l i g o d e o x y n u c l e o t i d e s w e r e a d m i n i s t e r e d to m i c e a l o n e or in c o m b i n a t i o n with d o x o r u b i c i n a n d their effect u p o n t u m o r g r o w t h a s s e s s e d . tumor  size,  Bcl-2  levels  in  antisense  treatment  groups  were  determined  during  o l i g o n u c l e o t i d e t r e a t m e n t a s w e l l a s in all t r e a t m e n t g r o u p s at t h e e n d of the s t u d y . protein l e v e l s in the t u m o r w e r e a l s o i n v e s t i g a t e d .  In addition to  the  course  of  A s w e l l , the B a x  T h e s u m of t h e s e r e s u l t s will h o p e f u l l y  indicate  potential of G e n a s e n s e a s a c h e m o s e n s i t i z i n g a g e n t a n d r e v e a l the i m p o r t a n c e of p r e - e x i s t i n g B c l - 2 level in solid t u m o r s in d e t e r m i n i n g r e s p o n s e to c h e m o t h e r a p y .  155  1.1.1  5.2 MATERIALS AND METHODS  Oligonucleotides (ODNs) P h o s p h o r o t h i o a t e O D N s ( 1 8 - m e r ) with a s e q u e n c e c o m p l e m e n t a r y for t h e first s i x c o d o n s of the o p e n r e a d i n g f r a m e of B c l - 2 m R N A : 5'-tct c c c a g e gtg c g c c a t - 3 ' w a s u s e d a s t h e a n t i s e n s e O D N ; G 3 1 3 9 to B c l - 2 a n d w a s a gift f r o m G e n t a Inc., S a n D i e g o , C A . R e v e r s e P o l a r i t y s e q u e n c e ( G 4 1 2 6 ) 5'-tac c g c gtg c g a c c c tct-3' w e r e u s e d a s c o n t r o l s ( G e n t a Inc.).  Cell lines and Xenografts T h e h u m a n b r e a s t c a n c e r c e l l line M D A - M B - 4 3 5 / L C C 6  w a s obtained from Dr. R. Clarke,  ( G e o r g e t o w n U n i v e r s i t y ) a n d t r a n s f e c t e d with either a v e c t o r o r p l a s m i d c o n t a i n i n g the bcl-2 c o d i n g region.  C l o n e s w e r e c r e a t e d f r o m t r a n s f e c t e d p o p u l a t i o n s a n d c h a r a c t e r i z e d a s d e s c r i b e d in C h a p t e r 2 .  In t h e s e e x p e r i m e n t s , high B c l - 2 - e x p r e s s i n g M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d M D A 4 3 5 / L C C 6 V e c t o r were used.  C e l l s w e r e g r o w n a s a d h e r e n t c u l t u r e s a n d m a i n t a i n e d in D M E M s u p p l e m e n t e d with 10 %  F B S at 3 7 ° C a n d 5 % C 0 . C e l l s w e r e u s e d in e x p o n e n t i a l g r o w t h p h a s e , u p to a m a x i m u m of 2 5 in vitro 2  passages.  F e m a l e S C I D - R A G 2 m i c e (4-6 w e e k - o l d , 1 8 - 2 2 g) w e r e o b t a i n e d f r o m t h e B C C a n c e r A g e n c y  J o i n t A n i m a l Facility b r e e d i n g c o l o n y a n d kept in a s e p t i c e n v i r o n m e n t s . A n orthotopic t u m o r m o d e l of M D A 4 3 5 / L C C 6 c e l l s in R A G 2 m i c e w a s e s t a b l i s h e d b y bilateral i m p l a n t a t i o n of 2 x 1 0 in vivo p a s s a g e d 6  M D A 4 3 5 / L C C 6 c e l l s into t h e m a m m a r y fat p a d . A l l a n i m a l p r o t o c o l s w e r e a p p r o v e d b y the B . C . C a n c e r Agency Animal Welfare Committee.  Western Blot Analysis of Bcl-2 Expression T u m o r t i s s u e w a s s o n i c a t e d in i c e - c o l d lysis buffer ( 1 5 0 m M N a C l , 1 % N P - 4 0 , 0 . 5 % s o d i u m d e o x y c h o l a t e , 0.1 % S D S , 2 . 5 m M E D T A , 0.1 % s o d i u m a z i d e ) , c o n t a i n i n g p r o t e a s e inhibitors ( C o m p l e t e Mini  R  p r o t e a s e inhibitor t a b l e t s , B o e h r i n g e r M a n n h e i m G m B H , G e r m a n y ) .  S a m p l e s w e r e c e n t r i f u g e d at  1 4 , 0 0 0 r p m for 1 5 m i n , a n d s t o r e d at - 7 0 ° C . P r o t e i n c o n t e n t in t h e l y s e d e x t r a c t s w a s d e t e r m i n e d u s i n g a d e t e r g e n t - c o m p a t i b l e B i o R a d a s s a y ( B i o R a d L a b s , H e r c u l e s , C A ) . E q u a l a m o u n t s of protein (20 p g / l a n e ) w e r e s u b j e c t e d to 12.5 % S D S - P A G E ( B i o r a d ) for 4 5 m i n a t 1 5 0 V in a g l y c i n e buffer (1.92 M g l y c i n e , 2 5 m M T r i s p H 8.3) c o n t a i n i n g 1 % S D S . G e l s w e r e t r a n s f e r r e d to n i t r o c e l l u l o s e m e m b r a n e s in a g l y c i n e  156 transfer buffer with 2 0 % m e t h a n o l for 1 h at 7 0 V .  M e m b r a n e s w e r e b l o c k e d o v e r n i g h t at 4 ° C with 5 %  s k i m milk, 0 . 0 5 % s o d i u m a z i d e in P B S (20 m M T r i s p H 8 . 2 , 1 3 7 m M s o d i u m c h l o r i d e ) .  M o u s e m A b to  h u m a n B c l - 2 ( D A K O , D e n m a r k ) , 1:3,000, h u m a n B a x ( U p s t a t e B i o t e c h ) 1 : 1 0 0 0 , or B a k 1:1000 ( U p s t a t e , B i o t e c h ) a n d m o u s e a n t i - h u m a n B-actin m A b ( S i g m a , S t . L o u i s , M O ) , 1 : 1 0 , 0 0 0 w e r e u s e d diluted in 1 % s k i m milk in T B S , c o n t a i n i n g 0 . 0 5 % T w e e n - 2 0 a n d 0 . 0 5 % s o d i u m a z i d e .  T h e m e m b r a n e s w e r e then  i n c u b a t e d for 1 h with 1:3,000 h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d a n t i - m o u s e I g G ( P r o m e g a , M a d i s o n , WI).  