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Checkpoint inhibition and cell cycle effects of 13-hydroxy-15-oxozoapatlin, ent-kaur-16-en-15-oxo-18-9oic… Rundle, Natalie T. 2003

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CHECKPOINT INHIBITION AND C E L L C Y C L E EFFECTS OF 13-HYDROXY-15-OXOZOAPATLIN, ^AT-KAUR-ie-EN-lS-OXO-lS-OIC ACID, AND ISOGRANULATIMIDE By NATALIE T. RUNDLE B. Sc. (Hons.), University of Toronto, 1994 M . Sc., McMaster University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Biochemistry and Molecular Biology) We accept this thesis as conforming to the required standard University of British Columbia May, 2003 © Natalie Rundle, 2003 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t hesis f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date $\4Ml. 2 , IjQOy Printed for Natalie Rundle (tsunami) ABSTRACT Cell cycle checkpoints are activated in response to D N A damage and cause arrest in G, and G 2 phase. Inhibitors of the G 2 checkpoint are sought because they can increase the effectiveness of D N A damaging cancer therapies against cells with mutant p53. These inhibitors also have potential value as tools for biochemical analysis of the checkpoint pathway. Our laboratory conducted a cell-based screen to identify chemical inhibitors of the G 2 D N A damage checkpoint in extracts from terrestrial plants, marine microorganisms, and marine invertebrates. Several new checkpoint inhibitors were identified, including 13-hydroxy-15-oxozoapatlin (OZ), ent-kaur-16-en-15-oxo-18-oic acid (OKA), and isogranulatimide (IGR). The goals of my research were i) to study the cell cycle effects of IGR, OZ, and O K A , and ii) to identify their molecular targets, i f possible. Most of the checkpoint inhibitors discovered in the cell-based screen were inhibitors of checkpoint protein kinases or protein phosphatases, but a small number acted against unknown targets. OZ is a member of the latter category.-OZ was a potent inhibitor of the G 2 checkpoint (IC 5 0 6 uM), but did not inhibit the checkpoint kinases A T M , ATR, Chk l , Chk2, or P lk l in vitro. OZ also showed no activity against purified PP1, PP2A, or Ser/Thr protein phosphatases in MCF-7 cell extracts. O K A is very similar in structure to OZ, suggesting that it also does not inhibit protein phosphatases or checkpoint kinases. We hypothesized that OZ and O K A act on a new checkpoint protein. Unlike other checkpoint inhibitors, OZ and O K A are also antimitotic agents. OZ- and O K A -treated cells arrested in a stage resembling prometaphase, in which bipolar spindles formed and chromosomes failed to congress. In contrast to other antimitotic agents, OZ and O K A did not exert a strong effect on tubulin polymerization in vitro. Nevertheless, live cell microscopy demonstrated that chromosome movement was greatly reduced in OZ- and OKA-treated cells. Immunofluorescence microscopy revealed that the intracellular localization of the mitotic motor protein CENP-E and an associated kinase, hBubRl, were altered after OZ or O K A treatment. These results suggest that OZ and O K A interfere with the activity of a mitotic motor, causing a block in chromosome alignment and stalling progression through mitosis. Considering that a target of OZ and O K A could play a role in both the checkpoint pathway and in chromosome congression, we became interested in identifying the molecular target(s) of these compounds. A biotinylated analogue of O K A (B-OKA) was synthesized and retained the ii principal biological activities of OZ and O K A . B - O K A bound covalently to several target proteins. These were purified by streptavidin affinity precipitation and six B-OKA-binding proteins were identified using mass spectrometry. One of these, RanBP2, was chosen for more detailed studies. RanBP2 and biotinylated proteins were purified from Xenopus laevis egg extracts treated with B - O K A . A biotinylated protein that co-migrated with full-length RanBP2 was detected. Fragments of biotinylated RanBP2 were also precipitated by RanBP2 polyclonal antibody and by streptavidin agarose. These results are consistent with a direct interaction between B - O K A and RanBP2, suggesting that modulation of RanBP2 activity results in checkpoint inhibition and/or prometaphase arrest. Many checkpoint inhibitors block protein kinase activity. IGR shares structural similarity with the protein kinase inhibitor staurosporine. In vitro kinase assays revealed that IGR was a selective inhibitor of GSK-3 p kinase activity. I tested whether checkpoint inhibition by IGR is caused by its action on GSK-3 p. The kinase was over-expressed by transfection, and the response of transfected cells to ionizing radiation was examined by immunofluorescence microscopy and flow cytometry. The checkpoint effects of a closely related compound, didemnimide A , were also examined. The results did not support a role for GSK-3 P in the checkpoint pathway. Taken together, these studies describe the cell cycle effects of two different types of checkpoint inhibitors: i) a type that has antimitotic effects, including OZ and O K A , and ii) the protein kinase inhibitor IGR. This research also exemplifies two different strategies for identifying targets of small molecule inhibitors. In the case of O K A , chemical modification of the compound allowed unbiased screening for interacting proteins from amongst all available cellular targets in vivo. In the case of IGR, a candidate target was selected on the basis of structural information and in vitro data, and tested for checkpoint effects in vivo. This research represents a first and important step in the characterization of novel small molecule inhibitors. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi PREFACE xiv ACKNOWLEDGEMENTS xv CHAPTER 1: INTRODUCTION 1 1 . 1 . I N T R O D U C T I O N T O T H E C E L L C Y C L E I 1 . 1 . 1 . T H E P H A S E S O F MITOSIS I 1 . 1 . 2 . F A C T O R S THAT C O N T R O L T H E O N S E T O F M I T O S I S 5 1 . 2 . C E L L C Y C L E C H E C K P O I N T S ACTIVATED BY D N A D A M A G E 6 1 . 2 . 1 . S E N S O R S O F D N A D A M A G E 7 I . 2 . 2 . E F F E C T O R S O F T H E C H E C K P O I N T PATHWAY I O I . 2 . 3 . D O W N S T R E A M T A R G E T S O F T H E D N A D A M A G E C H E C K P O I N T I I I . 2 . 4 . T H E R O L E O F P 5 3 IN T H E G , A N D G 2 D N A D A M A G E C H E C K P O I N T PATHWAYS I 2 I . 3 . T H E M I T O T I C S P I N D L E I 3 1 . 3 . 1 . S P I N D L E A S S E M B L Y I 3 I . 3 . 2 . D I R E C T I N G C H R O M O S O M E C O N G R E S S I O N T O W A R D S T H E M I D L I N E O F T H E S P I N D L E 1 6 1 . 3 . 3 . A N T I M I T O T I C A G E N T S THAT T A R G E T T H E S P I N D L E 1 8 I .3.A. T H E S P I N D L E C H E C K P O I N T I 9 I . 4 . G 2 C H E C K P O I N T INHIB ITORS 2 2 I . 4 . I . R A T I O N A L E F O R T H E U S E O F G 2 C H E C K P O I N T I N H I B I T O R S IN C A N C E R T R E A T M E N T 2 2 I . 4 . 2 . C U R R E N T G 2 C H E C K P O I N T INHIBITORS 2 3 1 . 5 . R E S E A R C H O B J E C T I V E S 2 6 CHAPTER 2: DISCOVERY OF CHECKPOINT INHIBITING AND ANTIMITOTIC ACTIVITIES OF 13-HYDROXY-15-OXOZOAPATLIN (OZ) 28 2 . 1 . S U M M A R Y 2 8 2 . 2 . I N T R O D U C T I O N 2 8 iv 2 . 3 . E X P E R I M E N T A L P R O C E D U R E S 3 0 2 . 3 . I . ISOLATION A N D IDENTIFICATION O F I 3 - H Y D R O X Y - I 5 - O X O Z O A P A T L I N 3 0 2 . 3 . 2 . I N D I R E C T I M M U N O F L U O R E S C E N C E L A B E L L I N G O F M I T O T I C C E L L S F O R F L O W C Y T O M E T R Y 3 1 2 . 3 . 3 . F L O W C Y T O M E T R Y A N D DATA A N A L Y S I S 3 1 2 . 3 . 4 . INHIBIT ION O F T H E G £ D N A D A M A G E C H E C K P O I N T 3 2 2 . 3 . 5 . I M M U N O F L U O R E S C E N C E M I C R O S C O P Y 3 2 2 . 3 . 6 . C E L L P R O L I F E R A T I O N A S S A Y S 3 3 2 . 3 . 7 . C H E M I C A L MODIF ICATION O F O Z 3 3 2 . 3 . 8 . R A D I O L A B E L L I N G O F I 3 - [ 3 H ] M E T H Y L - I 5 - O X O Z O A P A T L I N - B I N D I N G P R O T E I N S 3 4 2 . 4 - . R E S U L T S 3 5 2 . 4 . I . ISOLATION A N D IDENTIFICATION O F I 3 - H Y D R O X Y - I 5 - O X O Z O A P A T L I N ( O Z ) AS A G 2 C H E C K P O I N T INHIBITOR 3 5 2 . 4 . 2 . E F F E C T S O F O Z ON C E L L C Y C L E P R O G R E S S I O N 3 5 2 . 4 . 3 . E F F E C T S O F O Z ON MITOTIC M O R P H O L O G Y 4 I 2 . 4 . 4 . INTERACTION B E T W E E N O Z A N D O T H E R C H E C K P O I N T INHIB ITORS 4 4 2 . 4 . 5 . INHIBITION O F C E L L P R O L I F E R A T I O N BY O Z 4 4 2 . 4 . 6 . M E C H A N I S M O F A C T I O N O F O Z 4 6 2 . 4 . 7 . I 3 - [ 3 H ] M E T H Y L - I 5 - O X O Z O A P A T L I N L A B E L L I N G O F P R O T E I N T A R G E T S . . 4 8 2 . 5 . D I S C U S S I O N 5 2 2 . 6 . A C K N O W L E D G E M E N T S 5 5 CHAPTER 3: DISCOVERY OF THE ANTIMITOTIC AND CHECKPOINT INHIBITING ACTIVITIES OF .EVT-KAUR-16-EN-15-OXO-18-OIC ACID (OKA) 56 3 . I . S U M M A R Y 5 6 3 . 2 . INTRODUCTION 5 7 3 . 3 . E X P E R I M E N T A L P R O C E D U R E S 5 8 3 . 3 . 1 . C E L L C U L T U R E A N D G 2 C H E C K P O I N T INHIBITION A S S A Y S 5 8 3 . 3 . 2 . 3 - D C O N F O C A L M I C R O S C O P Y O F L I V E C E L L S E X P R E S S I N G G F P -H I S T O N E H I 5 8 3 . 3 . 3 . T U B U L I N P O L Y M E R I Z A T I O N A S S A Y S 5 9 3 . 3 . 4 . I M M U N O F L U O R E S C E N C E M I C R O S C O P Y 5 9 3 . 3 . 4 . A . INDIRECT I M M U N O F L U O R E S C E N C E L A B E L L I N G O F (3- O R Y" T U B U L I N 5 9 3 . 3 . 4 . B . I N D I R E C T I M M U N O F L U O R E S C E N C E L A B E L L I N G O F C E N P - E O R H B U B R I 6 0 3 . 3 . 4 . C . I N D I R E C T I M M U N O F L U O R E S C E N C E L A B E L L I N G WITH T H E 3 F 3 / 2 A N T I B O D Y 6 1 3 . 4 . R E S U L T S 6 2 3 . 4 . I . IDENTIFICATION O F O K A AS A G 2 C H E C K P O I N T INHIB ITOR 6 2 3 . 4 . 2 . O K A A R R E S T S C E L L S IN A S T A G E R E S E M B L I N G P R O M E T A P H A S E 6 5 3 . 4 . 3 . E F F E C T S O F O Z A N D O K A ON C H R O M O S O M E M O V E M E N T IN L IVE C E L L S 6 5 3 . 4 . 4 . E F F E C T S O F O Z A N D O K A O N T U B U L I N A S S E M B L Y IN VITRO 6 7 3 . 4 . 5 . E F F E C T S O F O Z A N D O K A ON I N T R A C E L L U L A R L O C A L I Z A T I O N O F C E N P - E 7 5 3 . 4 . 6 . I N T R A C E L L U L A R LOCALIZATION O F H B U B R I is A L T E R E D IN O Z - A N D 7 8 v O K A - T R E A T E D C E L L S 3 . 4 . 7 . Y " T U B U L I N M O R P H O L O G Y IN O Z - A N D O K A - T R E A T E D C E L L S 8 I 3 . 4 . 8 . I M M U N O F L U O R E S C E N C E M I C R O S C O P Y A N A L Y S I S O F 3 F 3 / 2 E P I T O P E E X P R E S S I O N 8 3 3 . 5 . D I S C U S S I O N 8 5 3 . 6 . A C K N O W L E D G E M E N T S 9 0 CHAPTER 4: INVESTIGATION OF THE INTRACELLULAR DISTRIBUTION AND MOLECULAR TARGETS OF OKA 91 4 . I . S U M M A R Y 9 1 4 . 2 . I N T R O D U C T I O N 9 1 4 . 3 . E X P E R I M E N T A L P R O C E D U R E S . . . . . : 9 4 4 . 3 . I . S T R E P T A V I D I N - A L E X A 5 9 4 AFF IN ITY L A B E L L I N G F O R F L U O R E S C E N C E M I C R O S C O P Y 9 4 4 . 3 . 2 . S T R E P T A V I D I N AFFINITY PURIF ICATION O F P R O T E I N S INTERACTING WITH B - O K A 9 4 4 . 3 . 3 . D E T E C T I O N O F B IOTINYLATED P R O T E I N S BY S T R E P T A V I D I N - H R P O V E R L A Y 9 6 4 . 3 . 4 . S I L V E R S T A I N I N G O F E L E C T R O P H O R E S E D P R O T E I N S 9 6 \ 4 . 3 . 5 . A N A L Y S I S O F B - O K A - B I N D I N G P R O T E I N S BY M A S S S P E C T R O M E T R Y . . . . 9 7 4 . 3 . 6 . P R E C I P I T A T I O N A N D B L O T T I N G M E T H O D S F O R T E S T I N G F O R AN INTERACTION B E T W E E N B - O K A A N D R A N B P 2 9 8 4 . 4 . R E S U L T S 9 9 4 . 4 . I . B IOTINYLATION D O E S N O T INHIBIT T H E C H E C K P O I N T A N D ANTIMITOTIC A C T I V I T I E S O F O K A 9 9 4 . 4 . 2 . I N T R A C E L L U L A R LOCALIZATION O F B - O K A I 0 3 4 . 4 . 3 . P U R I F I C A T I O N O F B - O K A IN C O M P L E X E S WITH P R O T E I N T A R G E T S I 0 5 4 . 4 . 4 . M A S S S P E C T R O M E T R Y IDENTIFICATION O F B - O K A - B I N D I N G P R O T E I N S . I 0 7 4 . 4 . 5 . T E S T I N G AN INTERACTION B E T W E E N B - O K A A N D T H E CANDIDATE T A R G E T R A N B P 2 I I 2 4 . 5 . D I S C U S S I O N I I 5 4 . 6 . A C K N O W L E D G E M E N T S I I 8 CHAPTER 5: INVESTIGATION OF A G 2 DNA DAMAGE CHECKPOINT ROLE FOR GSK-3 3 KINASE 119 5 . 1 . S U M M A R Y I 1 9 5 . 2 . I N T R O D U C T I O N I 1 9 5 . 3 . E X P E R I M E N T A L P R O C E D U R E S 1 2 2 5 . 3 . I . C E L L T R A N S F E C T I O N A N D GENERATION O F P O O L E D O R C L O N A L S T A B L E C E L L L I N E S 1 2 2 5 . 3 . 2 . W E S T E R N B L O T T I N G A N D IMMUNOAFFINITY PRECIP ITATION I 2 3 5 . 3 . 3 . M I T O T I C S P R E A D S I 2 4 5 . 3 . 4 . I M M U N O F L U O R E S C E N C E EXAMINATION O F G S K - 3 3 A N D H A E P I T O P E E X P R E S S I O N IN TRANSIENTLY T R A N S F E C T E D C E L L S I 2 4 5 . 3 . 5 . P R E P A R A T I O N O F T H E B IOTINYLATED G F - 7 A N T I B O D Y A N D ITS A P P L I C A T I O N IN F L O W CYTOMETRY I 2 5 5 . 3 . 6 . F L O W C Y T O M E T R I C EVALUATION O F T H E E F F E C T S O F O V E R -E X P R E S S I N G G S K - 3 3 O N T H E C E L L C Y C L E I 2 6 5 . 4 . R E S U L T S 1 2 7 vi 5 . 4 . I . E F F E C T S O F S T A B L E O V E R - E X P R E S S I O N O F G S K - 3 (3 ON G £ A R R E S T IN P O O L E D S T A B L E C E L L L I N E S I 2 7 5 . 4 . 2 . G E N E R A T I O N O F A S T A B L E C L O N A L C E L L L I N E O V E R - E X P R E S S I N G G S K - 3 (3 I 2 7 5 . 4 . 3 . E F F E C T S O F T R A N S I E N T T R A N S F E C T I O N O F G S K - 3 B ON T H E RADIATION R E S P O N S E I 2 9 5 . 4 . 4 . D E V E L O P M E N T O F F L O W C Y T O M E T R I C M E T H O D S F O R C E L L C Y C L E A N A L Y S I S IN T R A N S F E C T E D C E L L S I 3 5 5 . 4 . 5 . E F F E C T S O F O V E R - E X P R E S S I N G H A - G S K - 3 B ON T H E C E L L C Y C L E A F T E R D N A D A M A G E I 4 0 5 . 4 . 6 . C H E C K P O I N T E F F E C T S O F D I D E M N I M I D E A I 4 3 5 . 5 . D I S C U S S I O N 1 4 5 5 . 6 . A C K N O W L E D G E M E N T S 1 4 8 CHAPTER 6: DISCUSSION 149 6 . I . T H E S I S O V E R V I E W I 4 9 6 . 2 . T H E C H E M I C A L G E N E T I C S A P P R O A C H TO IDENTIFYING C H E C K P O I N T I N H I B I T O R S I 5 0 6 . 3 . C O N C L U S I O N S A N D F U T U R E D I R E C T I O N S I 5 3 REFERENCES 158 APPENDIX 1 177 vii LIST OF TABLES T A B L E I . I . M l C R O T U B U L E - A S S O C I A T E D M O T O R P R O T E I N S I N V O L V E D IN C H R O M O S O M E M O V E M E N T 1 7 T A B L E I .2. I N H I B I T O R S O F T H E G 2 D N A D A M A G E C H E C K P O I N T 2 4 T A B L E 4 . I . CANDIDATE T A R G E T S F O R INHIBITION BY B - O K A I I I viii LIST O F FIGURES F I G U R E I . I T H E P H A S E S O F T H E C E L L C Y C L E 2 F I G U R E 1 . 2 M O R P H O L O G Y O F C H R O M O S O M E S A N D T H E S P I N D L E D U R I N G MITOSIS. 4 F I G U R E 1 . 3 A M O D E L F O R C H E C K P O I N T C O N T R O L O F T H E G 2 T O M TRANSIT ION. . . . 8 F I G U R E I . 4 A M O D E L O F T H E F O R C E S C O N T R I B U T I N G TO C H R O M O S O M E M O V E M E N T D U R I N G MITOSIS I 4 F I G U R E I . 5 INHIBITION O F T H E M E T A P H A S E T O A N A P H A S E TRANSITION BY T H E S P I N D L E C H E C K P O I N T PATHWAY IN B U D D I N G Y E A S T 2 1 F I G U R E 2 . 1 I 3 - H Y D R O X Y - I 5 - O X O Z O A P A T L I N ( O Z ) 3 6 F I G U R E 2 . 2 G 2 C H E C K P O I N T INHIBITION A N D MITOTIC A R R E S T I N D U C E D BY O Z 3 8 F I G U R E 2 . 3 O Z T R E A T M E N T C A U S E S A P R O G R E S S I V E I N C R E A S E IN MITOTIC C E L L N U M B E R S 3 9 F I G U R E 2 . 4 O Z INHIBITS T H E G 2 D N A D A M A G E C H E C K P O I N T 4 0 F I G U R E 2 . 5 INHIBITION O F G 2 A R R E S T IN M C F - 7 M P 5 3 A N D H C T I I 6 - / - C E L L S BY O Z 4 2 F I G U R E 2 . 6 O Z - T R E A T E D M C F - 7 W T P 5 3 C E L L S A R R E S T IN A S T A G E R E S E M B L I N G P R O M E T A P H A S E 4 3 F I G U R E 2 . 7 O Z I N C R E A S E S C H E C K P O I N T INHIBITION BY I G R A N D D B H , B U T NOT C A F F E I N E O R U C N - O I 4 5 F I G U R E 2 . 8 O Z E N H A N C E S T H E EFFECTS O F D B H ON RADIATION- INDUCED C E L L K I L L I N G 4 7 F I G U R E 2 . 9 C H E M I C A L MODIFICATION O F O Z 4 9 F I G U R E 2 . I O T H E a,P-UNSATURATED C A R B O N Y L G R O U P O F O Z is R E Q U I R E D F O R C H E C K P O I N T INHIBITION 5 0 F I G U R E 2 . 1 1 L A B E L L I N G O F C E L L U L A R P R O T E I N S WITH I 3 - [ 3 H ] M E T H Y L - I 5 -O X O Z O A P A T L I N 5 I F I G U R E 3 . I S T R U C T U R A L F O R M U L A E O F EWT-KAUR- I 6 - E N - I 5-oxo-1 8-oic A C I D ( O K A ) A N D I 3 - H Y D R O X Y " I 5 " O X O Z O A P A T L I N ( O Z ) 6 3 F I G U R E 3 . 2 INHIBIT ION O F G 2 A R R E S T IN H C T I I 6 - / - C E L L S B Y O K A A N D O Z 6 4 F I G U R E 3 . 3 O K A A R R E S T S H C T I I 6 - / - C E L L S IN A S T A G E R E S E M B L I N G P R O M E T A P H A S E 6 6 F I G U R E 3 . 4 T H E S T A G E S O F MITOSIS IN M C F - 7 C E L L S O V E R - E X P R E S S I N G G F P - H I S T O N E H I 6 8 F I G U R E 3 . 5 C H R O M O S O M E MOTION IS S E V E R E L Y C U R T A I L E D IN O Z - A N D O K A -T R E A T E D C E L L S 7 0 F I G U R E 3 . 6 O Z A N D O K A DO NOT P O L Y M E R I Z E T U B U L I N IN VITRO 7 3 F I G U R E 3 . 7 P O L Y M E R I Z A T I O N O F T U B U L I N IN VITRO IS NOT INHIB ITED BY O Z O R O K A 7 4 F I G U R E 3 . 8 I N T R A C E L L U L A R LOCALIZATION O F C E N P - E T H R O U G H O U T T H E C E L L C Y C L E ( 2 P A G E S ) 7 6 F I G U R E 3 . 9 C E N P - E IS PRIMARILY AT O R NEAR T H E C E N T R O S O M E A F T E R O Z A N D O K A T R E A T M E N T 7 9 F I G U R E 3 . I O C E N P - E A S S O C I A T E S WITH M I C R O T U B U L E S IN N O C O D A Z O L E - T R E A T E D C E L L S 8 0 F I G U R E 3 . I I H B U B R I is L O C A L I Z E D A R O U N D T H E C E N T R O S O M E A F T E R O Z O R O K A T R E A T M E N T 8 2 ix F I G U R E 3 . 1 2 O Z A N D O K A D O NOT A F F E C T C E N T R O S O M A L Y - T U B U L I N LOCALIZATION 8 4 F I G U R E 3 . 1 3 T H E 3 F 3 / 2 E P I T O P E IS NOT EXTINGUISHED IN O Z - A N D O K A - T R E A T E D C E L L S 8 6 F I G U R E 4 . I C H E M I C A L MODIFICATION O F O K A WITH N-IODOACETYL-M B I O T I N Y L H E X Y L E N E D I A M I N E I O O F I G U R E 4 . 2 B l O T I N Y L A T I O N D O E S NOT P R E V E N T C H E C K P O I N T INHIBITION BY B - O K A 1 0 2 F I G U R E 4 . 3 B - O K A D I S T R I B U T E S F R E E L Y T H R O U G H O U T T H E C E L L I 0 4 F I G U R E 4 . 4 S T R E P T A V I D I N A G A R O S E AFFINITY PURIF ICATION O F B - O K A - B I N D I N G P R O T E I N S I 0 6 F I G U R E 4 . 5 S D S - P A G E ANALYSIS O F P R O T E I N S C O - P U R I F I E D WITH B - O K A I 0 8 F I G U R E 4 . 6 C H R O M O G E N I C L A B E L L I N G O F B - O K A - B I N D I N G P R O T E I N S P U R I F I E D . . . . I I O F I G U R E 4 . 7 R E C I P R O C A L PRECIPITATION O F B IOT INYLATED R A N B P 2 I 1 4 F I G U R E 4 . 8 C O - M I G R A T I O N O F R A N B P 2 AND A B - O K A - L A B E L L E D P R O T E I N I I 6 F I G U R E 5 . I S T R U C T U R A L F O R M U L A E F O R D E B R O M O H Y M E N I A L D E S I N E , I S O G R A N U L A T I M I D E , A N D DIDEMNIMIDE A 1 2 1 F I G U R E 5 . 2 S T A B L E O V E R - E X P R E S S I O N O F WILD-TYPE O R K 8 5 A G S K - 3 (3 D O E S NOT A F F E C T ENTRY INTO MITOSIS A F T E R IRRADIATION I 2 8 F I G U R E 5 . 3 IDENTIFICATION O F T H E 3 G 3 C L O N A L C E L L L I N E O V E R - E X P R E S S I N G W I L D - T Y P E G S K - 3 3 I 3 0 F I G U R E 5 . 4 T H E 3 G 3 C L O N E E X P R E S S E S H A - T A G G E D G S K - 3 3 1 3 1 F I G U R E 5 . 5 T H E 3 G 3 C L O N E IS SENSITIZED TO L O W D O S E S O F IONIZ ING RADIATION I 3 2 F I G U R E 5 . 6 T R A N S I E N T TRANSFECTION O F WILD-TYPE B U T NOT K 8 5 A G S K - 3 3 INHIBITS ENTRY INTO MITOSIS A F T E R D N A D A M A G E I 3 4 F I G U R E 5 . 7 A S C H E M E F O R A S S E S S I N G T H E E F F E C T S O F H A - G S K - 3 3 E X P R E S S I O N ON T H E C E L L C Y C L E , U S I N G BIOTINYLATED G F - 7 A N T I B O D Y I 3 6 F I G U R E 5 . 8 B I O T I N Y L A T E D G F - 7 ANTIBODY ( B - G F - 7 ) A L L O W S C O U N T I N G O F MITOTIC C E L L S IN F L O W CYTOMETRY I 3 8 F I G U R E 5 . Q A STRATEGY F O R L A B E L L I N G H A - G S K - 3 3-EXPRESSING C E L L S F O R C E L L C Y C L E ANALYSIS U S I N G RAT A N D M O U S E PRIMARY A N T I B O D I E S . . I 3 9 F I G U R E 5 . I O A N A L Y S I S O F F L O W CYTOMETRY DATA F O R M E A S U R I N G T H E E F F E C T S O F E C T O P I C G E N E E X P R E S S I O N ON T H E C E L L C Y C L E I 4 I F I G U R E 5 . 1 1 E X P R E S S I O N O F WILD-TYPE H A - G S K - 3 3 R E D U C E S ENTRY INTO MITOSIS A F T E R D N A DAMAGE I 4 4 F I G U R E 5 . 1 2 E F F E C T S O F DIDEMNIMIDE A ON C E L L P R O L I F E R A T I O N I 4 6 F I G U R E 6 . I F O R W A R D C H E M I C A L GENETICS A P P R O A C H T O IDENTIFYING C H E C K P O I N T INHIBITORS A N D T H E I R TARGETS I 5 2 F I G U R E 6 . 2 A M O D E L F O R T H E ACTION O F O Z A N D O K A ON R A N B P 2 1 5 5 X LIST OF ABBREVIATIONS 7-AAD: 7-aminoactinomycin D A-T: ataxia telangiectasia APC: anaphase promoting complex APC: adenomatous polyposis coli A T M : ataxia telangiectasia mutated ATR: ataxia telangiectasia and rad 3 related BSA: bovine serum albumin B-OKA: biotinylated e«f-kaur-16-en-15-oxo-18-oic acid BUB: budding uninhibited by benzimidazole CAF: caffeine CDK: cyclin-dependent kinase CDKI: cyclin-dependent kinase inhibitor CENP-E: centromeric protein E D B H : debromohymenialdisine D M E M : Dulbecco's modified Eagle media DMSO: dimethyl sulphoxide DNA-PK: DNA-dependent protein kinase DTT: dithiothreitol EDTA: ethylenediamine tetraacetic acid EGTA: ethylene glycol-bis(2-aminoethylether)-JV, N, N', N', tetraacetic acid ELICA: enzyme-linked immunocytochemical assay ESP1: extra spindle poles 1 FITC: fluorescein isothiocyanate xi FRAP: FKBP12-rapamycin associated protein Gy: Grays hMDM-2: human homologue of murine double minute-2 HRP: horseradish peroxidase IGR: isogranulatimide kDa: kiloDaltons MDM-2: murine double minute-2 M A D : mitotic arrest deficient mTOR: mammalian target of rapamycin MeOH: methanol MOPS: (3 - [N-Morpholino]propanesulfonic acid) MTT: 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium N.C.I.: National Cancer Institute NMR: nuclear magnetic resonance NOC: nocodazole NPC: nuclear pore complex O K A : ent-kaur-16-en-15-oxo-18-oic acid OZ: 13 -hydroxy-15 -oxozoapatlin PAGE: polyacrylamide gel electrophoresis PE: phycoerythrin PBS: phosphate-buffered saline PBS-T: PBS-Tween 20 PCNA: proliferating cell nuclear antigen PI-3K: phosphatidylinositol-3 kinase xii PI: propidium iodide PIPES: 1,4-piperazine diethane sulfonic acid PMSF: phenylmethylsulfonyl fluoride RFC: replication factor C SAB: standard azide buffer SDS: sodium dodecyl sulphate Tris: tris(hydroxymethyl)aminomethane TBS: Tris-buffered saline Tx-100: Triton X-100, t-Octylphenoxypolyethoxyethanol UCN-Ol: 7-hydroxystaurosporine xiii P R E F A C E This thesis is presented in six chapters. Chapters 2 through 5 were written in the format of a scientific journal article. The introduction, methods and discussion pertaining to the research are presented in the same chapter as the results themselves. The aim of this was to teach me to write in the academic style, and to facilitate any future publications from this thesis. Chapter 2 is almost entirely composed of a publication with co-authors Dr. Lin Xu, Dr. R. J. Andersen and Dr. Michel Roberge [155]. In this chapter, all flow cytometry data, time-course assays, checkpoint assays and immunofluorescence microscopy are my own work. The chemical preparation of OZ was performed in the laboratory of Dr. R. J. Andersen (Oceanography and Earth Ocean Sciences) by Lin Xu. The original ELICA assays that identified the checkpoint inhibiting activity of OZ were done before I joined the lab. Cell proliferation assays that were part of the paper were performed by Lianne McHardy. Also included in this chapter are time course assays and radiolabelling experiments that were not part of the paper. xiv A C K N O W L E D G E M E N T S There are many people I would like to thank for their contribution to this thesis. First is Dr. Michel Roberge, for his excellent supervision, teaching and inspiration. I'll miss our scientific discussions and working together. The members of my supervisory committee, Dr. Roger Brownsey and Dr. Calvin Roskelley, were very generous with their time, guidance and support. Many past, present, and honorary lab members have helped me along the way and made the lab a fun place to work in: Hilary Anderson, Cristina Bigg, Colleen Brown, Darko Curman, Geoff Karjala, Cameron Mackereth, Lianne McHardy, Delphine Reberioux, Chris Sturgeon, Tamsin Tarling and Yong-Jun Wang. Throughout this degree, my family has offered endless support. M y parents always showed an interest in my work and offered their encouragement. Thank you, most especially, to Howard, my husband and partner in all things. xv Chapter 1 INTRODUCTION 1.1. Introduction to the Cell Cycle The regulation of cellular growth and division is one of the fundamental issues in cell biology. The duplication of all the components of a cell, the faithful replication of the entire genome, and the coordination of these events to the birth of daughter cells, is referred to as the cell cycle and has fascinated biologists for many decades. Two parts of the cell cycle can be observed in light microscopy: mitosis and interphase. In the traditional classification, there are four subdivisions in the cell cycle: G x (gap 1), S (for DNA synthesis), G 2 (gap 2), and M phase that includes both mitosis and cytokinesis (Figure 1.1). Chromosomes and centrosomes (a type of microtubule organizing centre) are replicated during S phase. In mitosis, the mitotic spindle is formed, attaches to chromosomes, and delivers one copy of the genome to each of the incipient daughter cells. M phase concludes with cytokinesis in which the cell is divided into two. 1.1.1. The Phases of Mitosis Early studies of mitosis by biologists focussed on the morphological changes that were detectable by available dyes and light microscopy [1]. As a result, the conventional phases of mitosis are based upon the morphology of chromosomes and the mitotic spindle [2]. Hallmarks of the six major divisions of M phase, in order of appearance, are described below (reviewed in [3-5]) (Figure 1.2 [6]). 1 Figure 1.1 The phases of the cell cycle. The cell cycle is divided into four main phases. The two principal cell cycle transitions are entry into S phase (yellow) in which the genome is replicated, and entry into M (red), in which mitosis and cytokinesis are completed. These two intervals are separated by the gap phases G1 (blue) and G2 (green) in which cells grow and prepare for the upcoming transition. 2 Prophase: The first indication that cells have begun mitosis is the condensation of nuclear chromatin. This progresses from an initial stippled appearance of the nuclei in early prophase until fully condensed sister chromatids, joined.by centromeres, are visible by the end of prophase. Spindle microtubules can be seen stretching between the centrosomes, around which astral microtubules are arrayed. During this phase, the nuclear envelope, endoplasmic reticulum and Golgi apparatus disassemble. Prometaphase: In prometaphase, the mitotic spindle is completed and the centrosomes are well-separated. Microtubules reaching from each centrosome begin to enter what had been the nuclear space. There the microtubules encounter chromosomes and become stably attached to the kinetochores. Once each sister kinetochore has attached to microtubules emanating from each pole, chromosomes begin to migrate towards the equatorial plane of the cell. Metaphase: In this phase, chromosomes align at the midline of the cell. At this point, mitosis appears to pause as the cell checks that all chromosomes have aligned correctly. This process can last 30-45 min, making metaphase the longest phase of mitosis. Anaphase: Anaphase begins with the cleavage of cohesion proteins in the centromeric region joining sister chromatids. As anaphase A progresses, chromatids are drawn towards opposite poles as the microtubules bound to them begin to shorten. In anaphase B, the spindle elongates and draws centrosomes further apart. 3 Figure 1.2 Morphology of chromosomes and the spindle during mitosis. The conformation of the spindle and chromosomes are the features that define each phase of mitosis. DNA (blue), microtubules (green) and centrosomes (orange) were labeled in human U20S osteosarcoma cells and visualized in immunofluorescence microscopy. Reprinted with permission from the Encyclopedia of Life Sciences [6]. 4 Telophase: During the last phase of mitosis, chromosomes are gathered around each centrosome and de-condense. The nuclear envelope, Golgi, and endoplasmic reticulum begin to reform while the mitotic spindle is disassembled. Cytokinesis: This represents the final act of M phase, and proceeds concurrently with late anaphase and telophase. An actin and myosin ring assembles at the equatorial plane. As it constricts, a cleavage furrow becomes apparent and eventually pinches the cell in two. 1.1.2. Factors that Control the Onset of Mitosis The earliest experiments in the study of mitotic control began with amphibian eggs (reviewed in [3]). Immature frog oocytes are normally arrested in meiotic prophase, a state resembling G 2 . In 1971 Masui and Markert found that injection of cytoplasm from maturing oocytes into immature oocytes caused the nuclear envelope to break down, demonstrating that a cytoplasmic activity promotes cell cycle progression [7]. This cytoplasmic activity was named ^maturation-promoting factor" (MPF), and a search began for its components. MPF was soon found to represent a more general phenomenon, as its activity was also demonstrated in mitotic cells [8]. Its activity rose and fell with a periodicity matching the cell cycle, suggesting that MPF was a general controller of entry into mitosis [8]. MPF activity was purified from X. laevis eggs and contained two proteins of molecular mass 32 and 45 kDa [91 The 32 kDa protein was a kinase that phosphorylated the 45 kDa protein, histone HI and other substrates [9]. Work in sea urchin extracts had shown that the abundance of the 45 kDa protein, named cyclin, rose and fell with the same periodicity as the cell cycle, suggesting that cyclins regulate entry into mitosis [10]. At the same time as MPF 5 was being purified using biochemical methods, genetic approaches identified the cdc2 gene as a positive regulator of mitosis in S. pombellY\. These two fields converged when MPF purified from X. laeviswas recognized by a cdc2antibody [12]. Cdc2 is ubiquitous and well conserved, and the role of cyclin B-cdc2 in regulating entry into mitosis is now well-established [13]. Cdkl/cdc2, the master switch of mitosis, is regulated in several modes: by the synthesis or degradation of activating cyclins, by the expression of inhibitory proteins, and by multiple phosphorylations and de-phosphorylations [14, 15]. In part it is controlled by cyclins that are synthesized and degraded in a cell cycle-dependent manner. Cdkl is activated by binding of its partner cyclins A or B, and by phosphorylation on Thrl61. Activation of cdkl is not complete until two inhibitory phosphorylations on Thrl4 and Tyrl5 (added by Weel and/or Myt l kinases) are removed by cdc25 phosphatase. Cyclin B l is degraded at the end of mitosis, ending the active phase of cdkl kinase activity. The multiple levels of control allow cdkl/cdc2 to integrate information from several inputs, and means that there are many signal transduction opportunities for regulating cdkl activity. 1.2. Cell Cycle Checkpoints Activated by DNA Damage As the study of mitotic control progressed, and new types of cell division mutants were discovered, it became apparent that the cell cycle is regulated by checkpoints. These are required both to modulate the length of the cell cycle to suit prevailing conditions (such as food supply), and for stopping the cell cycle when its continuation would be destructive to the cell [16, 17]. Two major points of control in the cell cycle are duplication of DNA in S phase, and mitosis. Checkpoint pathways are activated by changes in osmolarity, cell size, 6 unreplicated DNA, spindle assembly and positioning, and damaged DNA. The research in this thesis concerns the checkpoint pathway activated by DNA damage. DNA damage occurs naturally in the cell as a by-product of DNA replication and is also generated by free radicals. External sources of DNA damage include ionizing and ultraviolet radiation and chemical agents. Exposure to these agents will activate a checkpoint and cause a delay in G1 or G 2 phase, allowing the cell time to repair the damage before replicating DNA or entering mitosis [17]. Loss of the DNA damage checkpoint pathway is oncogenic, most likely as a consequence of genetic instability [18]. The members of the G1 and G 2 DNA damage checkpoint pathways can be classified as sensors, effectors, and downstream targets. The general features of DNA damage checkpoints are well conserved across diverse eukaryotic species, although the individual contributions of sensors and effectors varies with the species and with the type of DNA damage. The research in this thesis pertains to the human pathway activated by double-stranded DNA breaks and resulting in arrest in G 2 phase. The pathway is complex, involving many regulators and ultimately converging upon the regulation of cdkl (cdc2) activity (Fig. 1.3) [19-23]. For the sake of clarity, the discussion will focus on the human pathway, although many of the original discoveries were made in 5. cerevisiae, S. pombe, or X. laevis (reviewed in [3-5]). 1.2.1. Sensors of DNA Damage The detection of DNA damage is an active area of research, and its mechanism is not yet understood. A current area of interest in this field stems from the observation that signalling proteins rapidly form foci on chromatin at the sites of double-stranded DNA breaks. Because ATM and the 9-1-1 complex localize to these foci and are necessary for the checkpoint 7 DNA REPAIR inactive active G 2 ARREST ENTRY INTO M Figure 1.3 A model for checkpoint control of the G 2 to M transition. The mammalian response to double-stranded DNA breaks is shown. For the sake of clarity, only the p53-independent initiation phase of G 2 arrest is shown. Kinases are depicted as rectangles, cyclins as ovals, and phosphatases as hexagons. Stimulatory regulatory events are indicated by (+), inhibitions by (— ) . Entry rito mitosis is prevented by inhibitory phosphorylation of cyclin B-cdk1 on Thr14 and Tyr15. DNA damage activates ATM, which phosphorylates Chkl and Chk2 kinases. Chkl enforces G 2 arrest by simultaneously activating Wee1 kinase (promoting phosphorylation on Tyr15) and by inhibiting cdc25C (protecting the phosphates from removal). Activation of the sensors of DNA damage also promotes DNA repair (not shown). Abbreviations: cdk, cyclin-dependent kinase; ATM, ataxia telangiectasia mutated; 9-1-1, Rad9-Hus1-Rad1 complex;, M/R/N, Mre11-Rad50-Nbs1 complex. 