P r o t e i n s w e r e d e t e c t e d by u s i n g a n e n h a n c e d c h e m i l u m i n e s c e n c e ( E C L , A m e r s h a m P h a r m a c i a  B i o t e c h , B u c k i n g h a m s h i r e , E n g l a n d ) m e t h o d , a n d v i s u a l i z e d after e x p o s u r e to K o d a k film. densitometry  (Molecular Dynamics, Sunnyvale, C A ) was  performed  to q u a n t i f y  band  Scanning  intensities  by  v o l u m e / a r e a integration. T h e a m o u n t of B c l - 2 protein in c e l l s / t u m o r w a s n o r m a l i z e d to their B-actin l e v e l s .  In Vivo Anti-Tumor Activity E f f i c a c y e x p e r i m e n t s w e r e c o n d u c t e d in R A G 2 m i c e b e a r i n g M D A 4 3 5 / L C C 6 V e c t o r or C l o n e 7 t u m o r s a s s i g n e d into 6 m i c e / g r o u p . T r e a t m e n t s w e r e A S ( G e n a s e n s e G 3 1 3 9 ) or R P O D N s (5 over a 3 w e e k regimen.  c o m m e n c e d on d a y 3 post-cell inoculation.  Bcl-2  m g / k g , i.p.) w e r e a d m i n i s t e r e d i.p., d a i l y for 5 d o s e s / w e e k d a y s  D o x o r u b i c i n ( F a u l d i n g , M o n t r e a l , Q u e ) at a c o n c e n t r a t i o n of 5.0 m g / k g w a s  a d m i n i s t e r e d i.v. v i a the tail v e i n o n d a y s 7, 14 a n d 21 p o s t - c e l l i n o c u l a t i o n .  In t h e e x p e r i m e n t s u s e d to  for W e s t e r n a n a l y s e s , d o x o r u b i c i n injections w e r e a d m i n i s t e r e d o n c e a w e e k for t h r e e w e e k s o n c e t u m o r s i z e r e a c h e d 0.1 g r a m s .  W h e n O D N s w e r e a d m i n i s t e r e d in c o m b i n a t i o n with d o x o r u b i c i n , the  drug  t r e a t m e n t w a s s t a g g e r e d 3 h after O D N treatment. M i c e w e r e o b s e r v e d d a i l y a n d m o u s e b o d y w e i g h t s a s w e l l a s s i g n s of s t r e s s ( e . g . lethargy, ruffed c o a t , a t a x i a , etc.) w e r e u s e d to d e t e c t p o s s i b l e toxicities. A n i m a l s with u l c e r a t e d t u m o r s or > 2 5 % l o s s of b o d y w e i g h t w e r e s a c r i f i c e d .  C a l i p e r m e a s u r e m e n t s of  t u m o r s w e r e c o n v e r t e d into m e a n t u m o r w e i g h t (g) u s i n g the f o r m u l a : V2 (length ( c m ) x [width (cm)] ). 2  An  a v e r a g e t u m o r w e i g h t / m o u s e w a s c a l c u l a t e d f r o m the m e a n of the two bilateral t u m o r s a n d w a s u s e d to c a l c u l a t e the g r o u p m e a n t u m o r w e i g h t ± S . E . M .  A d d i t i v e v e r s u s s y n e r g i s t i c cytotoxicity w a s d e t e r m i n e d  by c a l c u l a t i n g the e x p e c t e d t u m o r s i z e if the t r e a t m e n t s w e r e a d d i t i v e a n d c o m p a r i n g t h e m to o b s e r v e d tumor sizes.  E x p e c t e d t u m o r s s i z e s for additive c o m b i n e d t r e a t m e n t g r o u p s for a g i v e n t i m e point w e r e  determined as follows: size  D o x o r u b i c i n  ( A v e r a g e t u m o r size nasense aionJAverage t u m o r size ntroi) X ( A v e r a g e Ge  Co  / A v e r a g e t u m o r size ntroi) X A v e r a g e t u m o r size ntroi. Co  Co  tumor  T h i s r e s u l t i n g t u m o r s i z e w a s then  157 c o m p a r e d to the o b s e r v e d a v e r a g e t u m o r s i z e of t h e c o m b i n e d t r e a t m e n t f o r that time-point. values  indicated  additive  cytotoxicity  while  a smaller  observed  tumor  size  indicates  Identical synergistic  cytotoxicity.  Statistical Analyses Multiple c o m p a r i s o n s w e r e d o n e u s i n g o n e - w a y a n a l y s i s of v a r i a n c e ( A N O V A ) , a n d p o s t - t e s t s c o m p a r i n g different treatment m e a n s w e r e d o n e u s i n g T u k e y H S D test ( S t a t i s t i c a r e l e a s e 4 . 5 , S t a t S o f t Inc., C A ) ; p v a l u e s for e a c h t r e a t m e n t g r o u p in relation to control w e r e r e p o r t e d .  5.3 RESULTS  Protein Levels in Each Treatment Group ; T h e B c l - 2 protein level w a s a n a l y z e d at t h e termination point of e a c h t r e a t m e n t g r o u p (Figure 5.1) . P r o t e i n l e v e l s w e r e d e r i v e d f r o m a n i m a l s i m p l a n t e d with M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s o n d a y 4 6 a n d f r o m a n i m a l s injected with M D A - M B - 4 3 5 / L C C 6 V e c t o r o n d a y 6 4 . A t d a y 4 6 , B c l - 2 p r o t e i n s l e v e l s w e r e identical in a n i m a l s injected with M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s r e g a r d l e s s o f w h e t h e r t h e y h a d b e e n left u n t r e a t e d o r treated with a n t i s e n s e , r e v e r s e polarity, d o x o r u b i c i n o r a n y c o m b i n a t i o n of O D N with d r u g , indicating that n o n e of t h e s e t r e a t m e n t s altered B c l - 2 level o n a l o n g t e r m b a s i s . P r i o r to treatment, protein l e v e l s of B c l - 2 , B a x a n d B a k in M D A - M B - 4 3 5 / L C C 6 V e c t o r c e l l s w e r e c o m p a r e d with t h o s e in M D A - M B - 4 3 5 / L C C 6 W i l d - t y p e c e l l s a n d f o u n d to b e identical (data not s h o w n ) .  A n a l y s i s of P r o t e i n L e v e l s in R e s p o n s e to B c l - 2 a n t i s e n s e T r e a t m e n t In o r d e r to e s t a b l i s h t h e b a s i s for G e n a s e n s e to effectively s u p p r e s s t h e growth of e s t a b l i s h e d t u m o r s w e e x a m i n e d t h e c h a n g e s in B c l - 2 e x p r e s s i o n d u r i n g t h e c o u r s e o f a n t i s e n s e t r e a t m e n t ( F i g u r e . 5.2) .  M i c e i m p l a n t e d with e s t a b l i s h e d (0.1 - 0 . 