8 response, they are currently being explored as sensors of DNA damage. One candidate sensor is ATM (ataxia telangiectasia mutated) kinase. Mutation of the gene for ATM kinase causes ataxia telangiectasia (A-T), a recessive syndrome manifested by loss of balance, predisposition to cancer, and infertility, among other symptoms (reviewed in [24]). Early attempts to treat the cancers of these patients resulted in acute sickness and death [25, 26], revealing the crucial role of ATM in protecting the cell against DNA damage. ATM is a large (357 kDa) Ser/Thr protein kinase and a member of the PI-3K (phosphoinositol-3 kinase) superfamily, sharing homology with ATR (ataxia telangiectasia and rad 3 related), DNA-PK (DNA-dependent protein kinase), and mTOR/FRAP (mammalian target of rapamycin/FKBPl2-rapamycin associated protein) (reviewed in [27, 28]). Studies on A-T cell lines have clearly demonstrated that ATM is necessary for delaying mitosis after DNA damage [29]. ATM is activated by ionizing radiation and binds to DNA at damage-induced foci [30, 31]. The mechanism underlying ATM activation by DNA damage is unknown, but appears to result in fr-a/75-autophosphorylation of kinase dimers, causing the release of active monomers [32]. ATM activity leads to cell cycle arrest in a number of ways. It enhances the stability and activity of p53 by direct phosphorylation and by action upon HDM-2 (the human homologue of MDM-2 (murine double minute-2)) [30, 33, 34]. ATM also phosphorylates Chkl and Chl<2 [35], the major effector kinases of the checkpoint pathway (described below). Phosphorylation of p53 and full checkpoint activation requires 53BP1, an adaptor protein that may recruit substrates to ATM [36, 37]. A separate complex containing Mrel l -Rad50-Nbsl (M/R/N) is required for checkpoint activation and is phosphorylated by ATM (reviewed in [38, 39]). It is suggested that the M/R/N complex recruits ATM substrates to DNA damage sites and may coordinate damage detection with 9 activation of DNA repair [21]. Another potential sensor of DNA damage is a multiprotein complex containing Rad9, Radl, and Husl (9-1-1) that acts together with a heteropentameric complex containing Radl7 and four subunits of replication factor C (RFC) (Fig 1.3) (reviewed in [20, 40]). These two complexes are necessary for the checkpoint and associate with sites of DNA damage [413. 9-1-1 shares structural homology with the proliferating cell nuclear antigen (PCNA) complex [42]. The current model is that 9-1-1 and the Radl7-RFC complex interact in a similar way to PCNA and RFC in DNA replication: 9-1-1 is predicted to form a heterotrimeric sliding clamp, and Radl7-RFC could function as a clamp loader [42]. In a manner analogous to PCNA, 9-1-1 is proposed to increase the processivity of break repair enzymes. 9-1-1 may interact with checkpoint signalling proteins as an early sensor of DNA damage. Radl7-RFC could direct the substrate specificity of 9-1-1 towards double stranded breaks [42]. 1.2.2. Effectors of the Checkpoint Pathway Checkpoint kinases 1 and 2 (Chkl and Chl<2) are nuclear Ser/Thr kinases that have been found in every eukaryotic organism studied [43-45]. Chkl and Chl<2 are rapidly phosphorylated after DNA damage by ATM, but it is not clear whether this increases the activity of these kinases above basal levels [23, 43, 44]. Phosphorylated Chl<2 disperses rapidly throughout the nucleus and may promote a widespread response to localized double-stranded breaks in DNA [46]. Both Chkl and Chl<2 phosphorylate p53 on Ser20, increasing its activity and resulting in cell cycle arrest [47]. Chkl and Chl<2 phosphorylate cdc25A, cdc25B, and cdc25C in vitro [43, 44]. 10 The roles of Chkl and Chl<2 kinases in the checkpoint pathway have been explored by gene targeting. Disruption of the Chkl gene in mice is lethal at a very early embryonic stage [48]. Conditional targeting of the Chkl gene in these animals inhibited G 2 arrest after ionizing irradiation [48]. In the avian DT-40 cell line, cells remained viable after Chkl targeting but were unable to arrest in G 2 after ionizing irradiation [49]. Similarly, Chkl gene silencing in human HeLa cells prevented G 2 arrest after DNA damage [50]. The consequences of targeting the Chl<2 gene are less clear. In mouse embryonic stem cells, Chl<2-deficient cells were able to initiate, but not maintain, G 2 arrest after exposure to ionizing irradiation [47]. Conversely, mice that lacked Chl<2 showed no defect in either initiation or maintenance of G 2 arrest [51]. In these animals, preservation of the checkpoint response may be due to compensation by Chkl kinase. 1.2.3. Downstream Targets of the DNA Damage Checkpoint Cdc25C is a positive regulator of cdkl activity, and acts by removing the inhibitory phosphates on Thrl4 and Tyrl5 of cdkl (Fig 1.3). One of the principal effects of the checkpoint pathway is inhibition of cdc25C. A major site of phosphorylation on cdc25C in vitro and in vivo is Ser216 [43, 44, 52]. Phosphorylation at this site is essential for checkpoint arrest after DNA damage and creates a binding site for 14-3-3 proteins, which may prevent cdc25C from acting upon cdkl [52]. Surprisingly, disruption of the cdc25C gene has no effect on G 2 arrest after irradiation, suggesting that other cdc25 family members may play compensatory roles in cdkl regulation [50, 53]. Negative regulators of cdkl activity are also part of the checkpoint pathway. Once cyclin B l is synthesized and bound to cdkl , premature mitosis is prevented by two inhibitory 11 phosphorylations. hWeel is a nuclear tyrosine-specific kinase that phosphorylates cdkl on Tyrl5 [54]. hMytl is related to hWeel, but distinct in that it can phosphorylate cdkl on both Thrl4 and Tyrl5 and it is localized to Golgi membranes [55]. During a normal cell cycle, hWeel and Myt l phosphorylate cdkl on Thrl4 and Tyrl5 and inhibit cdkl-cyclin B l activity [54, 55]. When the DNA damage checkpoint pathway is activated, Weel and Myt l are stimulated, ensuring inhibition of cdkl . Weel from S. pombeor X. Iaevis\s phosphorylated by Chkl in vitro [56, 57]. Phosphorylation of X. /aev/'s Weel creates a binding site for 14-3-3 proteins and increases the activity of weel towards cdc2 (cdkl) [57]. The weel pathway is not sufficient to initiate arrest. In fission yeast, neither weel nor cdc25 is required for checkpoint inhibition, but the double mutant fails to arrest [58]. It appears that Chkl enforces checkpoint inhibition through two pathways: activation of weel and inhibition of cdc25. Cooperation of these two pathways may be necessary for full inhibition of cdkl [23]. 1.2.4. The Role of p53 in the G1 and G2 DNA Damage Checkpoint Pathways In many respects, arrest in G : after DNA damage uses the same molecular pathways outlined above for G 2 arrest. The G x checkpoint is dependent upon A T M , and uses the effector kinases Chkl and Chl<2. An important difference between arrest in G a and G 2 concerns the role of the tumour suppressor p53, a transcription factor that is normally found at low levels in the nucleus [59, 60]. After DNA damage, post-translational modifications stabilize p53 and increase its transcriptional activity. The net result is increased transcription of both the INK4 and-Cip/Kip classes of cyclin-dependent kinase inhibitors (CDKI), resulting in cell cycle arrest. The activity of p53 is essential for arrest in G : after DNA damage [61]. 12 The situation is somewhat different for the G 2 checkpoint, which does not require p53 to initiate arrest [62]. When p53 function is disrupted, irradiated cells arrest in G 2 but not in G1 [63, 64], reflecting the fact that p53 is indispensable for arrest in G x [61]. p53 does play a role in sustaining arrest in G 2 , however, by up-regulating transcription of the CDKI p21 [62]. 1.3. The Mitotic Spindle 1.3.1. Spindle Assembly During mitosis, the microtubule network is dismantled and re-assembles as a spindle. The main function of the spindle is to provide a structural framework for the organization of chromosomes and the generation of forces that regulate the correct partitioning of chromosomes to daughter cells. The general structural features of the spindle are illustrated in Figure 1.4. At each end of the spindle are centrosomes, which contain a pair of centrioles and are surrounded by an amorphous matrix containing y-tubulin ring complexes. In mitosis, the microtubule nucleating activity of the centrosome is greatly increased as y-tubulin is recruited [65]. There are three classes of microtubules: astral microtubules stretch towards the cell cortex, chromosomal fibres connect centrosomes to the arms or kinetochores of chromosomes, and polar spindle fibres overlap with microtubules emanating from the opposite pole. Microtubules are polymers of a and (3-tubulin heterodimers. The heterodimers assemble into hollow, dynamic microtubules with "plus" and "minus" ends. The dynamic properties of microtubules are characterized by the frequencies of rescue (or elongation) and catastrophe (or disassembly) phases, and their rates of growth and shrinkage (reviewed in [66]). Stochastic switching between growth and shrinkage is referred to as dynamic 13 Figure 1.4 A model of the forces contributing to chromosome movement during mitosis. Tubulin forms the mitotic spindle by assembling astral (a), polar (b) and kinetochore (c) microtubules. Microtubules originate with their minus-ends at the spindle poles and plus-ends extending to chromosomes (purple) or the cell cortex (orange). The directions of forces are indicated with red arrows. Microtubule flux generates a poleward force. Force is also exerted through microtubule-associated motor proteins. Chromokinesins (X) oppose the poleward force and move chromosome arms to the equator. Dynein (X) helps to position the spindle poles and pulls the kinetochores towards the poles. CENP-E (X) is a kinetochore motor that pulls chromosomes towards the equator. Eg5 ( ^ ) cross-links adjacent polar microtubules and assists with separation of the spindle poles. 14 instability. The mitotic spindle is a kinetic structure: its overall length fluctuates, many proteins are transported back and forth across'jits surface, and its microtubules continually probe the cellular space in cycles of growth and shrinkage. In addition to the influence of the centrosome on microtubule nucleation, there are two principal factors that influence spindle assembly: the dynamics of tubulin, and microtubule-associated motor proteins. Several aspects of microtubule dynamics are regulated during mitosis for the construction and function of the spindle [67]. During mitosis, the catastrophe rate is increased, reducing the average half-life of a spindle microtubule to 1 min, and allowing microtubules to probe space around the centrosome. Microtubules are stabilized at their minus ends by attaching to Y-tubulin at the centrosome and at the plus ends by kinetochores. Chromatin itself exerts a stabilizing influence on spindle assembly through Ran-GTP [68, 69]. Meiotic extracts can form spindles in the absence of centrosomes and kinetochores, indicating that microtubules and their associated proteins (MAPs) also have an innate capacity for spindle formation. There are many motor proteins that bind to the spindle microtubules. They are involved in spindle formation, transport of mitotic cargoes, and generation of forces necessary for mitosis. Microtubule-based motors of the BimC class, such as Eg5, generate a plus-end-directed sliding force between overlapping anti-parallel microtubules, separating the poles and elongating the spindle [70]. The dynein motor protein also contributes to spindle assembly. Dynein is found in microtubule-associated multiprotein-complexes at the kinetochore, on astral microtubules and along the spindle. Dynein is involved in diverse mitotic processes including nuclear envelope breakdown, delivery of cargo to the centrosome, chromosome movement during prometaphase and anaphase, and spindle assembly [71-74]. Dynein contributes to spindle assembly by cross-linking adjacent microtubules. Its minus-end-15 directed motor activity focusses parallel microtubule arrays into spindle poles. 1.3.2. Directing Chromosome Congression Towards the Midline of the Spindle Perhaps the most striking display in mitosis is the gathering of chromosomes at the cell midline and their eventual migration towards the poles of the spindle (reviewed in [73, 75, 76]). When a chromosome first captures a microtubule, it glides rapidly along its surface until it reaches a spindle pole, and is referred to as monooriented. Eventually it becomes b/oriented as it connects with a microtubule that has stretched across from the opposite pole, and the chromosome begins to move towards the midline of the cell. As it does, more microtubules are captured by kinetochores, resulting in a more stable attachment. Eventually the cell equator is reached. Both mono- and bioriented chromosomes oscillate back and forth, a sign of the opposing forces that are acting upon them. There are several models for the many forces acting upon congressing chromosomes, differing in the relative importance placed upon microtubule dynamics, molecular motors, or a combination of the two (reviewed in [1, 2]. On one hand, kinetochores are attached to the dynamic plus ends of microtubules, and as'microtubule ends grow and shrink they generate enough force to push and drag the chromosomes [77]. There is also a constant poleward force exerted-on kinetochores as tubulin subunits flux towards the poles, which could serve to align the chromosomes (Fig. 1.4). On the other hand, there are several motor proteins that reside at the kinetochore and move chromosomes along microtubules (Table 1.1) [78]. For example, CENP-E is a plus-end-directed kinetochore motor that transports chromosomes towards the midline [79-82]. CENP-E also acts as a coupler, maintaining a connection between kinetochores and microtubule plus ends as they 16 Table 1.1 Microtubule-associated motor proteins involved in chromosome movement. Several ATP-dependent molecular motors bind to microtubules and generate the forces necessary to move chromosomes during mitosis. These are classified according to the direction of their movement along the microtubule, towards the plus or minus end. Name: I)ircctionalii\ * , Function: CENP-E plus end chromosome alignment, spindle elongation during anaphase B, possible spindle checkpoint protein Dynein minus end chromosome alignment, chromosome segregation, spindle pole positioning, cargo transport to poles, nuclear envelope breakdown MCAK destabilizes microtubules, involved in prometaphase and anaphase chromosome movement Xkid plus end aligning chromosome arms at the equator 17 depolymerize and drag chromosomes towards the poles [83]. Poleward chromosome movement during prometaphase is assisted by dynein at the kinetochore [73, 74, 84]). Poleward chromosome movement during prometaphase is also partially driven by MCAK, a motor protein found at the kinetochore that destabilizes microtubules and promotes their shortening during prometaphase and anaphase [73]. Furthermore, chromokinesin motor proteins, such as XKid in X. laevis, move chromosome arms towards the cell midline and allow them to keep pace with the movement of the kinetochore [85-87]. The congression of chromosomes is a complex process that requires the regulation and balance of many simultaneous forces, and coordination between sister kinetochores. 1.3.3. Antimitotic Agents that Target the Spindle Unregulated growth is a sign of cancer, and historically this is the property that has been targeted by cancer treatments. Because the spindle is one hallmark of an actively dividing cell, the majority of antimitotic agents target the spindle and usually bind directly to tubulin. The effects of antimitotic agents are concentration-dependent [88, 89]. They act as polymerizing or depolymerizing agents when applied in high concentration, promoting the appearance of alternate forms or the dissolution of microtubules. At their lowest effective concentrations, they suppress dynamic instability by stabilizing the plus ends of microtubules [90]. Taxol (paclitaxel) has had some success in the treatment of ovarian, breast, lung, head and neck cancers (reviewed in [91]). It stabilizes microtubules, and at high concentrations results in microtubule bundles. Taxol binds to the tubulin heterodimer at a site distinct from GTP, colchicine, and the vinca alkaloids. Crystal structures of the taxol-tubulin complex reveal 18 that taxol binding causes conformational changes that stabilize lateral contacts between subunits [92]. Destabilizing agents such as the vinca alkaloids, colchicine, and nocodazole, inhibit polymerization of microtubules [66]. For example, nocodazole binds to tubulin heterodimers and sequesters them in the cytoplasm, lowering the free concentration of tubulin below the threshold for microtubule formation [66]. Colchicine binds covalently to tubulin between the a and 3 subunits and alters their conformation to one unfavourable for polymerization [88]. Vinca alkaloids also tend to destabilize microtubules and instead promote formation of spiral polymers [88]. An antimitotic agent that does not directly bind to tubulin has recently been discovered [70]. Monastrol inhibits the microtubule-associated motor protein hEg5 [93, 94]. hEg5 is essential for centrosome separation during spindle formation, thus monastrol-treated cells form monopolar asters and cannot complete mitosis. 