1 5 g) bilateral M D A - M B - 4 3 5 / L C C 6 t u m o r s w e r e treated  with either R e v e r s e P o l a r i t y o r G e n a s e n s e o l i g o d e o x y n u c l e o t i d e s .  L y s a t e s from tumor homogenates  w e r e p r o b e d with B c l - 2 a n d a c t i n a n t i b o d i e s . A s t h e s e a n t i b o d i e s w e r e d i r e c t e d a g a i n s t the h u m a n p r o t e i n s , this a l l o w e d for d e t e r m i n a t i o n of h u m a n B c l - 2 l e v e l s o b t a i n e d f r o m t u m o r cell s p e c i m e n s without  FIGURE 5.1 Western Analysis of Bcl-2 Levels in MDA-435/LCC6 Clone 7 and Vector Tumors in Various Treatment Groups. A . Bcl-2 protein levels in M D A - M B 435/LCC6 Clone 7 tumors (high Bcl-2) at day 46 B . Bcl-2 protein levels in MDA-MB-435/LCC6 Vector tumors (low Bcl-2) at day 64.  159  Reverse Polarity  Bcl-2 Antisense  A .  C.  Bcl-2 Antisense  Reverse Polarity  T  —  e,  ir,  ^  5"  S?  S?  t£  —  S"  £  j»  o  o  o  o —  — o  S>  >•  a  Q  >• Q  >• Q  Q  Q  £• D  Actin  W I  D.  MW"*  -*>*«m  """""""  Bax  »*»>>ww  A  Bcl-2 Antisense  Reverse Polarity  Actin  —  ,^  m  ^ ^  ^  ^  B a X  FIGURE 5.2 Western Analysis of Bcl-2 and Bax Levels in MDA-MB435/LCC6 Clone 7 and Vector Cells in Response to Treatment with Bcl-2 Antisense or Reverse Polarity Control. A. B c l - 2 protein l e v e l s in M D A - M B 4 3 5 / L C C 6 C l o n e 7 t u m o r s (high B c l - 2 ) B. B c l - 2 protein l e v e l s in M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s (low B c l - 2 ) C . B a x protein l e v e l s in M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s (high B c l - 2 ) D B a x protein l e v e l s in M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s (low B c l - 2 ) .  D a y l a b e l s indicate t h e  n u m b e r of d a y s b e t w e e n the start of o l i g o n u c l e o t i d e t r e a t m e n t a n d t u m o r h a r v e s t .  160 contamination from Bcl-2 derived from m o u s e cells. S y s t e m i c a d m i n i s t r a t i o n of G e n a s e n s e (5 m g / k g ) c a u s e d a d o w n - r e g u l a t i o n of B c l - 2 e x p r e s s i o n within 3 d a y s of initiating G e n a s e n s e t r e a t m e n t w h e r e B c l - 2 protein l e v e l s w e r e r e d u c e d b y a p p r o x i m a t e l y o v e r 9 5 % ) in c o m p a r i s o n to B c l - 2 l e v e l s in t u m o r s prior to t r e a t m e n t (Figure 5.2A and B).  N o down-  r e g u l a t i o n of B c l - 2 protein w a s o b s e r v e d with R e v e r s e P o l a r i t y t r e a t m e n t s (Figure 5.2A and B). W i t h t h e G e n a s e n s e treatment, B c l - 2 protein l e v e l s returned to p r e - t r e a t m e n t l e v e l s within s e v e n d a y s after t h e final O D N t r e a t m e n t of t h e first w e e k l y s c h e d u l e . O f particular interest w a s t h e o b s e r v a t i o n that t h e s o l i d tumors' w e r e i n s e n s i t i v e to s u b s e q u e n t c o u r s e s of G e n a s e n s e treatment.  Specifically, no detectable  d e c r e a s e in B c l - 2 e x p r e s s i o n w a s o b s e r v e d in the s e c o n d o r third w e e k l y c o u r s e of treatment. B a x protein l e v e l s w e r e a l s o d e t e r m i n e d in t u m o r s d e r i v e d f r o m M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d V e c t o r c e l l s prior to a n d d u r i n g treatment with G e n a s e n s e a n d R e v e r s e P o l a r i t y c o n t r o l s (Figure 5.2 C and D). A g a i n t u m o r l y s a t e s w e r e p r o b e d with h u m a n a n t i b o d i e s to e n s u r e d e t e r m i n a t i o n of l e v e l s of h u m a n B a x protein o n l y . R e s u l t s i n d i c a t e d n o significant c h a n g e in t h e B a x protein l e v e l s o v e r t h e c o u r s e of treatment with G e n a s e n s e .  Growth Rates of MDA-MB-435/LCC6 Clone 7 and Vector Tumors and Treatment with Doxorubicin In o r d e r to e v a l u a t e growth p r o p e r t i e s of t h e h i g h B c l - 2 - e x p r e s s i n g M D A - M B - 4 3 5 / L C C 6 C l o n e 7 and  low Bcl-2-expressing M D A - M B - 4 3 5 / L C C 6  Vector cells, cells were  implanted  bilaterally  in t h e  m a m m a r y fat p a d of R a g - 2 M m i c e a n d the g r o w t h of t h e t u m o r s m o n i t o r e d o v e r time (Figure 5.4). M D A M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s d i s p l a y e d a m u c h h i g h e r growth rate t h a n M D A - M B - 4 3 5 / L C C 6 V e c t o r c e l l with M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s b e i n g o n l y 7 0 % the s i z e of M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s at 5 0 d a y s p o s t - t u m o r i n o c u l a t i o n (p <0.05). T o e v a l u a t e t h e effect of c h e m o t h e r a p y treatment a l o n e o n M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d V e c t o r t u m o r s , t u m o r s w e r e g r o w n to a s i z e of a p p r o x i m a t e l y 0.1 g r a m s a n d treated with d o x o r u b i c i n (Figure 5.4). M i c e r e c e i v e d a total of three t r e a t m e n t s of d o x o r u b i c i n (5.0 m g / k g ) o n d a y s 3 3 , 4 0 , a n d 4 7 after t u m o r i n o c u l a t i o n .  D o x o r u b i c i n t r e a t m e n t s initially s l o w e d t h e g r o w t h o f both M D A - M B - 4 3 5 / L C C 6  C l o n e 7 a n d M D A - M B - 4 3 5 / L C C 6 Vector tumors between d a y s 3 7 a n d 4 4 post-tumor inoculation, while significant t u m o r growth w a s i m p a i r e d after 4 6 d a y s p o s t - t u m o r i n o c u l a t i o n (p < 0.05). W h i l e both a g e n t s r e s p o n d e d to d o x o r u b i c i n treatment a n d t u m o r growth w a s i m p a i r e d , h i g h B c l - 2 - e x p r e s s i n g M D A - M B -  161 4 3 5 / L C C 6 C l o n e 7 t u m o r s a p p e a r e d to b e slightly m o r e s e n s i t i v e to d o x o r u b i c i n t h a n a n d 4 3 5 / L C C 6 Vector tumors.  MDA-MB-  A t d a y 4 7 , M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s treated with d o x o r u b i c i n w e r e  6 8 % the s i z e of u n t r e a t e d M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s w h i l e M D A - M B - 4 3 5 / L C C 6 V e c t o r c e l l s treated with d o x o r u b i c i n w e r e 7 5 % the s i z e of M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s .  162  L6W Vector L6W Bcl-2 Transfected L6W Vector Free Dox (10 mg/kg) - • — L6W Bcl-2 Transfected Free Dox (10 mg/kg) •  0.600.550.500.450.400.350.30C  0.25-  B 0.20B  H  0.150.100.05-  0.00 10  15  20  25  30  135  <|0  45  j  Days Post-Tumour Innoculation  50  55  60 Doxorubicin { Treatments I (10 mg/kg)  FIGURE 5.3 Growth of MDA-435/LCC6 Cells Implanted in Female RAG-2M Mice and Treated with Doxorubicin: Comparison of Bcl-2 Transfected and Vector Controls. 2 x 10 cells were implanted in the left and right fat pads and mice treated with 5.0 mg/kg 6  doxorubicin once tumor sizes reached 0.1 - 0.15 grams.  163  Treatment of MDA-MB-435/LCC6 Clone 7 and Vector Tumors with Genasense in Combination with Doxorubicin W i t h d a t a indicating the effect of c h e m o t h e r a p y a l o n e o n t h e s e t u m o r s , s t u d i e s w e r e c o n d u c t e d to e v a l u a t e the effect of G e n a s e n s e treatment a l o n e or in c o m b i n a t i o n with c h e m o t h e r a p y d r u g s .  MDA-MB-  4 3 5 / L C C 6 C l o n e 7 a n d V e c t o r t u m o r s w e r e treated with G e n a s e n s e o r R e v e r s e P o l a r i t y o l i g o n u c l e o t i d e s a l o n e , d o x o r u b i c i n a l o n e , o r c o m b i n a t i o n s thereof, a n d c o m p a r e d with u n t r e a t e d c o n t r o l s {Figure 5.5). M i c e w e r e treated with G e n a s e n s e or R e v e r s e Polarity o l i g o n u c l e o t i d e s o n d a y s 3 - 7 , 1 0 - 1 4 , a n d 17-21 p o s t - t u m o r i n o c u l a t i o n a n d d o x o r u b i c i n w a s a d m i n i s t e r e d o n 7, 14 a n d 21 d a y s after t u m o r - i n o c u l a t i o n . A s with p r e v i o u s f i n d i n g s , M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s d i s p l a y e d h i g h e r growth rates t h a n all MDA-MB-435/LCC6  Vector tumors:  A l l treatment  groups  of M D A - M B - 4 3 5 / L C C 6  Clone 7  tumors  d i s p l a y e d h i g h e r growth rates t h a n all M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s , a n d at d a y 4 6 , M D A - M B 4 3 5 / L C C 6 V e c t o r c o n t r o l s w e r e o n l y 5 0 % t h e s i z e of M D A - M B - 4 3 5 / L C C 6 C l o n e 7 C o n t r o l s (Figure 5.5) In r e g a r d  to treatment,  MDA-MB-435/LCC6  Clone 7 tumors  r e s p o n d e d to treatment  with  G e n a s e n s e a n d t u m o r growth w a s i m p a i r e d to a g r e a t e r d e g r e e w h e n G e n a s e n s e w a s c o m b i n e d with d o x o r u b i c i n (p< 0.05);  at d a y 4 6 , G e n a s e n s e treated t u m o r s w e r e 8 3 % t h e s i z e of c o n t r o l s w h i l e t u m o r s  treated with G e n a s e n s e c o m b i n e d with d o x o r u b i c i n w e r e 6 9 % the s i z e of u n t r e a t e d controls.(Figure  5.6).  L i k e w i s e , t r e a t m e n t of G e n a s e n s e a l o n e i m p a i r e d t h e growth of M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s a n d this w a s i n c r e a s e d w h e n G e n a s e n s e w a s c o m b i n e d with d o x o r u b i c i n (p<0.05).  At day 46, G e n a s e n s e  treated t u m o r s w e r e 3 8 % t h e s i z e of controls w h i l e t u m o r s treated with G e n a s e n s e c o m b i n e d with d o x o r u b i c i n w e r e 3 1 % t h e s i z e of untreated  c o n t r o l s (Figure 5.7).  R e v e r s e P o l a r i t y treated  mice  c o n t a i n e d c o m p a r a b l e t u m o r s i z e s to untreated c o n t r o l s in both cell l i n e s a n d t u m o r s i z e s f r o m m i c e treated  with R e v e r s e Polarity in c o m b i n a t i o n  with d o x o r u b i c i n  were  s i m i l a r to t h o s e treated  with  doxorubicin alone. In c o m p a r i n g t h e effects of treatment r e g i m e n s b e t w e e n e a c h c e l l line, t h e d e g r e e of growth i m p a i r m e n t for M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d M D A - M B - 4 3 5 / L C C 6 V e c t o r w a s identical for  all treatment  regimens  except two:  G e n a s e n s e alone  a n d G e n a s e n s e in c o m b i n a t i o n  with  doxorubicin. T r e a t m e n t of M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s with G e n a s e n s e i n c u r r e d a g r e a t e r r e s p o n s e than w h e n this treatment w a s a p p l i e d to M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s . A t d a y 4 6 , G e n a s e n s e treatment a l o n e of M D A - M B - 4 3 5 / L C C 6 V e c t o r c e l l s p r o d u c e d t u m o r s that w e r e 3 8 % t h e s i z e of controls  164 w h i l e in M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s the s a m e treatment r e s u l t e d in t u m o r s that w e r e 8 3 % the s i z e of c o n t r o l s . L i k e w i s e , c o m b i n e d treatment h a d a g r e a t e r effect in M D A - M B - 4 3 5 / L C C 6 V e c t o r t u m o r s than M D A - M B - 4 3 5 / L C C 6 C l o n e 7 t u m o r s a s t u m o r s i z e s in m i c e r e c e i v i n g c o m b i n e d t r e a t m e n t w a s 3 1 % of control for the f o r m e r , a n d 6 9 % for the latter.  