1.3.4. The Spindle Checkpoint Once cells have reached metaphase, they pause for a significant length of time (30-45 min). During this interval, the cell checks the alignment of chromosomes at the midline. The failure of even a single chromosome to come into position is sufficient to activate a spindle checkpoint that delays the onset of anaphase [95]. Premature anaphase and division results in aneuploidy, which leads to cell death or in some cases cancer. Indeed, defects in checkpoint pathway genes have been found in several cancers {e.g. [96-99]). The spindle checkpoint is a complex signalling pathway (reviewed in [100, 101]). The mechanism is best understood in budding yeast, although a homologous pathway is present in 19 mammalian cells. Anaphase is activated by Espl (extra spindle poles, homologous to separin in humans). Binding of Pdsl (homologous to securin in humans) sequesters and inhibits Espl. During a normal transition from metaphase to anaphase, the APC (anaphase promoting complex) E3 ubiquitin ligase and its co-activator cdc20 become activated and target Pdsl/securin for degradation. This allows Espl/separin to become activated and promote anaphase entry. This process is inhibited in response to spindle damage or the presence of an unattached kinetochore (Fig 1.5) [101-103]. The activating signal originates from kinetochores on mislocalized chromosomes [95]. It is not clear what kinetochore signal is detected; it may be reduced attachment of microtubules, a lack of tension across the kinetochore, or perhaps both. Seven genes are essential for the checkpoint pathway in S. cerevisiae. MPS1, MAD1-3 (mitotic arrest deficient), and BUB1-3 (budding uninhibited by benzimidazole). All of these proteins localize to kinetochores during prometaphase and metaphase. The net result of checkpoint activation is inhibition of cdc20. How does cdc20 become inhibited? Early studies showed that Mad2 binds to cdc20 when the APC is inhibited, suggesting that Mad2 becomes modified at the kinetochore, enabling its inhibition of the APC. Recently, it has been shown in human cells that the kinetochore kinase hBubRl (a homologue of Bubl) also binds to the APC and potently inhibits its activity [104-106]. These findings are especially intriguing because hBubRl binds to the kinetochore motor protein CENP-E [107], raising the possibility that CENP-E senses kinetochore tension and transmits this information to the APC through hBubRl [104-106, 108]. 20 Mad2| ( M a d ? ) ANAPHASE Fig 1.5 Inhibition of the metaphase to anaphase transition by the spindle checkpoint pathway in budding yeast. Anaphase is activated by Esp1, which is normally sequestered and inhibited by Pds1. During a normal exit from metaphase, Pds1 is targeted for degradation by the APC (anaphase promoting complex) and its co-activator cdc20. During prometaphase or in response to spindle damage or an unattached kinetochore, Bub1, Bub3, Mad 1-3 and Mps1 load onto the kinetochore and delay anaphase onset. This may be achieved in part through inhibitory effects of Mad2 on cdc20, preventing activation of the APC. Adapted from [100-103]. 21 1.4. G 2 Checkpoint Inhibitors 1.4.1. Rationale for the Use of G 2 Checkpoint Inhibitors in Cancer Treatment Protection against DNA damage is crucial to the cell. One sign of this is the sensitivity of the cell to this type of injury: a single double-stranded break is sufficient to arrest the cell cycle [109]. Extensive unrepaired DNA damage is dangerous to the cell: if lesions are not repaired and become fixed in the genome, the outcome for the cell is typically death by senescence, apoptosis, or mitotic catastrophe [110]. The mutation rate is much higher in cancers than in normal cells [111], and one likely mechanism for creating genetic heterogeneity is the elimination of the DNA damage checkpoint [112]. Loss of checkpoint function predisposes cells to genomic instability, permitting the substantial translocations, mutations, and aneuploidy observed in cancer [18, 113]. It is estimated that 50 - 70 % of cancer cells lack p53 function [114, 115]. While the realization that many cancers have defective DNA damage checkpoints provides a partial understanding of mechanisms underlying carcinogenesis, it also offers a potential way to target cancer cells for specific treatment [18]. As described in section 1.2.4., p53 activity is essential for arrest in G y and cells that are deficient for p53 arrest only in G 2 after irradiation [64]. If irradiation-induced arrest in G 2 could also be prevented by chemical inhibitors, irradiated cancer cells would be crippled: without a mechanism for arrest and damage repair before entering mitosis, they will eventually die [116, 117]. What is especially attractive about this strategy is that the anticipated effects of checkpoint inhibitors towards cells with wild-type p53 are minimal. This could be the most important benefit of checkpoint inhibitors as cancer therapy. There are dozens of ways to kill a cancer cell, the challenge is to develop one that spares normal cells. Many of the cancer treatments already 22 in use are DNA damaging agents (ionizing radiation, DNA-damaging chemotherapies). G 2 checkpoint inhibitors may sensitize cancer cells to these agents, lowering the dose of radiation or chemotherapy necessary and thus reducing the toxicity burden to the patient. 1.4.2. Current G2 Checkpoint Inhibitors Over the years, several inhibitors of the G 2 checkpoint have come to light. As might be expected, considering the importance of phosphorylation to the checkpoint pathway, these block either protein kinases or phosphatases (Table 1.2). The inhibitors that have been characterized to some extent in the literature are described below. New inhibitors found in our laboratory, namely 13-hydroxy-15-oxozoapatlin (OZ), 6,^-kaur-16-en-15-oxo-18-oic acid (OKA), debromohymenialdisine (DBH), and isogranulatimide (IGR) are introduced later in the thesis. Caffeine was the first G 2 checkpoint inhibitor to be described. At the time, the importance of cAM P-dependent signalling pathways was emerging, and caffeine is an inhibitor of cAMP phosphodiesterases [118]. Prevention of mitotic delay after ionizing irradiation by caffeine has since been confirmed in many studies (e.g. [118-121]), and caffeine enhances the toxicity of radiation (e.g. [122-124]). This radiosensitizing effect is selective for cells that lack p53 function (e.g. [125-127]). Caffeine inhibits ATM kinase, and this is likely to account for its effects on the checkpoint [128]. Pentoxifylline is a derivative of caffeine that causes a synergistic increase in cell killing by DNA damage in breast, lung, squamous cell, or melanoma cancer cell lines that lack p53 function [129-131]. Pentoxifylline is already approved for clinical use in the treatment of circulatory problems, and has been applied in Phase II and III clinical trials as a 23 Table 1.2 Inhibitors of the G2 DNA damage checkpoint. Listed beloware some of the currently known checkpoint inhibitors. Not included in the list are the checkpoint inhibitors DBH, IGR, OZ, and OKA, discovered by our laboratory and described in detail in later chapters. For each inhibitor, the putative or identified target is indicated. Checkpoint Inhibitor: Targets: Reference: Protein Kinase Inhibitors: staurosporine many kinases, some P K C specificity 137 UCN-01 Chkl kinase 138 SB-218078 Chkl kinase 139 ICP-1 Chkl kinase, Chk2 kinase 136 caffeine diverse targets, including A T M kinase 128 PD0166285 Weel kinase, Myt l kinase 147 Protein Phosphatase Inhibitors: okadaic acid PP1 andPP2A 144 fostriecin PP1 andPP2A 146 24 radiosensitizing agent for cancer treatment, with limited success [132, 133]. 2-aminopurine is a protein kinase inhibitor that also blocks G 2 arrest after DNA damage [121, 134, 135]. Staurosporine is a broad-based kinase inhibitor, with some degree of specificity towards PKC. Staurosporine and the structurally related compounds UCN-01, SB-218078, and ICP-1 prevent arrest in G 2 after ionizing irradiation [136-139]. UCN-01 and SB-218078 sensitize cells to DNA damaging agents [138, 139], and in the cases of UCN-01 and ICP-1 the decreases in survival were shown to be p53-selective [136, 138]. UCN-01, SB-218078 and ICP-1 are in vitro inhibitors of Chkl kinase [128, 136, 139, 140], although actions on other kinases are not excluded [50, 139]. Inhibition of Chkl could enable cdc25C activation and also inactivate Weel kinase, releasing cdkl from inhibition [141]. Crystal structures of Chkl in complex with UCN-01, staurosporine or SB-218078 revealed that these three checkpoint inhibitors all bind to the ATP-binding site of the kinase [142]. UCN-01 has recently completed Phase 1 clinical trials [143]. It bound very tightly to a plasma protein, reducing the free concentration of the drug, extending the drug clearance time, and resulting in complex pharmacokinetics. The most common side effects of UCN-01 were hyperglycemia and nausea. Phosphatase inhibitors are also known to inhibit G 2 arrest, reflecting the important role that regulation of phosphorylation plays in the checkpoint pathway. Okadaic acid and fostriecin, inhibitors of types 1 and 2A protein phosphatases, release cells from arrest in G 2 [144-146]. The effectiveness of G 2 checkpoint inhibitors has been proven in vitro, where they are radiosensitizing agents and their effects are selective against mammalian cells that lack p53 25 function {e.g. [147, 148]). However, there are several hurdles to overcome before an in vitro activity is useful to patients. In some cases, the relatively weak activity of checkpoint inhibitors means that checkpoint effects would probably not be achieved at the maximum dose tolerable for patients. For example, caffeine has a number of other cellular targets, and its effective dose would be too toxic. Distribution of U CN-01 to the plasma compartment and its effects on other kinases could affect its applicability in the clinic. For these reasons, it is clear that the potential therapeutic benefit of G 2 checkpoint inhibitors has not yet been fully achieved, and a search for new checkpoint inhibitors may yield more promising compounds. 1.5. Research Objectives In this thesis, I explore the effects and mechanisms of action of two classes of compounds identified by our laboratory: isogranulatimide (IG R) and a family of compounds including 13-hydroxy-15-oxozoapatlin (OZ) and e/7M<aur-16-en-15-oxo-18-oic acid (OKA). In all cases, these agents prevented arrest in G 2 after irradiation, suggesting their potential utility as biochemical tools for the study of the checkpoint pathway. The two main objectives of my research were: i) to characterize the effects of these compounds on the cell cycle and ii) to identify their molecular targets, if possible. I explored the effects of these inhibitors on the cell cycle in irradiated and non-irradiated cells using immunofluorescence microscopy and flow cytometry. In the course of this work, I applied a number of approaches for identifying molecular targets. In the case of IG R, prior evidence had suggested G S K-3 (3 kinase as a candidate target. I transfected cells with this kinase and examined the resulting effects on the checkpoint pathway in immunofluorescence microscopy and flow cytometry. For my studies on OZ and the related compound OKA, I developed target labelling methods and 26 purification strategies for isolation of potential targets. I chose a single candidate, RanBP2, for subsequent testing by secondary methods. This research contributes to an understanding of the mechanisms of action of the novel checkpoint inhibitors IG R, OZ and OKA. 27 Chapter 2 DISCOVERY OF CHECKPOINT INHIBITING AND ANTIMITOTIC ACTIVITIES OF 13-HYDROXY-15-OXOZOAPATLIN (OZ) 2.1. SUMMARY Using a cell-based assay for G2 checkpoint inhibitors, we have screened extracts from the N.C.I. National Institutes of Health Natural Products Repository and have identified 13-hydroxy-15-oxozoapatlin (OZ) from the African tree Parinari curatellifolia. Flow cytometry with a mitosis-specific antibody showed that checkpoint inhibition by OZ xoas maximal at 10 /uM, which released 20 % of irradiated MCF-7 mp5 3 cells, and 30 % of irradiated HCT116-/-cellsfrom G2 arrest. OZ additively increased the response to the checkpoint inhibitors isogranulatimide and debromohymenialdesine, but it did not enhance the effects of UCN-01 or caffeine. Unlike other checkpoint inhibitors, OZ did not inhibit ATM, ATR, Chkl, Chkl, Plkl or Ser/Thr protein phosphatases in vitro. Treatment with OZ also caused G2-arrested and cycling cells to arrest in mitosis in a state resembling prometaphase. In these cells, the chromosomes were condensed and scattered over disordered mitotic spindles. These results demonstrate that OZ is both a G2 checkpoint inhibitor and an antimitotic agent. 2.2. INTRODUCTION In response to DNA damage, cell cycle progression pauses to allow time for DNA repair. This checkpoint response helps to maintain the integrity of the genome, and loss of checkpoints may enable the accumulation of mutations during carcinogenesis [18, 112]. Cell 28 cycle arrest is achieved by inhibiting the activities of cyclin-dependent kinases that govern entry into S phase or mitosis. In Q>1 phase, DNA damage leads to stabilization and nuclear localization of the p53 transcription factor, resulting in up-regulation of p 2 i W a f l / C i p l / an inhibitor of G : cyclin-dependent kinase activities [22, 149-151]. The G 2 checkpoint operates through a different mechanism, and the details of this pathway are beginning to emerge [23]. In the current model, regulation is exerted through a phosphorylation cascade and by control of the subcellular localization of enzymes and their substrates. ATM and ATR kinases play a role in the early signalling of DNA damage, and phosphorylate Chkl and Chl<2 kinases [38, 39, 43, 152, 153]. Phosphorylation of the phosphatase Cdc25C by Chkl and Chl<2 creates a 14-3-3 binding site and inhibits its phosphatase activity in vitro, preventing its activation of Cdc2 kinase, the master regulator of mitosis [44, 52, 154]. Cdc2 activity is also regulated by inhibitory phosphorylation by Weel and Myt l kinases [54, 55]. Inhibitors of the G 2 checkpoint such as caffeine, pentoxifylline, staurosporine and UCN-01 have been studied extensively in tissue culture model systems [121, 130, 135, 137, 138]. More recently, we have identified isogranulatimide (IGR) and debromohymenialdesine (DBH) as checkpoint inhibitors from marine invertebrate extracts [155, 156]. In vitro, G 2 checkpoint inhibitors can sensitize cancer cells lacking p53 function, which have a defective G : checkpoint, to the effects of DNA-damaging cancer chemotherapeutic agents and ionizing radiation [125-127, 129, 138, 155]. This suggests that these inhibitors are of potential benefit in the treatment of cancer, and clinical trials of UCN-01 are underway [143]. To find new inhibitors of the G 2 checkpoint, we have screened plant extracts from the National Cancer Institute (N. C. I.) Natural Products Repository. From this screen, we have identified 13-hydroxy-15-oxozoapatlin (OZ) and describe its activity as a checkpoint inhibitor and as 29 an an t im i to t i c agent. 2.3. EXPERIMENTAL PROCEDURES 2.3.1. Isolation and Identification of 13-hydroxy-15-oxozoapatlin A methanol ex t rac t of Parinan curatellifolia bark f r o m the N.C. I , was in i t ia l l y f rac t ionated by a modi f ied Kupchan pa r t i t i on ing scheme (Dr. L. X u , labora tory of Dr. R. J . Andersen, Departments of Chemistry and Oceanography-Ear th and Ocean Sciences) [ 1 5 7 ] . The ex t rac t was dissolved in 5 0 0 ml of M e O H / H 2 0 (1:4) and sequent ial ly ex t rac ted w i t h hexane (3 x 2 0 0 ml ) , ch lo ro fo rm (3 x 2 0 0 m l ) , and ethyl acetate (3 x 2 0 0 m l ) . G 2 checkpoint inh ib i t ion assays were per formed on the hexane and ch lo ro fo rm soluble f rac t ions as described previously [ 1 5 5 ] . Br ie f ly , M C F - 7 cells lacking p53 funct ion ( M C F - 7 m p 5 3 ) were arrested in G 2 phase by ioniz ing i r r a d i a t i o n . T rea tmen t w i t h a checkpoint inh ib i t ing f rac t i on and nocodazole caused cells to escape G 2 a r res t and b lock in mi tos is . M i t o t i c cells were then detected by enzyme-l inked immunocy tochemica l assay ( E L I C A ) using an ant ibody t h a t recognizes a phosphorylated epitope in mi tosis (TG-3) . The ch lo ro fo rm f rac t ion was subjected to si l ica-gel f lash ch romatography (eluent: ethyl acetate: hexane, 2 0 : 8 0 ) to y ie ld two compounds. Thei r structures were determined by  1H and 1 3 C N M R and mass spect rometry and found to be identical w i t h publ ished values f o r 13-hydroxy-15-oxozoapat l in and an analogue tha t di f fers by the add i t ion of two hydrogen atoms [ 1 5 8 , 1 5 9 ] . The f i r s t compound demonstrated higher act iv i ty and was chosen f o r fu r the r study. 30 2.3.2. Indirect Immunofluorescence Labelling of Mitotic Cells for Flow Cytometry DNA and mitotic epitopes were labelled by modification of the Braylan procedure for dual labelling of DNA and cell surface markers [160, 161]. Cells were collected by trypsinization, washed in standard azide buffer ((SAB), PBS containing 1 % fetal calf serum (FCS) and 0.1 % sodium azide), fixed in 70 % ethanol, and stored at 4 °C overnight. Cells were then washed twice in Tw-SAB (SAB containing 0.5 % Tween 20), incubated in the same buffer for 30 min on ice to block non-specific binding sites, and then re-suspended at a concentration of 10 x lOVml in Tw-SAB containing 5 % additional FCS. Mitotic epitopes were labelled by incubation with TG-3 (recognizes phosphorylated nucleolin) or GF-7 antibodies (recognizes uncharacterized mitotic phosphoepitopes) (1:100 dilution in Tw-SAB; generously provided by Dr. P. Davies, Albert Einstein College of Medicine, Bronx, NY) on ice for 30-90 min [162]. After washing twice in Tw-SAB, cells were re-suspended as for primary antibody labelling and incubated with FITC-conjugated goat anti-mouse (IgG + IgM, H + L) secondary antibody (Pierce, diluted 1:150 in Tw-SAB) for 30 min on ice in darkness. Cells were then washed twice in Tw-SAB and treated with RNase A (Roche Diagnostics, 500 U/ml in 4 mM sodium citrate buffer, pH 8.4) for 30 min at 37 °C. To label DNA, an equal volume of 50 |Jg/ml propidium iodide (PI) in sodium citrate buffer was added and the incubation was continued for 20 min. Cells were finally re-suspended at a concentration of 1 x 10 6 /ml in dilute PI solution (25 u.g/ml in sodium citrate buffer) and stored in darkness at 4 °C overnight. 