165  •o © •a  LO CO  c S  m  m  O  Ic i  I- Hh-AH  o c/> 0 o >, o.  1H  i s 2  ft. §  •a ^  %-  cu <N  s«  .2 OQ — •—  E  H-  d o  • i-H  — cu  CJ  o a o g  ' u "3 cs  3 o X O  <N o  — o CQ -o 3 cj a >  o  o  C o C .H  E 3 0 C  0  B  q  ? s 1 "5  O  OH  •g 1 1 o g « >a  •o *S  CS  3  o  is a a  C-  a  u  JS <=> OD JS  U  CU  0g  <  1)  •2  .a  s *  m  M  —  0  §  ca  3  «  o  i«s 5 "3 ra (_> D. O  — 1 H  <u E l* 3  0  •  C/3 —  u (U  <u cE CJ  2 x °° "  -a  1 "g•5 - § SH C 1 = 1 te H §  U 00 .S  166  0 o -3 & CD  St  I- H  11 s8  O CD Efl  —  £^ 9 0  HH  -  LO LO  5S K  fZ  O  O  I •  f B K life  \\\ \  LO  CO  i  +  I* S  S2  o  CO  •c  + o  I§  •I I s  LO CM  s oj o + + 3 + q + a +p o o o  CNJ  \\t ]I CD  O  O  LO  :  nr un o  •  C  8 I' s -  RI K  11 "B o  a «c •a E *- u  « «  C/)  o  P3  >^  o  CN  i .s  CT)  O  U  U  o  ds  t. Q  cd  tS 2 K  o  >>>>>> ;  o o c  05  <U PH . 1)  g  TO  Q.  D  „ 1) 00  u -o •  O LO  LO  "-T3  tt B  cj  O  > 5 U £ fe u  f  l  a  It  * co jo  <N § —  s <0 "O  d  co  d  CM  d  o  d  o a  w -  (6) 8 Z ( s jnoujni  9  1§ ID  |  S  C  o  3 O  CQ -a a > DO  lift'll) E E  167  o S3 fc  •a & 9  §.  ca cu -  3  O  I  •a c  c  i  o  Q  o  —  1  03 O  o  c _c  C/5  O  CL  C/)  >^  03  Q  TJ £ -2 a.  I1 at  «  —  u  u _ IN  X CN  U  =  sd rl  J5  51 •s II  of o  u& u £  * -r — a <N i a.o -a3 o ra  S I I I I I I I I I I I I I I o m o i o o L o o u o o L o o L O O L O o 1  s  o  1  ffl  o  1  ©  o  1  iq  o  1  w  o  1  ^  o  1  o  ^  1  o  «  1  o  «  1  o  OJ  1  o  1  w  o  1  r  1  r  q  °  q  -1-  ca  CM  i 0  T3  ? -1 8 £ Sf g oc U o  (6) ezis jnoujni  v5 = •3 g <u 03 -a S  cu  o. >  CA W) 60  E  e  168  5.4 DISCUSSION  T h e s t u d i e s d e s c r i b e d h e r e w e r e u n d e r t a k e n to i n v e s t i g a t e the i n f l u e n c e o f B c l - 2 e x p r e s s i o n o n sensitivity o f c e l l s to G e n a s e n s e a n d d o x o r u b i c i n a n d the r e l a t i o n s h i p o f a b s o l u t e B c l - 2 e x p r e s s i o n v e r s u s B c l - 2 to B a x ratios  in this  regard  a s well.  W h i l e t h e effect  of c o m b i n i n g  B c l - 2 a n t i s e n s e with  c h e m o t h e r a p y d r u g s h a s b e e n i n v e s t i g a t e d in other in vivo s t u d i e s , t h e effect o f i n h e r e n t B c l - 2 level in r e g a r d to c o m b i n e d c h e m o t h e r a p y a n d B c l - 2 a n t i s e n s e t r e a t m e n t h a s not b e e n e v a l u a t e d to the s a m e extent.  W i t h this in m i n d , w e a s s e s s e d t h e effect o f G e n a s e n s e a l o n e a n d in c o m b i n a t i o n  with  d o x o r u b i c i n in two c e l l lines e x p r e s s i n g v a r y i n g l e v e l s o f B c l - 2 . T h e results p r e s e n t e d h e r e i n d i c a t e that B c l - 2 l e v e l s in both t u m o r s a r e d o w n - r e g u l a t e d in a s e q u e n c e s p e c i f i c m a n n e r o v e r the c o u r s e o f the five d a y s o f t r e a t m e n t with G e n a s e n s e , after w h i c h time protein l e v e l s return to a m o u n t s f o u n d in control t u m o r s .  T h e s e e m i n g l y t r a n s i e n t n a t u r e o f the d o w n -  r e g u l a t i o n o f B c l - 2 in both c e l l lines a n d the s u b s e q u e n t d e v e l o p m e n t o f t u m o r s r e s i s t a n t to G e n a s e n s e m a n i p u l a t i o n m a y b e e x p l a i n e d b y a n u m b e r o f c o n s i d e r a t i o n s . F o r e m o s t m a y b e the s e l e c t i o n o f c e l l s in the t u m o r m a s s naturally r e s i s t a n t to G e n a s e n s e , w h i c h c o u l d b e t h e result o f i m p a i r e d u p t a k e a n d transport m e c h a n i s m s within the c e l l o r alterations in the B c l - 2 m R N A w h i c h r e n d e r it l e s s s e n s i t i v e to binding b y the antisense molecule.  O t h e r e x p l a n a t i o n s m a y lie in t h e fact that a s t h e t u m o r  size  i n c r e a s e s , d e c r e a s e d u p t a k e of the o l i g o n u c l e o t i d e m a y o c c u r d u e to p o o r a c c e s s d u e to i n a d e q u a t e l y d e v e l o p e d v a s c u l a t u r e . T h u s , the p h a r m a c o k i n e t i c s a n d u p t a k e o f the a n t i s e n s e m o l e c u l e m a y c h a n g e o v e r t h e c o u r s e o f treatment.  In s p i t e o f t h e s e c o n d i t i o n s , o u r r e s u l t s i n d i c a t e that e v e n transient d o w n -  regulation o f B c l - 2 w a s sufficient to i m p a i r t u m o r growth. T r e a t m e n t of M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d V e c t o r t u m o r s with G e n a s e n s e d i s p l a y e d direct antitumor  activity a n d r e s u l t e d in t h e i m p a i r m e n t  studies  ; f u r t h e r m o r e , this i m p a i r m e n t w a s i n c r e a s e d w h e n c o m b i n e d with d o x o r u b i c i n .  