2.3.3. Flow Cytometry and Data Analysis Cells fixed and labelled as described above were analyzed in a Becton-Dickinson FACScan with standard laser and filter configurations. Data was collected for a minimum of 20,000 Pl-triggered events on the low flow rate setting (approximately 100 events/s). The 31 parameters measured were: forward and side scatters; FITC intensity; PI intensity; PI pulse area and width. The FITC signal was compensated for PI emission overlap and recorded in 4-decade logarithmic mode. All other parameters were measured in linear mode. The data were saved in listmode format and analyzed in WinMDI freeware. Clumps and debris were categorized on the basis of DNA pulse width and scatter measurements, respectively, and excluded from the data analysis. 2.3.4. Inhibition of the G2 DNA Damage Checkpoint MCF-7 cells expressing a dominant negative mutant p53 (mp53) or HCT116 cells in which the p53 gene was removed by targeted deletion (HCT116-/-) were seeded into 10 cm 2 dishes (2.2 x 10 6 cells/dish). MCF-7 mp53 or HCT116-/-cells were irradiated the next day with 10 or 12 Gy, respectively, from a 6 0 Co source (Gammacell 200, Atomic Energy Commission of Canada). Cells were subsequently cultured for 16 h to allow the population to fully arrest in G 2 [155]. They were then exposed for 8 h to checkpoint inhibitors and/or nocodazole at 100 or 300 ng/ml, respectively, to block cells in mitosis. To assess the degree of G 2 checkpoint inhibition, mitotic cells were then detected by flow cytometry using the mitosis-specific antibody G F-7. 2.3.5. Immunofluorescence Microscopy Cells were seeded onto poly-L-lysine-coated cover slips (225,000 cells in 1.5 ml) in 6-well tissue culture plates. On the following day, cells were treated with DMSO or OZ (10 UM, 8 h). After fixation in formaldehyde (3.7 % in TBS, 30 min), the cover slips were washed twice in TBS. Cells were permeabilized in blocking solution (1% BSA in TBS) containing 0.1 % 32 Triton X-100 and then incubated for 1 h with E7 R-tubulin monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa), diluted l : 2 0 i n blocking solution. After washing twice in TBS, cells were incubated with Alexa 488-conjugated secondary antibody (Molecular Probes; diluted 1:500 in blocking solution) for 50 min. Cover slips were washed twice in sodium citrate buffer (40 m M , pH 8.4) and incubated with RNase A (Roche Diagnostics, 500 U/ml) in citrate buffer for 1 h at 37 °C. After washing twice in TBS, DNA was stained by incubation with T0T0-3 (Molecular Probes, 2 U.M) for 20 min in darkness. After washing twice in TBS, the cover slips were mounted in 90 % glycerol in PBS containing 0.2 M n-propyl gallate. Cells were visualized with a BioRad Radiance 2000 confocal on a Zeiss Axiovert S100TV microscope with a 63 X oil immersion objective lens (N. A. 1.4). Samples were excited with 488 nm and 638 nm light from argon and red diode lasers. The emission filters were HQ515/30 and HQ660L, respectively. Red and green signals were collected sequentially to avoid bleedthrough, in sections spaced 0.15 (Jm apart. Images were projected using NIH Image and Adobe Photoshop software. 2.3.6. Cell Proliferation Assays Inhibition of cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay described previously [156 ] . These experiments were performed by Lianne McHardy and Michel Roberge. 2.3.7. Chemical Modification of OZ OZ was modified by reaction with (3-mercaptoethanol to form a 13-hydroxy-15-oxozoapatlin-R-mercaptoethanol adduct according to published methods [158 ] . A 20 mM 33 solution of OZ in tetrahydrofuran was prepared. 150 nmoles of OZ was mixed with a five-fold molar excess of (3-mercaptoethanol in 1 ml reaction buffer (0.2 mM cacodylate, pH 7.4). Reaction progress was followed by monitoring absorbance at 230 nm. 2.3.8. Radiolabelling of 13-[3H]methyl-15-oxozoapatlin-Binding Proteins 13-C3H]methyl-15-oxozoapatlin was prepared by Dr. M. Roberge. 15-oxozoapatlin (100 nmoles) was dissolved in anhydrous tetrahydrofuran containing IMaH. An equimolar amount of [3H]methyl iodide in toluene was added (7.5 mCi total, specific activity of 85 Ci/mmol, Amersham). After stirring the reaction overnight at room temperature, the toluene layer was collected and concentrated by evaporation. Incorporation of [ 3H] was verified by thin layer chromatography and liquid scintillation counting. MCF-7 mp53 cells were seeded into 10 cm 2 dishes and irradiated (0 or 10 Gy) (Section 2,3.4.). Cell pellets were collected by trypsinization, washed twice in PBS containing 0.2 mM PMSF and flash-frozen. After incubating cell pellets in lysis buffer (20 mM MOPS, pH 7.2, 50 mM glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, trace okadaic acid, 5 mM EGTA, 2 mM EDTA, 1 ug/ml aprotinin, 1 ug/ml leupeptin, 1 mM PMSF) for 30 min on ice, lysates were centrifuged for 30 min (4 °C, 13,000 g). Cellular proteins were labelled with 13-[3H]methyl-15-oxozoapatlin using two different methods. In the first, after soluble cellular proteins (10 ul, 100 ug) were incubated with 13-[3H]methyl-15-oxozoapatlin (2 ul) for 25 min, the labelling reaction was terminated by addition of Laemmli sample buffer (50 mM Tris-CI, pH 6.8, 100 mM DTT, 2 % SDS, 0.1 % bromophenol blue, 10 % glycerol). Proteins were separated by SDS-PAG E and fixed in the gel by incubation with de-stain solution (40 % methanol, 10 % glacial acetic acid). 34 In the second method, proteins separated by SDS-PAGE were refolded before incubation with 13-C 3H]methyl-15-oxozoapatlin. Briefly, gels were incubated in three changes (20 min each) of the following three buffers: wash buffer (0.5 M Tris, pH 8.0, 20 % isopropanol), followed by denaturing buffer (50 mM Tris, pH 8.0, 6 M guanidine hydrochloride), and re-naturing buffer (50 mM Tris, pH 8.0, 5 mM (3-mercaptoethanol, 0.04 % Tween 20). Gels prepared using either method were incubated with EI\1[3H]AI\ICE for 30 min. Dried gels were exposed to f i lm for 1 month at -80° C. 2.4. RESULTS 2.4.1. Isolation and Identification of 13-hydroxy-15-oxozoapatlin (OZ) as a G2 Checkpoint Inhibitor A cell-based assay for G 2 checkpoint inhibitors was used to screen extracts from the N.C.I. Natural Products Repository [155 ] . Bark extract from the South African tree Parinari curatellifolia contained a checkpoint-inhibiting activity that was purified by chromatographic techniques (Experimental Procedures). By analysis of its mass spectrometry and NMR data, the active compound was identified as the previously described ewf-kaurene diterpenoid 13-hydroxy-15-oxozoapatlin [158 ] (Fig. 2.1). 2.4.2. Effects of OZ on Cell Cycle Progression OZ was originally identified as a compound with broad-spectrum cytotoxic activity towards human cancer cell lines and causing cell cycle arrest in G 2 /M [ 1 5 8 ] . For this reason, I f irst examined the effects of OZ on the cell cycle using flow cytometry. Treatment of exponentially growing MCF-7 mp53 cells with 10 |JM OZ for 8 h decreased the proportion of G : phase cells from 33 % to 19 ± 2 % (mean ± S. D., n = 2), caused little 35 Figure 2.1 13-hydroxy-15-oxozoapatlin (OZ) 36 or no change in the number of S phase cells, and increased the proportion of G 2 /M cells from 35 ± 4 % to 52 ± 1 % (Fig. 2.2 A). To determine whether the increase occurred in G 2 or M phase, cells were also labelled with the mitosis-specific monoclonal antibody GF-7 (Fig. 2.2 B). This revealed that the main effect of the compound was an increase in the number of mitotic cells from 3 % to 14 ± 4 %. Similar results were observed in HCT116-/- cells (not shown). The increase in mitotic cell numbers after OZ treatment was explored by treating non-irradiated MCF-7 mp53 cells with 10 UM OZ for up to 12 h (Fig.2.3). The number of cells in mitosis steadily increased over this time period, indicating that a proportion of the cycling cells became arrested in mitosis as a consequence of OZ treatment. I next studied the effects of the compound on cells arrested at the G 2 checkpoint by ionizing radiation (Fig. 2.2 C, D). Consistent with earlier work [155], treatment with 10 Gy caused about 90 % of the cells to arrest in G 2 phase by 16 h after irradiation (88 ± 2 %, n = 5). Cells that did not receive further treatment remained arrested in G 2 during the next 8 h. Treatment with 10 UM OZ at 16 h after irradiation caused 21 % of the cells to escape G 2 . The cells released from G 2 arrest did not progress into Q>1 but became blocked in mitosis. That the cells were indeed in mitosis was confirmed by counting mitotic figures under the microscope (data not shown). Taken together, these results show that OZ inhibits the G 2 checkpoint and. also arrests cells in mitosis. The effects of OZ on cells arrested in G 2 were also examined in an enzyme-linked immunocytochemical assay (ELICA) with a second mitosis-specific monoclonal antibody, TG-3 [155] (Fig. 2.4). 16 h after irradiation, MCF-7 mp53 cells were treated with a range of OZ concentrations for 8 h, and entry into mitosis evaluated by ELICA assay. Checkpoint 37 A Non-Irradiated no drug OZ > I G 2 + M > I M I y "LLI 1023 DNA (PI) DNA (PI) G Irradiated no drug OZ CO c > L U c > 111 DNA (PI) DNA (PI) B o 1^ -I LL O Non-Irradiated no drug o OZ 1 £01 (Oil IM LL S • CD © J DNA (PI) no drug Irradiated DNA (PI) OZ O | Is Sb 2 1 1 • LL 2 CD LL 2 CD o © DNA (PI) DNA (PI) Fig. 2.2 G2 checkpoint inhibition and mitotic arrest induced by OZ. The effects of OZ were tested on non-irradiated MCF-7 mp53 cells (A, 8) and cells irradiated with 10 Gy ( C D ) . 16 h after irradiation, cells were cultured in the presence of 10 JJ.M OZ or DMSO for 8 h. DNA was stained with PI and mitotic cells were detected by FITC-GF-7 immunofluorescence labelling (Experimental Procedures). A and C, his-tograms of the DNA profiles. 8 and D, the mitotic FITC-GF-7 fluorescence plotted against the DNA PI signal. The number of events associated with a coordinate on the plot is indicated by the colour scale (blue indicating low density and red indi-cating high density). 38 0 3 6 9 12 Time (h) Fig. 2.3 OZ treatment causes a progressive increase in mitotic cell numbers. The effects of O Z (10 u.M) t reatment o n entry of n o n -i r radiated M C F - 7 m p 5 3 cells into mitosis was eva luated over a 12 h t ime pe r iod . After the indicated incubat ion p e r i o d , cells were label led with PI a n d the mitosis-specif ic an t ibody G F - 7 (Experimental Procedures). Mi to t ic cells were counted in f low cytometry a n d are expressed as percentages of the total number of cells. Results are typical fo r two separate experiments. 39 0.5 H 0 0.1 1 10 100 [OZ] (u.M) Fig. 2.4 OZ inhibits the G2 DNA damage checkpoint. 16 h after irradiation and addition of nocodazole (100 ng/ml), G 2-arrested MCF-7 mp53 cells were treated with the indicated concentrations of O Z for 8 h. Entry into mitosis was detected in an enzyme-linked immunocytochemical assay with the mitosis-specific antibody TG-3, and is indicated by an increase in A 4 0 5 [155]. Results are means ± S.D. for triplicate assays. 40 inhibiting activity was observed between 1 and 20 |JM, with an apparent peak at 6 |JM. Next, irradiated MCF-7 mp53 or HCTI 16 -/- cells were treated with a range of OZ concentrations and analyzed by flow cytometry as described above (Fig. 2.5). In agreement with earlier results (Fig. 2.2 C, D), at 10 U.M the compound inhibited G 2 arrest after irradiation in approximately 20 % of MCF-7 mp53 and 30 % of HCTI 16-/- cells (17 ± 1 %, n = 3 and 32 ± 4 %, n = 2, respectively). Entry into mitosis was strongly reduced at higher concentrations of OZ). 2.4.3. Effects of OZ on Mitotic Morphology I investigated the mitotic arrest caused by OZ by examining its effects on chromosome condensation and mitotic spindle formation, two morphological hallmarks of mitosis. Cycling MCF-7 wtp53 cells were treated with 10 |JM OZ for 8 h and immunostained with a P-tubulin monoclonal antibody to examine the mitotic spindle and counterstained with the DNA dye T0T0-3 to examine the chromosomes (Fig. 2.6). The nuclei and microtubules of interphase OZ-treated cells were indistinguishable from the no-drug controls. However, after OZ treatment, no prophase, anaphase or telophase cells were observed. Al l mitotic cells had a similar and atypical appearance in which the spindle poles were separated but the overall appearance of the spindles was disorganized in comparison to controls. The chromosomes resembled those in normal metaphase, except that they had failed to align at the spindle equator. Dual-labelling images showed that the chromosomes co-localized with the spindles, implying that the chromosomes were in contact with the spindle apparatus. The overall appearance of the OZ-treated mitotic cells suggests that they were arrested in a stage resembling prometaphase. Similar effects were observed in MCF-7 mp53, HCT116-/- and 41 [OZ] (,iM) Fig. 2.5 Inhibition of G2 arrest in MCF-7 mp53 and HCT116-/- cells by OZ. 1 6 h a f ter i r r a d i a t i o n a n d n o c o d a z o l e t r e a t m e n t , G 2 - a r r e s t e d cel ls w e r e c u l t u r e d in the pres-e n c e o f the i n d i c a t e d c o n c e n t r a t i o n s o f O Z f o r 8 h. A f te r F I T C - G F - 7 a n d PI d u a l l a b e l l i n g , m i to t i c cel ls w e r e c o u n t e d in f l ow cy tomet ry (Exper imenta l Procedures) . M i t o t i c cel l c o u n t s f o r H C T 1 1 6-1- (hollow triangles) a n d M C F - 7 m p 5 3 (so//d tri-angles) a r e expressed as p e r c e n t a g e s o f the to ta l n u m b e r o f ce l ls . D a t a f o r M C F -7 m p 5 3 a n d H C T 1 1 6 - / - cel ls w e r e co l l ec ted separate ly , a n d in e a c h case a re typ ica l f o r t w o s e p a r a t e exper imen ts . 42 P-tubulin DNA Merge Fig. 2.6 OZ-treated MCF-7 wtp53 cells arrest in a stage resembling prometaphase. Mitotic spindles and chromosomes were examined by immunofluorescence microscopy after an 8 h treatment with DMSO (top) or OZ (1 0 u M , bottom). Spindles were labelled with P-tubulin and goat-anti-mouse-Alexa 488 second-ary antibodies, and DNA was stained with TOTO-3. The P-tubulin (left) and DNA (middle) signals were visualized in confocal microscopy (Experimental Pro-cedures). Bar, 1 0 um. 43 H CT116+/+ cells, and were unchanged by prior irradiation of cells (not shown). 2.4.4. Interaction Between OZ and Other Checkpoint Inhibitors Flow cytometry had shown that OZ inhibited the G 2 checkpoint in only 15-20 % of G 2 -arrested MCF-7 mp53 cells. I next asked whether the extent of checkpoint inhibition could be increased by combining OZ with other checkpoint inhibitors. G2-arrested MCF-7 mp53 cells were treated for 8 h with 10 UM OZ and/or optimal checkpoint-inhibiting concentrations of caffeine (2 mM), UCN-01 (100 nM), debromohymenialdesine (DBH, 40 UM), or isogranulatimide (IG R, 20 UM), in the presence of nocodazole to arrest cells in mitosis. Cells were labelled with GF-7-FITC and PI, and mitotic cells were counted by flow cytometry (Fig. 2.7). UCN-01 and caffeine alone released 59 ± 8 % and 65 ± 13 % (means ± S. D., n = 2) of cells, respectively, from G2arrest. The effects of caffeine and UCN-01 were not increased by co-incubation with OZ. IGR and DBH alone induced a more moderate response, releasing 23 ± 3 % and 30 ± 1 % of cells, respectively. However, this response was increased to 43 ± 3 % and 52 ± 4 %, respectively, when OZ was added, almost reaching the levels attained by caffeine or UCN-01. Therefore, the effects of OZ were additive with those of IGR or DBH. 2.4.5. Inhibition of Cell Proliferation by OZ One measure of the therapeutic potential of checkpoint inhibitors is their ability to increase cell death after irradiation. To test whether OZ has this property, we first examined its effects on proliferation in non-irradiated cells. MCF-7 mp53 cells were incubated with different concentrations of OZ for 24 h, the compound was washed away and cell 44 80 A NoOZ O Z ( 1 0 L I M ) Fig. 2.7 OZ increases checkpoint inhibition by IGR and DBH, but not caffeine or UCN-01. G 2 -a r res ted i r radiated M C F - 7 m p 5 3 cells were treated for 8 h with nocodazo le (100 ng /ml ) and G 2 checkpoint inhibi tors, in the presence of D M S O (left) o r O Z (right, 1 0 uM). Checkpo in t inhibitors were at the fo l lowing concent ra t ions: white, no other d r u g ; horizontal stripes, IGR (20 | l M ) ; grey, DBH (40 uM); cross-hatch, UCN-01 (100 n M ) ; black, caffeine (2 m M ) . Mi to t ic cells were counted in f low cytometry after FITC-GF -7 a n d PI dual labe l l ing , a n d are expressed as percentages of the total number of cells for each t reatment (Experimental Procedures). Results are the averaged means f rom two separate experiments ± S. D.. 45 proliferation was measured 2 days later using the MTT assay (Lianne McHardy, laboratory of Dr. M. Roberge) [156]. OZ was a potent inhibitor of cell proliferation, with an IC 5 0 of 0.8 UM and IC 9 0 of 2 UM (data not shown). Cells were next treated with ionizing radiation followed immediately by a range of OZ concentrations for 24 h and proliferation was assessed as above. OZ did not kill irradiated cells more potently than non-irradiated cells (data not shown). Considering that a combination of OZ with DBH showed enhanced G 2 checkpoint inhibiting activity, we investigated whether inhibition of cell proliferation was also enhanced by this combination (Fig. 2.8). In non-irradiated cells, 30 UM DBH inhibited proliferation to 58 ± 12 % of no-drug controls, consistent with published values [156]. OZ (0.6 U.M) inhibited cell proliferation to 81 ± 11 %, and this was reduced in the presence of 30 U.M DBH to 24 ± 15 %. Similarly, in irradiated cells, treatment with DBH and OZ strongly inhibited proliferation in an additive manner. However, the survival of irradiated cells after this combination of treatments was not significantly lower than that of non-irradiated cells. 