1 3 , 1 4  of tumor  growth, a s h a s b e e n reported  in other  W e c o m p a r e d t u m o r s i z e s f r o m t h o s e m i c e treated with c o m b i n e d t h e r a p y with t u m o r s i z e s of m i c e treated  with e a c h a g e n t  a l o n e o r left u n t r e a t e d  c h e m o s e n s i t i z e d c e l l s to d o x o r u b i c i n (see  to d e t e r m i n e  whether  Bcl-2 down-regulation h a d  Materials and Methods for calculations).  O u r analyses  i n d i c a t e d that n o c h e m o s e n s i t i z a t i o n o c c u r r e d in either M D A - M B - 4 3 5 / L C C 6 V e c t o r or M D A - M B - 4 3 5 / L C C 6  169 Clone 7 tumors as combined treatments correlated with results that would be found if these two treatments as impaired tumor growth in an additive manner, rather than in a synergistic manner.  In mice  containing MDA-MB-435/LCC6 Vector cells, combined treatment should have resulted tumor sizes of 0.10 grams at day 46 if these treatments were simply additive; the average tumor size of this group at that time was 0.09 grams. Similar results were found with the MDA-MB-435/LCC6 Clone 7 groups, where combined treatment should have resulted in tumor sizes of 0.43 grams if the treatments were additive. The average tumor size observed on day 46 for MDA-MB-435/LCC6 Clone 7 cells that had received both treatments was, indeed, 0.43 grams. While we cannot conclusively rule out a chemosensitizing effect of Genasense, the results presented here provide no evidence that this is the case, and these results concur with our findings in vitro. In spite of this observation, the high degree of variability observed in tumor groups due to treating mice soon after tumor inoculation (early treatment regimen) obscure the ability to accurately delineate between additive and synergistic cytotoxicity of doxorubicin and Genasense.  Furthermore, the fact that  Genasense induces cytotoxicity in this animal model obscures the relationship between these two agents. When a putative chemosensitizing agent induces cytotoxicity on its own, or alters the growth rate without inducing significant cytotoxicity, increased inhibition of tumor growth may be the result of the enhanced cytotoxicity of the sensitizing agent and not an increase in chemoresponsiveness; thus, difficulties exist in delineating chemosensitivity from additive cytotoxicity in vivo. Other studies involving the combination of Bcl-2 antisense with chemotherapy drugs indicated an absence of cytotoxicity by the antisense molecule alone, and thus greater impairment of tumor growth could definitively be attributed to increased sensitivity as a result of Bcl-2 down-regulation  15,16  .  Another factor which must be taken into account when assessing chemosensitivity data are interactions which may take place between the two drugs during the combined treatment. Differences in sensitivity may occur due to altered pharmacokinetics, tissue distribution and metabolism of doxorubicin by Genasense, or vice verse, which may complicate the results of in the combined therapy. Indeed, other studies have shown that Genasense increases the tissue distribution of doxorubicin and decreases the amount of drug in the plasma . This may be attributable to increased tumor permeability caused by 17  Genasense, the formation of complexes between doxorubicin and Genasense that may modify drug uptake, or alterations of DNA integrity in cells induced into apoptosis by the oligonucleotide that increase doxorubicin toxicity.  170 O n e consideration when using antisense molecules w a y of C p G motifs, m a y affect o b s e r v e d results.  in vivo  is the effect that i m m u n e f u n c t i o n , b y  U n m e t h y l a t e d C G s e q u e n c e s p r e s e n t in the a n t i s e n s e  m o l e c u l e m a y s t i m u l a t e i m m u n e c e l l s , s p e c i f i c a l l y natural killer c e l l s , w h i c h m a y p o s s i b l y affect the growth of x e n o g r a f t e d t u m o r s  1 8 , 1 9  .  H o w e v e r , growth i m p a i r m e n t in both M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d V e c t o r  t u m o r s o c c u r r e d in a s e q u e n c e s p e c i f i c m a n n e r , w h i c h is significant a s both G e n a s e n s e a n d R e v e r s e Polarity o l i g o n u c l e o t i d e s c o n t a i n C G motifs; t h u s , r e d u c t i o n s in t u m o r s i z e a s the result of i m m u n e effects a r e unlikely.  F u r t h e r m o r e , other s t u d i e s involving the u s e of t h e s e s e q u e n c e s d i s p l a y e d s e q u e n c e -  s p e c i f i c activity in m i c e deficient for natural killer cell activity, t h e r e b y s u p p o r t i n g the result that function of t h e s e m o l e c u l e s is d e p e n d e n t u p o n their s p e c i f i c activity a n d not e x t r a n e o u s i m m u n e e f f e c t s . 20  In r e g a r d to the ability of inherent B c l - 2 l e v e l to contribute to the c h e m o r e s p o n s i v e n e s s of the two cell lines utilized, the f i n d i n g s p r e s e n t e d h e r e indicate that i n c r e a s e d B c l - 2 l e v e l d o e s c o n f e r s o m e r e s i s t a n c e to G e n a s e n s e a l o n e a n d G e n a s e n s e in c o m b i n a t i o n with d o x o r u b i c i n , but not d o x o r u b i c i n alone.  W h i l e this effect of B c l - 2 level b e t w e e n two cell lines h a s not b e e n i n v e s t i g a t e d  in vivo, in vitro  a n a l y s i s h a s b e e n u n d e r t a k e n a n d the r e s u l t s of t h e s e s t u d i e s a r e i n c o n c l u s i v e . In s o m e i n s t a n c e s B c l - 2 level a p p e a r s to c o r r e l a t e well with l a c k of r e s p o n s e to c h e m o t h e r a p y d r u g s , w h i l e in o t h e r s it h a s n o effect, a l t h o u g h c o m p a r i s o n s a r e c o m p l i c a t e d b y the fact that in m o s t c a s e s c o m p a r i s o n w e r e m a d e b e t w e e n different cell lines or b e t w e e n d r u g resistant a n d d r u g s e n s i t i v e v a r i a n t s of the s a m e cell line 21,22,23,24  v j tj ar  a  ons  b e t w e e n different cell lines or a c q u i r e d r e s i s t a n c e m e c h a n i s m s i n d e p e n d e n t of B c l - 2  f o u n d in d r u g resistant v a r i a n t s m a y contribute to the conflicting r e s u l t s i n d i c a t e d by t h e s e s t u d i e s . In r e g a r d to the cell lines u s e d in t h e s e e x p e r i m e n t , there a p p e a r s to b e n o difference in sensitivity b e t w e e n  MDA-MB-435/LCC6  However, MDA-MB-435/LCC6  C l o n e 7 a n d V e c t o r c e l l s to c h e m o t h e r a p y a g e n t s  alone.  C l o n e 7 t u m o r s a p p e a r to b e m o r e r e s i s t a n t to the c y t o t o x i c effects of  G e n a s e n s e a l o n e a n d in c o m b i n a t i o n with d o x o r u b i c i n  in vivo  than M D A - M B - 4 3 5 / L C C 6 Vector tumors as  t u m o r s i z e s of m i c e treated with c o m b i n e d t h e r a p y in M D A - M B - 4 3 5 / L C C 6 V e c t o r s w e r e m u c h s m a l l e r in c o m p a r i s o n to V e c t o r c o n t r o l s than w e r e M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c o m b i n e d treatment g r o u p s in c o m p a r i s o n to C l o n e 7 c o n t r o l s . T h i s result i n d i c a t e s that the e f f i c a c y of G e n a s e n s e treatment a l o n e m a y d e p e n d o n the extent to w h i c h B c l - 2 is o v e r - e x p r e s s e d relative to B a x a n d o t h e r p r o - a p o p t o t i c p r o t e i n s . R e g a r d i n g the i n c r e a s e d r e s i s t a n c e a p p a r e n t in the M D A - M B - 4 3 5 / L C C 6 C l o n e 7 cell line to c o m b i n e d t h e r a p y , a g a i n the r e s i s t a n c e is m o s t likely d u e to the s a m e m e c h a n i s m a s with the s i n g l e agent G e n a s e n s e treatment -  B c l - 2 d o w n - r e g u l a t i o n v i a G e n a s e n s e h a s a g r e a t e r effect in M D A - M B -  171 4 3 5 / L C C 6 V e c t o r c e l l s b y virtue of a l o w e r inherent B c l - 2 l e v e l . It is n o t e w o r t h y that M D A - M B - 4 3 5 / L C C 6 V e c t o r a n d M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s d o not differ in their sensitivity to d o x o r u b i c i n a s a s i n g l e a g e n t - o n l y w h e n it is c o m b i n e d with G e n a s e n s e d o e s r e s i s t a n c e o c c u r .  F u r t h e r m o r e , there w a s no  variation in the relative t u m o r s i z e of e a c h g r o u p treated with c o m b i n e d t h e r a p y in c o m p a r i s o n to G e n a s e n s e a l o n e ( e a c h a p p r o x i m a t e l y 8 3 % of t u m o r s i z e s in m i c e treated with G e n a s e n s e a s a s i n g l e agent).  S i n c e m i c e i n o c u l a t e d with M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d M D A - M B - 4 3 5 / L C C 6 V e c t o r w e r e  treated with the s a m e a m o u n t s of G e n a s e n s e a n d d o x o r u b i c i n , d i f f e r e n c e s in d r u g r e s i s t a n c e a r e attributable  to the inherent B c l - 2 level a n d r e s p o n s e to G e n a s e n s e , not d o x o r u b i c i n or a n y altered  p h a r m a c o k i n e t i c s of this d r u g s d u e to the p r e s e n c e of G e n a s e n s e . L a s t l y , o n e c h a r a c t e r i s t i c that is a p p a r e n t b e t w e e n the M D A - M B - 4 3 5 / L C C 6 C l o n e 7 a n d M D A M B - 4 3 5 / L C C 6 V e c t o r c e l l lines is t h e growth rate. T h o u g h this w a s not o b s e r v e d with t h e s e c e l l lines in  vitro,  M D A - M B - 4 3 5 / L C C 6 C l o n e 7 c e l l s , i n c l u d i n g all t r e a t m e n t g r o u p s , g r e w at a f a s t e r rate than M D A -  M B - 4 3 5 / L C C 6 V e c t o r c e l l s , i n d i c a t i n g , at least in this t u m o r m o d e l , that B c l - 2 c o n f e r s a g r o w t h a d v a n t a g e . D a t a c o n c e r n i n g the effect of B c l - 2 l e v e l s o n growth rates  in vivo  is not w e l l e s t a b l i s h e d , a l t h o u g h a n o t h e r  s t u d y u s i n g B c l - 2 s e n s e a n d a n t i s e n s e in M C F - 7 c e l l s g r o w n in n u d e m i c e i n d i c a t e d n o g r o w t h a d v a n t a g e with h i g h e r B c l - 2 l e v e l s . 2 5  In g e n e r a l , variation in growth rates m a y c o m p l i c a t e t h e e v a l u a t i o n of d a t a  c o n c e r n i n g d r u g sensitivity, a s the c y t o t o x i c effect of c h e m o t h e r a p y d r u g s is d e p e n d e n t u p o n g r o w t h rate and cell c y c l e .  