2.4.6. Mechanism of Action of OZ This compound is unusual among checkpoint inhibitors because it has an a, (3-unsaturated carbonyl group that makes it reactive to nucleophiles. As a result, OZ has the potential to covalently link to cysteine residues in proteins through Michael-type addition. I tested whether this reaction is required for G 2 checkpoint inhibition by OZ by reacting the unsaturated group with P-mercaptoethanol (Fig. 2.9). The reaction product did not retain any G 2 checkpoint inhibitory activity (Fig. 2.10). This lack of effect was not likely due to any interference exerted by unreacted P-mercaptoethanol in the reaction mixture, as an 46 3 1 H 1 1 1 1 1 1 — i 1—;—i 1 0 3 6 9 Ionizing Radiation Fig. 2.8 OZ enhances the effects of DBH on radiation-induced cell killing. The effects of O Z a n d DBH on cell survival were examined over a range of rad ia t ion doses. M C F - 7 m p 5 3 cells were g iven O Z (0.6 U.M, white circles), DBH (30 uM, b/ack circles), o r O Z + DBH [black squares) at the t ime of i r rad ia t ion. Cel l pro l i ferat ion was evaluated using the MTT assay after 2-4 d incubat ion with the drugs (Experimental Procedures). Results are means ± S. D. fo r quadrup l i ca te assays, a n d are normal ized to the no -d rug controls f o r each rad ia t ion dose. Data courtesy of Lianne McHardy . 4 7 equivalent amount of G-mercaptoethanol does not block checkpoint inhibition by caffeine (L. McHardy, personal communication). In the next investigation, we tested OZ for activity against known checkpoint proteins. In vitro, OZ did not inhibit the checkpoint kinases Chk l , Chl<2, P l k l , ATM and ATR (Dr. S. Lees-Miller, Dr. T. Holstrom, Dr. P. R. Clarke, and Dr. E. Nigg, personal communications). Furthermore, it did not inhibit the activities of purified PP1 and PP2A or the activity of protein Ser/Thr phosphatases present in MCF-7 cell extracts (Rebecca Osborne, data not shown), suggesting that OZ inhibits the G 2 checkpoint by a mechanism distinct from that of other checkpoint inhibitors. 2.4.7. 13-[3H]methyl-15-oxozoapatlin Labelling of Protein Targets Next, I performed labelling experiments to identify protein targets of OZ. Init ial attempts to attach a label to OZ were made by Dr. Lin Xu (laboratory of Dr. R .J . Andersen, Departments of Chemistry and Oceanography-Earth Ocean Sciences, UBC), and focussed on opening the lactone ring for attachment of a fluorochrome, but unfortunately this was not successful. One modification of 15-oxozoapatlin that had proven possible was methylation at position 13 (where a hydroxyl group is attached in OZ). The checkpoint activity of 13-methyl-15-oxozoapatlin was approximately 100-fold weaker than OZ. We prepared 13-C 3H]methyl-15-oxozoapatlin in the hope that this compound would radiolabei a target of OZ. 16 h after irradiation (0 or 10 Gy), proteins were extracted from MCF-7 mp53 cells and incubated with 13-[ 3H]methyl-15-oxozoapatl in. Radiolabeled proteins were separated by SDS-PAGE and detected by autoradiography (Fig. 2.11 A). An alternate approach was also used in an 48 2.5 0 2 4 6 Time (min) Fig. 2.9 Chemical modification of OZ. O Z was mod i f ied by react ion wi th p -mercap toe thano l accord ing to publ ished methods [ 1 5 8 ] . 1 5 0 nmoles of the c o m p o u n d were reacted with a 5- fo ld m o l a r excess of P -mercaptoethanol (solid black circles) o r buffer a lone (hollow circles) in 0 .2 m M cacodylate buffer (Experimental Procedures). M o d i f i c a t i o n of the a ,P-carbonyl g r o u p of O Z was measured by mon i to r ing absorbance at 2 3 0 n m . 49 A (1) (2) Fig. 2.10 The ct,p-unsaturated carbonyl group of OZ is required for checkpoint inhibition. (A) The a,p-unsaturated carbonyl group of OZ (1) was modified by reaction with excess P-mercaptoethanol to form the adduct product (2). (8) 16 h after irradiation (10 Gy), MCF-7 mp53 cells were treated with nocodazole (100 ng/ml) and compounds (1) or (2) (each at 5 pM). Mitosis was detected by ELICA assays, and results expressed as percentages of the positive controls (caffeine, 2 mM, 8 h). Data are the averaged means of triplicate assays from two separate experiments ± S. D. of the means. 50 A B M , C Y C Y M R Fig. 2.11 Labelling of cellular proteins with 13-[3H]methyl-15-oxozoapatlin. Proteins in MCF-7 mp53 cells were radiolabelled by incubation with [3H]-methyl-15-oxozoapatlin. 1 6 h after irradiation (0 Gy, C; 10 Gy, y), proteins were extracted and radiolabelled either before (A) or after (8) irradiation. In 8, proteins were refolded in the gel before radiolabelling (Experimental Procedures). Autoradiographs were exposed for 1 month at -80 °C. M , relative molecular mass (x 103) 51 attempt to improve the specificity of the labelling technique. Proteins extracted from irradiated or non-irradiated MCF-7 mp53 cells were separated by SDS-PAGE and refolded before incubating the gels in 13-[ 3H]methyl-15-oxozoapatl in (Fig. 2.11 B). No difference was detected between the labelling pattern of irradiated and non-irradiated cells. Unfortunately, both methods labelled numerous protein bands, indicating that the specificity of this compound is low in vitro. This 3 H labelling approach did not suggest any checkpoint or mitotic targets, and was not pursued further. 2.5. DISCUSSION 13-hydroxy-15-oxozoapatlin was f irst described in 1996 [158 ] as a compound that inhibited the proliferation of a number of human cancer cell lines and triggered an increase in the G 2 /M peak of the cell cycle profile. This effect was attributed to an arrest in G 2 phase rather than M because OZ failed to block a microtubule-dependent change in the morphology of rat astrocytes that has been used as an assay to detect antimitotic agents [163-165] . We rediscovered this compound while screening extracts from the N.C.I. Natural Products Repository for inhibitors of the G 2 checkpoint. The use of a mitosis-specific monoclonal antibody in flow cytometry allowed us to show that OZ exerts two main cell cycle effects: it inhibits the G 2 DNA damage checkpoint, and it causes mitotic arrest. As a G 2 checkpoint inhibitor, OZ acts in the low micromolar range, comparing favourably to other checkpoint inhibitors, which act in the mil l imolar range (caffeine [120, 121 , 123] , 2-aminopurine [119, 135]) , low micromolar range (DBH [ 1 5 6 ] , IGR [155] ) , or nanomolar range (okadaic acid [ 1 4 4 ] , staurosporine [137 ] , UCN-01 [138, 155]) . However, the efficacy of OZ is low. A t jts optimal concentration, it released 15-20 % of MCF-7 mp53 cells from G 2 52 arrest, compared to 30-40 % for DBH and IGR and 60-70 % for caffeine and UCN-01. The most efficacious checkpoint inhibitors found so far inhibit either DNA damage-proximal targets such as ATM for caffeine [128 ] , or multiple targets such as Chk l and Weel for UCN-01 [128, 140, 1 4 1 , 166] or Chkl and Chl<2 for DBH [ 1 5 6 ] . An attractive possibility is that OZ targets a discrete branch of the checkpoint pathway. The property of a G 2 checkpoint inhibitor that may be of value in the clinical setting is its ability to sensitize cells with mutated p53 to DNA damaging agents. OZ is toxic to non-irradiated cells at concentrations that inhibit the checkpoint. This fact, and the narrow active concentration range of the compound, make OZ unsuitable as a drug candidate. However, less toxic analogues of OZ could be synthesized. Lee eta/. (1996) found that the P-mercaptoethanol adduct of OZ was as toxic as the unreacted compound [158 ] . We found that the same reaction prevented checkpoint inhibition, indicating that the regions of OZ responsible for checkpoint inhibition and toxicity may be separable. For several reasons, the action of OZ as a checkpoint inhibitor is unusual. The G 2 checkpoint inhibitors identified to date act upon checkpoint kinases (caffeine, pentoxifylline, 2-aminopurine, DBH and UCN-01) [ 1 2 1 , 128, 134, 156] or Ser/Thr protein phosphatases (fostriecin, okadaic acid) [144, 146] . OZ is not an in vitro inhibitor of checkpoint kinases or Ser/Thr protein phosphatases. Second, the chemical structure of OZ does not resemble that of other checkpoint inhibitors. Furthermore, the requirement of an unsaturated a, P-carbonyl group for the checkpoint-inhibiting activity of OZ suggests that the drug interacts with cysteine residues on its target, a mechanism that has not been reported for checkpoint inhibitors. Agents that block cells in mitosis typically interact directly wi th tubulin, impeding the 53 formation and function of the mitotic spindle. For example, vinblastine and nocodazole cause microtubule depolymerization, while paclitaxel stabilizes microtubules (reviewed in [88] ) . In this respect, the arrest caused by OZ is unusual, because in contrast to cells treated with microtubule stabilizing or de-polymerizing agents, OZ-treated cells assembled recognizable mitotic spindles. Individually, the chromosomes of OZ-treated cells did not appear different from those in normal metaphase. However, their scattered distribution along the spindle apparatus suggests a defect in their movement to the midline. The phenotype of OZ-treated cells resembles that of X. laevisextracts lacking the chromokinesin X k i d l [85, 86] , with the exception that the chromosomes of OZ-treated cells do not appear stretched. Additionally, a mitotic kinesin is inhibited by the antimitotic agent monastrol [ 7 0 ] . However, monastrol treatment results in monopolar spindles, unlike OZ. I t is possible that OZ inhibits a spindle-associated motor protein that is required for chromosome congression. Alternatively, OZ may interfere with attachment of chromosome kinetochores to the l< fibres of the spindle and thus prevent their equatorial movement. The anti-proliterative effects of OZ occur at much lower concentrations than its antimitotic effects, suggesting that prometaphase arrest is not the main mechanism underlying cell death. We chose to screen a natural product collection because these contain diverse and complex compounds, increasing our chances of finding drugs that act in new ways to inhibit the G 2 checkpoint. This work suggests that OZ does not act through the checkpoint kinases or phosphatases known at present. We have found also that OZ-treated cells arrest in mitosis, but that unlike most antimitotic agents, OZ is not a spindle poison. Although we cannot exclude the possibility that OZ interacts with more than one target, an attractive concept is that OZ inhibits an activity that is part of the checkpoint pathway and that also plays a role 54 in chromosomal movement during mitosis. This hypothesis is currently under investigation in experiments designed to identify the target of OZ. 2.6. ACKNOWLEDGEMENTS We thank Gordon M. Cragg for access to the Open Repository program of the N.C.I., Geoff Osborne for expert advice on flow cytometry, Elaine Humphrey for help with confocal microscopy, Lianne McHardy and Cristina Bigg for technical assistance, Tim Holstrom for P l k l kinase assays, Susan Lees-Miller for ATM, ATR, and Chk l kinase assays, P. R. Clarke for Chkl and Chl<2 kinase assays, and Bert Vogelstein for HCT116 cell lines. 5 5 Chapter 3 DISCOVERY OF THE ANTIMITOTIC AND CHECKPOINT INHIBITING ACTIVITIES OF ENT-KAUR-16-EN-15-OXO-18-OIC ACID (OKA) 3.1. SUMMARY High-throughput cell-based screening detected a G2 checkpoint inhibiting activity in a Coccoloba acuminata extract in the Natural Products Repository of the N.C.I.. NMR studies identified the purified activity as ent-kaur-16-en-15-oxo-18-oic acid (abbreviated OKA), a molecule with significant structural similarity to OZ. In this chapter, the activities ofOZ and OKA are compared. OKA potently inhibited the G2 checkpoint (IC50 6 yM), and at its most effective concentration (12 /JM) released 47 ±17 % ofHCT116-/- cells from G2 arrest, compared to 25 ±13% for OZ (10 juM). In MCF-7 mp53, HCT116+/+ and HCT116-/- cells, OKA caused arrest in mitosis with condensed chromosomes scattered around a bipolar mitotic spindle. In contrast to many antimitotic agents, neither OZ nor OKA exerted striking effects on tubulin polymerization in vitro. Nevertheless, in cells arrested by either OZ or OKA treatment, chromosome movement was severely curtailed. Consequently, a possible inhibitory effect of these compounds on kinetochore or centrosome function was explored by immunofluorescence microscopy of hBubRl, CENP-E, 3F3/2, and y-tubulin. These studies revealed that OZ and OKA treatment increased CENP-E and hBubRl localization around the centrosomes. These compounds had no detectable effects on 3F3/2 or y-tubulin. While these studies did not identify any targets ofOZ or OKA, the results suggest that OZ and OKA represent two members of a new class of compounds that inhibit both the G2 DNA damage checkpoint and exit from the 56 prometaphase stage of mitosis. 3.2. INTRODUCTION Approximately two years after the checkpoint activity of OZ was identified, cell-based screening and subsequent NMR analysis identified the checkpoint activity of e/7M<aur-16-en-15-oxo-18-oic acid (OKA), a compound that is closely related in structure to OZ. Two properties of OZ make it an attractive tool for biochemical studies of the cell cycle: the prometaphase phenotype of arrested cells suggests that OZ may affect early events of mitosis, and the failure of OZ to inhibit many of the known components of the G 2 checkpoint suggests that the target of OZ may be a new checkpoint protein. The fact that OZ is both a checkpoint inhibitor and an antimitotic agent raises the possibility that a target of OZ plays a role in both of aspects of the cell cycle. For these reasons, it was important to establish whether OKA is a second compound with checkpoint and antimitotic effects. Agreement between the effects of OZ and OKA would suggest that OZ was the f irst representative of a class of related chemical compounds that share similar cell cycle effects. The existence of such a group would encourage the hope that one of these compounds may prove more tractable than OZ to modification for the identification of targets. Furthermore, comparisons between the related compounds could yield information on the structural features that are important for activity. Consequently, I began to investigate the activities of both OZ and OKA in parallel. In this chapter, several different experimental approaches were used for the study of OZ and OKA, with two major goals: a comparison of their effects, and tests for possible target identification. The effects of OZ and OKA on both the G 2 checkpoint and on mitosis were compared. As the major known checkpoint proteins had been ruled out as targets for OZ, I 57 focussed instead on possible causes of the mitotic arrest phenotype. Confocal microscopy of live cells revealed that the prometaphase arrest in OZ- and OKA-treated cells was attributable to a failure in chromosome movement. To explore whether inhibition of chromosome motion was a consequence of direct inhibition of microtubule dynamics, we tested the effects of OZ and OKA on polymerization of purified tubulin in vitro. Possible effects of OZ and OKA on kinetochore function were tested by immunofluorescence microscopy of the kinetochore motor protein CENP-E, the kinetochore kinase hBubRl , and the 3F3/2 marker for attachment or tension across the kinetochore. Centrosomes in treated cells were also examined using y-tubulin antibodies. The results indicate that OZ and OKA do not exert discernible effects on tubulin polymerization, nor do they significantly affect y-tubulin at the centrosome. The localization of hBubRl and CENP-E was altered after OZ and OKA treatment, resulting in a prominent localization around the centrosome. These studies did not identify any direct targets for OZ and OKA; however, the complete duplication of all effects of OZ by OKA strongly argues that identification of targets for one of these compounds wi l l also identify the targets of the other. 3.3. EXPERIMENTAL PROCEDURES 3.3.1. Cell Culture and G2 Checkpoint Inhibition Assays Cell culture and G 2 checkpoint assays were performed as described in Chapter 2. 3.3.2. 3-D Confocal Microscopy of Live Cells Expressing GFP-Histone H1 To visualize chromosomes in live cells, I used a MCF-7 cell line that stably over-expressed a histone H l -GFP fusion protein (gift of Dr. J . Th'ng, Lakehead University, Thunder Bay, 58 ON) [167 ] . Cells were seeded onto glass-bottomed culture wells (Tekware, 150,000 cells/1.5 ml), cultured until 50 % confluent, and treated with DMSO, OZ (10 |JM) or OKA (20 UM) for 6 h before microscopy. Temperature was maintained at 37 °C during microscopy with a thermal insulating enclosure (created in-house). During each 2 min recording cycle, a stack of 20 sections (total thickness 4 | jm) was collected at the approximate centre of the cell. Stacks for individual t ime points were projected and animated in ImageJ software (NIH). 3.3.3. Tubulin Polymerization Assays An in vitro assay tested for stimulatory or inhibitory effects of OZ and OKA on polymerization of tubulin subunits. Purified bovine brain tubulin (Cytoskeleton, Denver, CO) was dissolved in composition A (80 mM Pipes pH 6.9, 1 mM MgCI 2 , 1 mM EGTA, 10 % glycerol, 1 mM GTP in General Tubulin Buffer(http://www.cytoskeleton.com/main.html)) at 10 mg/ml and kept on ice. The final concentration of tubulin was adjusted to 1 mg/ml for drug-induced polymerization assays, and 3 mg/ml in assays for inhibition of spontaneous polymerization. After addition of DMSO (0.3 % ) , 0Z (20 OKA (20 (JM), taxol (10 UM) or nocodazole (30 p M ) in pre-chilled composition A, samples were immediately transferred to 37 °C and polymerization of tubulin subunits was monitored in a spectrophotometer at 340 nm. 3.3.4. Immunofluorescence Microscopy 3.3.4.A. Indirect Immunofluorescence Labelling of 3- or y-Tubulin Immunostaining of (3- or y-tubulin was carried out in the same manner. Cells were seeded onto poly-L-lysine coated cover slips (150,000 cells/1.5 ml) in 6-well tissue culture plates and were treated on the following day with DMSO, OZ (10 UM), or OKA (20 |JM) for 8 h. 59 After fixation in formaldehyde (3.7 % in TBS, 30 min), cells were washed twice in TBS, permeabilized in blocking solution (1 % BSA in TBS) containing 0.1 % Triton X-100 (Tx-100) for 15 min, and then incubated for 1 h with E7 P-tubulin monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) or y-tubulin antibody (Sigma), diluted 1:20 or 1:1,000, respectively, in TBS containing 1 % BSA. After washing twice in TBS, cells were incubated with Alexa 488-conjugated secondary antibody (Molecular Probes, diluted 1:500 in TBS containing 1 % BSA) for 30 min and washed once more in TBS. Chromosomes were then stained with the DNA dyes Hoechst 33258 or T0T0-3. Samples stained with Hoechst 33258 were incubated with the dye for 7 min (20 ng/ml in TBS), washed twice in TBS and then mounted in Fluormount (90 % glycerol in PBS containing 0.2 M /7-propyl gallate). Samples stained with T0T0-3 were f irst incubated with RNase A (Roche Diagnostics, 500 U/ml) in TBS for 30 min at 37 °C, followed by addition of T0T0-3 (Molecular Probes, 2 mM) for 20 min in darkness. After washing twice in TBS, the cover slips were mounted in DABCO. 3.3.4.B. Indirect Immunofluorescence Labelling of CENP-E or hBubRl HCT116-/- cells were seeded onto cover slips (150,000 cells/1.5 ml), followed by irradiation the next day (0 or 12 Gy). 16 h after irradiation, cells were treated with DMSO, NOC (300 ng/ml), OZ (10 (JM), or OKA (20 UM) for 8 h. Labelling of CENP-E or h B U B R l was carried out according to the method of Jablonski etal. [ 1 6 8 ] . Cells were extracted in MTSB (4 M glycerol, 0.1 M PIPES pH 6.9, 1 mM EGTA) containing 0.5 % Tx-100 for 1 min, washed 2 min in MTSB, and fixed by incubation in PBS, pH 6.8, containing 3.7 % formaldehyde for 7 min. After washing twice in KB (50 mM Tris-HCl, pH 7.4, 150 mM 60 NaCl, 0.1 % BSA), cover slips were incubated with rabbit CENP-E or h B U B R l antibodies (gifts from Dr. G. Chan, University of Alberta, diluted 1:1,000 in KB) for 1 h. After washing twice in KB, cover slips were incubated in Cy3-conjugated goat anti-rabbit secondary antibody (Sigma, diluted 1:1,000 in KB) for 30 min. After two washes in KB, DNA was stained according to the method described in section 3.3.4.A. 3.3.4.C. Indirect Immunofluorescence Labelling with the 3F3/2 Antibody Immunostaining for the 3F3/2 kinetochore epitopes was performed according to the method of Gorbsky etal. [1693. HCTI 16-/- cells were seeded and irradiated as described in section 3.3.4.B. 16 h after irradiation, cells were treated with DMSO, NOC (300 ng/ml), OZ (10 p M ) , or OKA (20 |JM) for 8 h. After rinsing twice in PH EM (60 mM Pipes, 25 mM Hepes, pH 7.0, 10 mM EGTA, 4 mM MgS0 4 ) , cells were extracted for 5 min in PH EM containing 0.5 % Tx-100 and 100 nM microcystin (Calbiochem) and fixed in PHEM containing 1 % formaldehyde for 15 min. After rinsing in MBST (10 mM MOPS, pH 7.3, 150 mM NaCl, 0.05 % Tween 20) slips were blocked for 30 min in M BS (M BST without Tween-20) containing boiled normal goat serum (20 % ) . Kinetochore epitopes were labelled by incubation with 3F3/2 antibody (gift of Dr. G. Gorbsky, University of Oklahoma, diluted to 1:20,000 in M BS containing 5 % boiled goat serum) for 45 min. After three washes in M BST, Alexa 488-conjugated goat anti-mouse secondary antibody (diluted 1:500 in M BS containing 5 % boiled goat serum) was applied for 45 min. Slips were washed a further three times in MBST, DNA was stained with Hoechst 33258 and the slides mounted. Cells were photographed with a Qimaging Microimager I I digital camera and images prepared in PhotoShop 6.0 software [170 ] . 61 3.4. RESULTS 3.4.1. Identification of OKA as a G2 Checkpoint Inhibitor High throughput cell-based screening of the Natural Products Repository of the N.C.I, by our laboratory identified a G 2 checkpoint inhibiting activity in an extract from Coccoloba acuminata, a fruit ing shrub native to the Southern United States, Central and Southern America [ 1 7 1 ] . The structure was analyzed in NMR and mass spectrometry, and comparison with published values revealed that it was identical with e/7f-kaur-16-en-15-oxo-18-oic acid, a diterpenoid originally discovered in the Brazilian shrub Croton argyrophylloides (OKA, Fig. 3.1 A) [ 172 ] (J. Nelson, laboratory of Dr. R. J . Andersen, Departments of Chemistry and Oceanography-Earth Ocean Sciences, U.B.C.). The effects of OKA on the checkpoint were initially characterized in ELICA checkpoint assays (section 2.4.1.) [ 155 ] . OKA was a potent checkpoint inhibitor ( IC 5 0 6 (JM), with activity peaking at a concentration of 25 (JM (L. McHardy, personal communication). Similar results were obtained when entry into mitosis was evaluated by mitotic spreads in the p53-defective cell lines MCF-7 mp53 and HCT116-/- (not shown). I compared the checkpoint effects of OZ and OKA by treating irradiated HCT116-/- cells with a range of OZ and OKA concentrations and counting mitotic cells in flow cytometry (Fig. 3.2). In agreement with earlier results (Chapter 2), OZ released approximately 25 % of cells f rom G 2 arrest at its most effective concentration (10 (JM). OKA inhibited the G 2 checkpoint at concentrations between 5 and 20 (JM. A t the most effective concentrations tested (12 and 16 |JM), OKA released 47 ± 17 % and 39 ± 7 % (means ± S. D., n = 3), respectively, of cells from G 2 arrest. OKA had a greater effect than OZ, which released 20 ± 7 % and 12 ± 2 % of arrested cells at 12 and 16 (JM, respectively (means ± S. D., n = 3). 62 Figure 3.1 Structural formulae of enf-kaur-16-en-15-oxo-18-oic acid (A, OKA) and 13-hydroxy-15-oxozoapatlin (B, OZ). 63 75 0 1 10 100 [compound] (u,M) Figure 3.2 Inhibition of G2 arrest in HCT116-/- cells by OKA and OZ. 16 h after i r rad ia t ion (12 Gy) , G 2 -a r res ted cells were cu l tured with nocodazo le ( 3 0 0 ng /m l ) a n d the indicated concent ra t ions of O K A (black circles) o r O Z (white circles) f o r 8 h. After G F - 7 a n d Pl-dual labe l l ing , mitot ic cells were counted in f low cytometry (Experimental Procedures, Chap te r 2 ) , a n d expressed as percentages of the tota l n u m b e r of cells. Results are the averaged means f r o m three separate exper iments ± S.D.. 64 Taken together, these results indicate that OKA inhibits the G 2 checkpoint over a range of concentrations similar to that for OZ, but with considerably greater effects. 3.4.2. OKA Arrests Cells in a Stage Resembling Prometaphase Because OKA is similar to OZ in structure and is also a checkpoint inhibitor, I investigated whether it causes the same type of mitotic arrest as OZ. HCT116-/- cells were immunostained with anti-(3-tubulin antibody and the DNA dye T0T0-3. After 8 h treatment with OKA, approximately 20 - 30 % of cells were arrested in mitosis with condensed chromosomes scattered across bipolar spindles (Fig. 3.3), a greater effect than observed for OZ (approximately 10 % ) . In contrast with OZ, there were rare examples of normal metaphase and anaphase cells after OKA treatment (approximately 1 in 50 mitotic cells), suggesting that OKA-induced arrest may be leaky. Similar antimitotic effects of OKA were seen in H C T 1 1 6 + / + , MCF-7 mp53 and MCF-7 wtp53 cells (not shown). Prior irradiation of cells did not alter the phenotype observed, but increased the number of arrested cells (not shown). Thus, OKA, like OZ, inhibits progression through mitosis and arrests cells in a state resembling prometaphase. 3.4.3. Effects of OZ and OKA on Chromosome Movement in Live Cells One can imagine at least two causes for the prometaphase arrest observed in immunofluorescence microscopy of OZ- and OKA-treated cells. The movement of mitotic chromosomes could be inhibited, giving them a scattered appearance as they become fixed in place wherever they attached to the spindle. Alternatively, the chromosomes may be able to move freely, but a failure in regulation of this motion could prevent their congression at the 65 p-tubulin DNA Merge Figure 3.3 OKA arrests HCT116-/- cells in a stage resembling prometaphase. M i t o t i c s p i n d l e s a n d c h r o m o s o m e s w e r e e x a m i n e d by i n d i r e c t i m m u n o f l u o r e s c e n c e m i c r o s c o p y a f te r t r e a t m e n t w i t h D M S O (top) o r O K A ( 2 0 u M , bottom) f o r 8 h. S p i n d l e m i c r o t u b u l e s w e r e l a b e l l e d w i th E-7 P-tubulin a n d A l e x a 4 8 8 - c o n j u g a t e d g o a t a n t i - m o u s e a n t i b o d i e s , a n d D N A w a s s t a i n e d w i th T O T O - 3 . T h e P-tubulin (left) a n d D N A (middle) s i gna ls w e r e v i s u a l i z e d by c o n f o c a l m i c r o s c o p y ( C h a p t e r 2 , E x p e r i m e n t a l P r o c e d u r e s ) . Ba r , 5 u m . 66 midline. To test whether chromosome movement was detectable in OZ- and OKA-treated cells, I used a stable cell line that over-expressed a GFP-histone H I fusion protein (gift from Dr. J . Th'ng). In immunofluorescence microscopy, cells are fixed before labelling. The GFP-histone-H l fusion protein makes chromosomes fluorescent, circumventing the need for fixation and staining and allowing microscopy of live cells. Cells were examined by time-lapse 3-D confocal microscopy while being cultured at 37 °C in the presence of DMSO (Fig. 3.4). Cells passed rapidly from prophase to metaphase within 20 min. Prometaphase was brief, lasting a maximum of 14 min. Metaphase was the longest-lasting phase of mitosis, extending for 28 min. Once anaphase began, chromosomes separated rapidly and cells reached telophase within 6 min. When cells were cultured in the presence of OZ (10 U.M) or OKA (20 U.M), a much different pattern was observed (Fig. 3.5). Over the entire observation period (30-60 min), very little chromosome movement was detected, and cells remained in a state resembling prometaphase. These results indicate that the failure of OZ- and OKA-treated cells to reach metaphase stems from a lack of chromosome movement. 3.4.4. Effects of OZ and OKA on Tubulin Assembly in vitro The strong reduction of chromosome movement by OZ and OKA could result from an effect of these compounds on the dynamics of microtubule assembly in the mitotic spindle. A microtubule is a hollow, cylindrical structure composed of a variable number of 67 Figure 3.4 The stages of mitosis in MCF-7 cells over-expressing GFP-histone H1 (following page). C o n d e n s a t i o n a n d m o v e m e n t o f c h r o m o s o m e s d u r i n g m i t o s i s w e r e m o n i t o r e d in f l u o r e s c e n c e m i c r o s c o p y o f l ive ce l l s . C h r o m o s o m e s in M C F - 7 ce l l s w e r e m a d e f l u o r e s c e n t by o v e r - e x p r e s s i o n o f a G F P - h i s t o n e H I f u s i o n p r o t e i n . C e l l s w e r e m o n i t o r e d by t i m e - l a p s e 3 - D c o n f o c a l m i c r o s c o p y w h i l e b e i n g c u l t u r e d a t 3 7 ° C in t h e p r e s e n c e o f D M S O ( E x p e r i m e n t a l P r o c e d u r e s ) . I m a g e s w e r e c o l l e c t e d e v e r y 2 m i n , a n d a r e p r o j e c t i o n s o f s tacks c o n t a i n i n g 2 0 s e c t i o n s . Bar, 5 ( Jm. 68 0 min " ;| 4 min 6 m m 8 min 10 min 12 min 14 min 16 mm 18 min 20 mm 22 min 24 mm 26 min 28 m m 30 mm 32 min 34 mm 36 min 38 m m 40 mm 42 min 44 m m 46 mm 48 m m 50 mm min 54 mm 56 mm 58 mm 60 min 62 min 64 mm 66 m m 68 m m 72 mm 74 mm 76 min 78 mm 80 m m 82 m m 84 min 86 m m 88 mm 90 mm 92 min 94 mm 69 Figure 3.5 Chromosome motion is severely curtailed in OZ- and OKA-treated cells (following page). M C F - 7 cells over-expressing GFP-histone H I were cu l tured at 3 7 °C in the presence of O Z (1 0 U M , top) o r O K A (20 U M , bot tom) fo r 6 h before microscopy of live cells began . Image co l lect ion a n d analysis was per formed as fo r Fig. 3 .4 . Results are typical fo r three separate record ings. Bar, 5 [Jm. 70 71 of a and P tubulin subunits. During mitosis, the cytoplasmic microtubule network re-assembles and forms the mitotic spindle. Spindle microtubules are dynamic structures that continually add and lose subunits in cycles of growth and collapse [ 6 7 ] . Many antimitotic agents act by influencing the dynamics of spindle microtubules, resulting in their stabilization (e.g. taxol) or disruption (e.g. colchicine, nocodazole) (reviewed in [88, 89]) . Immunofluorescence microscopy studies had shown that bipolar spindles were present after OZ or OKA treatment, indicating that neither compound exerts a strong depolymerizing effect on microtubules. Considering the inhibition of chromosome movement by these compounds, however, it was important to test whether they have a direct effect on microtubule polymerization. First, we tested for a stimulation of polymerization by OZ or OKA. Purified tubulin (1 mg/ml) was incubated with DMSO (0.3 % ) , OZ (20 UM), OKA (20 UM), or taxol (10 U.M) for 1 h at 37 °C (Fig. 3.6). As expected, a strong increase in turbidity was observed when tubulin was incubated with taxol, reflecting its stabilizing effect on microtubules. In contrast, after OZ or OKA treatment tubulin turbidity was indistinguishable from the DMSO control. Next, we tested whether OZ or OKA could prevent polymerization of microtubules. In these assays, the tubulin concentration was increased to promote spontaneous microtubule assembly at 37 °C (Fig. 3.7). Microtubules polymerized in the presence of OZ or OKA. A slight reduction in comparison to the DMSO control was noted, but was well within experimental error for this assay. Only the depolymerizing agent nocodazole prevented polymerization of microtubules. Taken together, these results indicate that OZ and OKA do not stabilize or prevent polymerization of microtubules in vitro. While I cannot rule out a subtle effect of these compounds on microtubules dynamics in vivo, these results suggest that 72 0.035 0.030 j 0.025 : o 0.020 co < 0.015 i n nni i TTTT Time (min) Figure 3.6 OZ and OKA do not polymerize tubulin in vitro. Purif ied bovine brain tubu l in (1 m g / m l ) was incubated with D M S O (0.3 %, white circles), O Z (20 u.M, black triangles), O K A (20 p M , black circles) o r taxol (1 0 p M , crosses) to test fo r a polymer iz ing effect. At 0 m in , addi t ions were m a d e to pre-chi l led tubu l in a l iquots a n d samples were rapidly b rought to 3 7 ° C . An increase in turbidi ty (A340) cor responds to assembly of micro tubu les . Assay per fo rmed with assistance f r o m C. Bigg (Experimental Procedures). 73 0.10 ZT Time (min) Figure 3.7 Polymerization of tubulin in vitro is not inhibited by OZ or OKA. Spontaneous micro tubu le polymer izat ion was st imulated by incubat ing tubul in (3 m g / m l ) at 3 7 ° C . Addi t ions of D M S O (0.3 %, white circles), O Z (20 u M , black triangles), O K A (20 u,M, black circles) o r nocodazo le (30 U.M, crosses) were m a d e at 0 m in . Assay per fo rmed with assistance f r o m C . Bigg (Experimental Procedures). 74 OZ and OKA inhibit chromosome movement though a tubulin-independent mechanism. 3.4.5. Effects of OZ and OKA on Intracellular Localization of CENP-E OZ and OKA could potentially inhibit chromosome movement by acting upon a chromosome-associated motor protein. Centromeric protein E (CENP-E) is a kinesin-like motor protein that plays an essential role in chromosome congression [ 8 1 , 82 ] . Inhibition of CENP-E by immunodepletion, antibody microinjection, or over-expression of dominant interfering mutants results in cell cycle arrest in prometaphase with condensed chromosomes and bipolar spindles [79, 80 ] . As this phenotype closely resembles that of OZ- and OKA-treated cells, I tested whether these agents affect CENP-E abundance or localization in immunofluorescence microscopy. First, I examined the localization of CENP-E throughout the cell cycle. HCT116-/- cells were treated with DMSO for 8 h and then immunostained with CENP-E and P-tubulin antibodies and counterstained with T0T0-3 to visualize chromosomes (Fig. 3.8). In agreement with published studies, the localization and abundance of CENP-E was dynamic and varied throughout the cell cycle [ 8 1 , 82, 168, 173-175] . During interphase, CENP-E was found at a single point, lying outside of the nucleus and coinciding with microtubules, that may represent the centrosome. Centrosomal CENP-E was clearly observed from prophase through to metaphase. A strong CENP-E signal was detected on kinetochores • beginning in prometaphase and waning during anaphase. In addition, CENP-E was found on the mitotic spindle during metaphase, the midzone during anaphase and telophase, and on the midbody in cytokinesis. The same pattern was observed in MCF-7 mp53 cells (not shown). 75 DNA p-tubulin CENP-E merge no primary antibody interphase prophase prometaphase Figure 3.8 Intracellular localization of CENP-E throughout the cell cycle. The distribution of CENP-E throughout the cell cycle was examined in non-irradiated HCTI 1 6-1- cells. After treatment of cells with DMSO for 8 h, spindle microtubules were labelled with E-7 P-tubulin and goat anti-mouse Alexa 488 antibodies. CENP-E was detected by rabbit polyclonal and goat anti-rabbit-Cy3 antibodies, and DNA was stained with TOTO-3 (Experimental Procedures). Bar, 5 U r n . 76 DNA p-tubulin CENP-E merge metaphase ' magM 1 •1 anaphase telophase 2 cytokinesis • Figure 3.8 Intracellular localization of CENP-E throughout the cell cycle, cont... 77 Next, I compared the abundance and distribution of GENP-E in cells that were in naturally occurring prometaphase with those that were arrested in this stage as a result of OZ or OKA treatment (Fig. 3.9). HCT116-/- cells were treated.with DMSO, OZ (10 UM) or OKA (20UM) for 8 h before immunostaining for CENP-E and P-tubulin and counterstaining of chromosomes. In DMSO-treated prometaphase cells, CENP-E was found on kinetochores near spindle fibres and centrosomes, in approximately equal brightness. In six independent experiments, I observed a consistent effect of OZ and OKA treatment. Kinetochore-associated CENP-E was diminished in most cells (approximately 80 % ) , while CENP-E became very bright around the centrosomes. These effects were also observed in H C T 1 1 6 + / + and MCF-7 mp53 cells. Prior irradiation only increased the number of prometaphase cells with this phenotype. That CENP-E localization was truly around the centrosome was confirmed in HCT116-/- cells by co-immunolabelling of CENP-E and the centrosomal protein y-tubulin (not shown). I next examined the CENP-E pattern in cells arrested in mitosis by nocodazole treatment (300 ng/ml, 8 h). In these cells, P-tubulin formed numerous small, astral-shaped polymers. CENP-E was found in discrete foci that coincided with P-tubulin and DNA (Fig. 3.10), suggesting that under these conditions CENP-E remains associated wi th microtubules and possibly kinetochores. These results indicate that a shift in CENP-E localization from the kinetochore to around the centrosome is not simply a consequence of mitotic arrest. 