H o w e v e r , o u r results indicate that t h e M D A - M B - 4 3 5 / L C C 6 C l o n e 7 cell lines w e r e  more  r e s i s t a n t to G e n a s e n s e a n d c o m b i n e d t h e r a p y t h a n M D A - M B - 4 3 5 / L C C 6 V e c t o r c e l l in s p i t e of h a v i n g a h i g h e r growth rate, w h i c h o n l y s u p p o r t s the p r e s e n c e of r e s i s t a n c e in this c e l l line. T h e finding that G e n a s e n s e d o e s not a p p e a r to i n c r e a s e the c h e m o r e s p o n s i v e n e s s of t u m o r s to c h e m o t h e r a p y t r e a t m e n t m a y detract f r o m its u s e a s a c h e m o s e n s i t i z i n g a g e n t . Genasense  p r o d u c e d s e q u e n c e - s p e c i f i c cytotoxicity  and  impaired  the  growth  H o w e v e r , the fact that of t u m o r s ,  and  that  c o m b i n e d G e n a s e n s e a n d d o x o r u b i c i n treatment w a s e v e n m o r e effective t h a n e a c h s i n g l e a g e n t a l o n e ( e v e n if this cytotoxicity w a s additive), i n d i c a t e s that G e n a s e n s e m a y p r o v e to b e u s e f u l in the clinic. C l i n i c a l s t u d i e s h a v e i n d i c a t e d that this a g e n t d o e s not p r o d u c e e x c e s s i v e toxicity, a n d its e f f i c a c y in c o m b i n a t i o n with v a r i o u s c h e m o t h e r a p y d r u g s is p r e s e n t l y u n d e r w a y . T h e d a t a p r e s e n t e d h e r e i n d i c a t e s that G e n a s e n s e is u s e f u l a s a t h e r a p e u t i c a g e n t , a n d the r e s u l t s of t h e s e c l i n i c a l s t u d i e s will h o p e f u l p r o v e c o n f i r m this f i n d i n g .  172  5.5 REFERENCES J u l i e n T , F r a n k e l B , L o n g o S , K y l e M , G i b s o n S , Shillitoe E , R y k e n T. A n t i s e n s e - m e d i a t e d inhibition o f the b c l - 2 g e n e i n d u c e s a p o p t o s i s in h u m a n m a l i g n a n t g l i o m a . Surg Neurol. 2 0 0 0 A p r ; 5 3 ( 4 ) : 3 6 0 - 8 . 1  P e p p e r C , T h o m a s A , H o y T, C o t t e r F, B e n t l e y P . A n t i s e n s e - m e d i a t e d s u p p r e s s i o n of B c l - 2 highlights its pivotal role in f a i l e d a p o p t o s i s in B - c e l l c h r o n i c l y m p h o c y t i c l e u k a e m i a . Br J Haematol. 1 9 9 9 Dec;107(3):611-5.  2  C a m p o s L, S a b i d o O , R o u a u l t J P , G u y o t a t D. E f f e c t s o f G e n a s e n s e o l i g o d e o x y n u c l e o t i d e s o n in vitro proliferation a n d s u r v i v a l o f n o r m a l m a r r o w p r o g e n i t o r s a n d l e u k e m i c c e l l s . Blood. 1 9 9 4 J u l 1 5 ; 8 4 ( 2 ) : 5 9 5 600.  3  R e e d J C , C u d d y M , H a l d a r S , C r o c e C , N o w e l l P , M a k o v e r D, B r a d l e y K. B C L 2 - m e d i a t e d t u m o r i g e n i c i t y of a h u m a n T - l y m p h o i d cell line: s y n e r g y with M Y C a n d inhibition b y B C L 2 a n t i s e n s e . Proc Natl Acad Sci USA. 1990 May;87(10):3660-4.  4  Z a n g e m e i s t e r - W i t t k e U , L e e c h S H , O l i e R A , S i m o e s - W u s t A P , G a u t s c h i O , L u e d k e G H , Natt F, H a n e r R, Martin P , H a l l J , N a l i n C M , S t a h e l R A . A n o v e l b i s p e c i f i c a n t i s e n s e o l i g o n u c l e o t i d e inhibiting both b c l - 2 a n d b c l - x L e x p r e s s i o n efficiently i n d u c e s a p o p t o s i s in t u m o r c e l l s . Clin Cancer Res. 2 0 0 0 J u n ; 6 ( 6 ) : 2 5 4 7 55.  5  K o t y P P , Z h a n g H , Levitt M L . A n t i s e n s e b c l - 2 t r e a t m e n t i n c r e a s e s p r o g r a m m e d cell d e a t h in n o n - s m a l l cell l u n g c a n c e r cell l i n e s . Lung Cancer. 1 9 9 9 F e b ; 2 3 ( 2 ) : 1 1 5 - 2 7 .  6  Z i e g l e r A , L u e d k e G H , F a b b r o D, A l t m a n n K H , S t a h e l R A , Z a n g e m e i s t e r - W i t t k e U . Induction of a p o p t o s i s in s m a l l - c e l l lung c a n c e r c e l l s b y a n a n t i s e n s e o l i g o d e o x y n u c l e o t i d e t a r g e t i n g the B c l - 2 c o d i n g s e q u e n c e . J Natl Cancer Inst. 1 9 9 7 J u l 1 6 ; 8 9 ( 1 4 ) : 1 0 2 7 - 3 6 . 7  J a n s e n B , S c h l a g b a u e r - W a d l H , B r o w n B D , B r y a n R N , v a n E l s a s A , M u l l e r M , W o l f f K, E i c h l e r H G , P e h a m b e r g e r H . G e n a s e n s e t h e r a p y c h e m o s e n s i t i z e s h u m a n m e l a n o m a in S C I D m i c e . Nat Med. 1 9 9 8 Feb;4(2):232-4.  8  G l e a v e M E , M i a y a k e H, Goldie J , Nelson C , Tolcher A . Targeting bcl-2 g e n e to delay androgeni n d e p e n d e n t p r o g r e s s i o n a n d e n h a n c e c h e m o s e n s i t i v i t y in p r o s t a t e c a n c e r u s i n g a n t i s e n s e b c l - 2 o l i g o d e o x y n u c l e o t i d e s . Urology. 1 9 9 9 D e c ; 5 4 ( 6 A S u p p l ) : 3 6 - 4 6 .  9  M i y a k e H , M o n i a B P , G l e a v e M E . 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T a r g e t i n g b c l - 2 g e n e to d e l a y a n d r o g e n i n d e p e n d e n t p r o g r e s s i o n a n d e n h a n c e c h e m o s e n s i t i v i t y in p r o s t a t e c a n c e r u s i n g a n t i s e n s e b c l - 2 o l i g o d e o x y n u c l e o t i d e s . Urology. 1 9 9 9 D e c ; 5 4 ( 6 A S u p p l ) : 3 6 - 4 6 . 1 6  L o p e s d e M e n e z e s D E , H u d o n N , M c i n t o s h N , M a y e r L D . M o l e c u l a r a n d pharmacokinetic properties a s s o c i a t e d with the t h e r a p e u t i c s of b c l - 2 a n t i s e n s e o l i g o n u c l e o t i d e G 3 1 3 9 c o m b i n e d with free a n d l i p o s o m a l d o x o r u b i c i n . Clin Cance