3.4.6. Intracellular Localization of hBubRl is Altered in OZ- and OKA-Treated Cells I then asked whether OZ and OKA have effects on other cell cycle checkpoint proteins. During the metaphase to anaphase transition, the cell monitors the alignment of 78 DNA fj-tubulin CENP-E Merge Figure 3.9 CENP-E is primarily at or near the centrosome after OZ and OKA treatment. After i ncuba t ion with D M S O , O Z (10 u M ) o r O K A (20 u M ) fo r 8 h, H C T I 16-1-cells were i m m u n o s t a i n e d with B-tubul in a n d CENP-E ant ibod ies a n d c h r o m o s o m e s were label led with Hoechst 3 3 2 5 8 (Experimental Procedures). Images were co l lec ted by con foca l microscopy. Bar, 5 u rn . 79 p-tubulin CENP-E DNA Merge DMSO NOC Figure 3.10 CENP-E associates with microtubules in nocodazole-arrested cells. The effects of mitot ic arrest o n CENP-E local izat ion were invest igated in nocodazo le - t rea ted HCT1 1 6 - / - cells. M ic ro tubu les , CENP-E a n d c h r o m o s o m e s were label led fo r immuno f luo rescence after 8 h incubat ion with D M S O (top) or nocodazo le ( N O C , 3 0 0 n g / m l , bo t tom) . M ic ro tubu les were label led with p-tubulin a n d g o a t an t i -mouse Alexa 4 8 8 an t ibod ies , CENP-E was detected with rabbi t po lyc lona l CENP-E a n d g o a t an t i -rabbi t Cy3 an t ibod ies , a n d c h r o m o s o m e s were stained with the D N A dye T O T O - 3 (Experimental Procedures). The P - tubul in, CENP-E, a n d D N A signals were visualized by con foca l microscopy. Bar, 10 u.m. SO chromosomes at the spindle midline. Failure of a single chromosome to congress is sufficient to activate the spindle checkpoint and delay entry into anaphase (reviewed in [95, 100, 102] . - i One of the early activating kinases in this signalling pathway is hBubRl [99, 106, 176] . hBubRl loads onto kinetochores during prophase, where it binds to CENP-E [168 ] . A high amount of hBubRl on the kinetochore corresponds to misalignment of the chromosome (occurring during natural prometaphase or as a result of mitotic defects) and also indicates activation of the spindle checkpoint pathway [168 ] . HCTI 16-/- cells were irradiated and treated with DMSO or OKA (20 |JM) for 8 h before immunostaining with hBubRl and B-tubulin antibodies and counterstaining DNA with TOTO-3 (Fig. 3.11). DMSO-treated cells in prometaphase exhibited bright hBubRl staining on the kinetochores and near the centrosomes. The brightness of the signal was comparable to that observed in nocodazole-arrested cells (not shown), and appeared to be equal in brightness in both intracellular locations. In each of six independent experiments, the hBubRl signal was stronger at or near the centrosomes and weaker on the kinetochores in the majority of prometaphase-arrested cells (approximately 70 %) (Fig. 3.11). In a minority of cells, localization and brightness of hBubRl were similar to that observed in natural prometaphase (approximately 30 % ) . OZ and OKA exerted the same effects in both irradiated and non-irradiated cells, and similar results were observed in H C T 1 1 6 + / + , MCF-7 wtp53 and MCF-7 mp53 cells (not shown). These results indicate that OZ and OKA treatment alter the intracellular localization of hBubRl in a manner similar to that observed for CENP-E. 3.4.7. Y-Tubulin Morphology in OZ-and OKA-treated cells Although OZ and OKA treatment allowed for formation of bipolar spindles, their 81 Figure 3.11 hBubRl is localized around the centrosome after OZ or OKA treatment. 1 6 h af ter i r r a d i a t i o n (12 G y ) , H C T 1 1 6-1- cells w e r e t rea ted wi th D M S O (top) o r O K A ( 2 0 u M , bottom) f o r 8 h be fo re i m m u n o s t a i n i n g . M i to t i c sp ind les a n d h B u b R l were labe l l ed wi th m o u s e B- tubul in a n d rabb i t h B u b R l a n t i b o d i e s , a n d D N A was c o u n t e r s t a i n e d wi th T O T O - 3 (Exper imenta l Procedures) . Images were co l l ec ted in c o n f o c a l m ic roscopy . Bar, 1 0 u r n . 82 appearance was different in some respects from that of normal spindles. In many instances, the spindle poles were ragged and unfocussed, with disarrayed astral microtubules attaching the centrosome to the plasma membrane. This suggested that perhaps OZ and OKA caused structural alterations in the centrosomes. For this reason, I tested whether localization of the centrosomal protein y-tubulin was altered after OZ and OKA treatment. Following an 8 h incubation with DMSO, OZ (10 UM), or OKA (20 UM) HCT116-/- cells were labelled with y-tubulin and Alexa 488-conjugated goat-anti-mouse antibodies and chromosomes stained with T0T0-3. In all cells, a diffuse cytoplasmic background was observed, most likely resulting from cytoplasmic y-tubulin (Fig. 3.12). Interphase cells had one or two foci of y-tubulin, corresponding to centrosomes before and after replication, y-tubulin was also observed at the spindle poles. Localization in OZ- and OKA-treated cells was indistinguishable from controls, indicating with respect to y-tubulin, no striking effect on the centrosome could be detected. 3.4.8. Immunofluorescence Microscopy Analysis of 3F3/2 Epitope Expression One possible cause for the failure of OZ and OKA-treated chromosomes to align during metaphase is a defect in attachment of kinetochores to spindle microtubules [75 ] . To explore this possibility I turned to the 3F3/2 monoclonal antibody. This antibody labels kinetochores in a manner dependent upon attachment or tension across the kinetochores [177 ] . The 3F3/2 antibody labels two phosphoepitopes on the anaphase promoting complex, and appearance of the 3F3/2 epitope corresponds to activation of the spindle checkpoint pathway [178-180] . HCT116-/- cells were immunostained with 3F3/2 antibody and chromosomes counterstained with Hoechst 33258. In agreement with published studies, the 3F3/2 signal was bright in 83 Y-tubulin DNA Merge Figure 3.12 OZ and OKA do not affect centrosomal y-tubulin localization. HCT116-/-cells were incubated with DMSO, OZ (10 uM) o r OKA (20 uM) for 8 h before immunosta in ing with y- tubul in [left) a n d Alexa 4 8 8 - c o n j u g a t e d goa t ant i -mouse ant ibodies a n d countersta in ing chromosomes with TOTO-3 [middle) (Experimental Procedures). Images were col lected by confoca l microscopy. Arrows indicate cent rosomal y- tubul in in interphase cells, ar rowheads indicate y- tubul in in mitot ic cells. Bar, 1 0 u m . 84 prometaphase, when kinetochores are unattached and not under tension [169 ] (Figure 3.13). This was also the case when cells were arrested in mitosis by nocodazole treatment. As chromosomes reached the midline during metaphase, the 3F3/2 signal was extinguished. After treatment with OZ or OKA, bright foci of 3F3/2 staining were observed in arrested cells, comparable to the level observed in prometaphase and nocodazole-arrested cells. These results are consistent wi th an activated spindle checkpoint in OZ- and OKA-treated cells. 3.5. DISCUSSION Continued screening of the N.C.I. Natural Product Repository yielded a second compound, OKA, with similar structure and activities to OZ. The structural differences between OKA and OZ include a lack of a hydroxyl at C13, a methyl group at C20, and opening of the lactone ring of OZ (C10-C18) (Figure 3.1). Although the two compounds are active over a similar range of concentrations, at its most effective concentration (12 U.M), OKA released 50 % of cells from G 2 arrest compared to approximately 25 % for OZ. The structural differences between OZ and OKA have enhanced the effectiveness of OKA as a checkpoint inhibitor. OKA also retained the antimitotic properties of OZ, and was more potent in this respect as well. After OZ and OKA treatment, chromosome movement was greatly reduced, which may be a factor contributing to the prometaphase arrest observed in immunofluorescence microscopy. Inhibition of microtubule dynamics is a common mechanism of action for antimitotic agents, and suppression of dynamics could prevent chromosome congression. However, we have found no evidence for a strong effect of OZ or OKA on tubulin polymerization or depolymerization. CENP-E was observed around the centrosome during interphase and prometaphase in untreated cells. This localization of CENP-E has not been reported in the literature, however 85 3F3/2 DNA DMSO prometaphase DMSO metaphase NOC OZ OKA Figure 3.13 The 3F3/2 epitope is not extinguished in OZ and OKA-treated cells. H C T 1 1 6 - / - ce l l s w e r e i n c u b a t e d w i t h D M S O , n o c o d a z o l e ( N O C , 3 0 0 n g / m l ) , O Z (1 0 uM) o r O K A ( 2 0 uM) f o r 8 h b e f o r e l a b e l l i n g w i t h m o u s e m o n o c l o n a l 3 F 3 / 2 (left) a n d A l e x a 4 8 8 - c o n j u g a t e d g o a t a n t i - m o u s e s e c o n d a r y a n t i b o d i e s a n d s t a i n i n g o f c h r o m o s o m e s w i t h H o e c h s t 3 3 2 5 8 (middle) ( E x p e r i m e n t a l P r o c e d u r e s ) . I m a g e s w e r e c o l l e c t e d by d i g i t a l p h o t o m i c r o s c o p y . Bar, 1 0 urn. 86 other motor proteins including hEg5 and the kinetochore motor protein dynein have been observed at the centrosome. I t must be noted that both of the cell lines I studied are cancer cell lines, which often have abnormal centrosomes (reviewed in [181 ] ) . I t would be interesting, therefore, to test whether CENP-E is found at the centrosome in non-cancerous cells. A new generation of antimitotic agents is emerging that targets microtubule-based mitotic motor proteins, rather than tubulin. For example, the antimitotic agent monastrol inhibits the activity of the plus-end-directed motor protein hEg5. Likewise, it is possible that the antimitotic effects of OZ and OKA result from inhibition of CENP-E activity. CENP-E is a plus-end directed microtubule motor protein that is essential for chromosome congression [79-81] . The phenotype of cells arrested by OZ or OKA treatment is indistinguishable from that of cells in which CENP-E is inhibited by antibody microinjection [ 7 9 ] . Direct inhibition of its movement towards the plus end by OZ or OKA could explain its localization near the centrosome, which anchors the minus ends of microtubules. Alternatively, as the localization of CENP-E and its association with microtubules is dynamic and subject to regulation [ 8 1 , 82, 168, 173-175, 182] , indirect effects through a regulatory pathway could inhibit its activity and also result in its relocalization. In either case, the loss of CENP-E from the kinetochore could deprive chromosomes of the force necessary to drive them towards the cell midline and exit f rom prometaphase. hBubRl normally binds to CENP-E at the kinetochore [107 ] . A continued interaction between these proteins after OZ or OKA treatment could explain the centrosomal localization of hBubRl . The effects of OZ and OKA on CENP-E and hBubRl were not universal, as cells with normal localization were observed (20 - 30 % ) . While prometaphase arrest can be caused by 87 malfunction in CENP-E, it is also possible that OZ and OKA cause prometaphase arrest through a completely different mechanism. In naturally occurring prometaphase, CENP-E was observed at the centrosome. Perhaps extended arrest in prometaphase increases this localization. The altered distribution of CENP-E could therefore be a consequence, rather than a cause, of prometaphase arrest. The range of OZ and OKA effects that I observed could therefore be a consequence of the length of time a cell has been arrested in prometaphase. I am unable to distinguish between CENP-E relocalization to the centrosome being a cause or a consequence of prometaphase arrest on the basis of my experimental results. I t would be informative to test if CEN P-E localization is altered in a similar way when cells are arrested in prometaphase by a different treatment. Also, if inhibition of CENP-E activity is responsible for arrest, over-expression of this protein may rescue chromosome movement. The time course studies presented in Chapter 2 indicated that the mitotic arrest in OZ-treated cells is sustained for several hours at least. In the event of a spindle defect or the failure of a chromosome to align, the spindle checkpoint pathway becomes activated and delays the metaphase to anaphase transition. Activation of the spindle checkpoint also occurs during prometaphase and metaphase during a normal mitosis, and ensures that anaphase does not begin prematurely. Bright labelling of cells with the 3F3/2 antibody correlates with activation of this pathway [177, 179] . The bright 3F3/2 labelling observed in OZ- and OKA-treated cells is consistent with activation of the spindle checkpoint in these prometaphase-arrested cells. Activation of this pathway could be responsible for sustaining the prometaphase arrest. In every instance examined, the effects of OKA have been identical to OZ, with the exception that OKA is more potent. Both compounds are checkpoint inhibitors and 88 antimitotic agents. Both OZ and OKA resulted in similar changes in localization of CENP-E and hBubRl . Neither OZ nor OKA strongly affected y-tubulin localization or tubulin dynamics. Finally, in both OZ- and OKA-treated cells chromosome movement was greatly reduced. The complete duplication by OKA of every tested activity of OZ leads to the hypothesis that some or all of the targets of OZ are acted upon by OKA as well. In Chapter 2, I showed that the a,P-unsaturated carbonyl group of OZ is required for checkpoint activity. In OKA, this group is retained, whereas the region around the lactone ring (joining CIO to C18) in OZ is opened in OKA. The conservation of the a,P-unsaturated carbonyl group in these two compounds suggests that it is essential for the activities of OZ and OKA, whereas alterations around the lactone ring and carbons 1 through 6 are tolerated. A further modification of the carboxylic acid group may therefore be tolerated as well. Carboxylic acid groups are more reactive than lactone rings, suggesting that OKA may be suitable for chemical modification. I t is of interest that these two compounds come from extracts of plants from different continents and taxonomic families, yet appear to converge with respect to structure and activity. The work presented here suggests that OZ and OKA are members of a family of compounds in which the a,P-unsaturated carbonyl group and mult i-r ing structure are hallmark features. In support of this concept, recent screening for checkpoint inhibitors by our laboratory identified psylostacchin compounds from the common ragweed. The psylostacchins also have an a,P-unsaturated carbonyl and multi-r ing structure, and exert checkpoint and mitotic effects very similar to those of OZ and OKA (C. Brown, C. Bigg, C. Sturgeon, personal communications). 89 3.6. A C K N O W L E D G E M E N T S I would like to thank Dr. G. Gorbsky for the 3F3/2 antibody, Dr. G. Chan for CENP-E and hBubRl antibodies, Dr. J . Th'ng for the GFP-histone H I cell line, Cristina Bigg for assistance with microtubule polymerization assays, and Lianne McHardy for ELICA checkpoint data. 90 Chapter 4 INVESTIGATION OF THE INTRACELLULAR DISTRIBUTION AND MOLECULAR TARGETS OF OKA 4.1. SUMMARY In this study, we modified the checkpoint inhibitor and antimitotic agent OKA by attaching a biotin group. The reaction product, abbreviated B-OKA, retained checkpoint inhibiting activity. Cells treated with B-OKA resembled OKA- and OZ-treated cells: they arrested in a stage resembling prometaphase, with enhanced localization of CENP-E and hBubRl around the centrosomes. We performed fluorescence affinity labelling to investigate the intracellular localization of B-OKA, and found that it was widely distributed throughout the cell, with the possible exception of mitotic chromosomes. We were also able to label, purify and identify a number of the proteins that bind to B-OKA. I tested whether B-OKA binds directly to one of these candidate targets, the nuclear transport protein RanBP2. In both RanBP2 immunoaffinity precipitations and streptavidin affinity precipitations from X . laevis egg extracts, biotinylated fragments ofRanBP2 were observed. After adjusting experimental conditions to reduce proteolysis, a biotinylated protein ivas observed in B-OKA-treated extracts that co-migrated with full-length RanBPl. The results strongly suggest that RanBP2 is a target of B-OKA. 4.2. INTRODUCTION We have previously discovered two checkpoint inhibitors, OZ and OKA. Identifying the target(s) of these compounds would be desirable for several reasons. OZ, and presumably 91 OKA, do not inhibit protein phosphatases or kinases known to play a role in the DNA damage checkpoint pathway, suggesting that their action is on a new member of this signalling pathway. OZ and OKA are also both antimitotic agents, and in contrast to most members of this class of compounds, do not act directly on tubulin (reviewed in [88]). A new class of antimitotic agents has recently been identified. These are inhibitors of mitotic motor proteins, e.g. the hEg5 inhibitor monastrol, and have potential value as cancer treatments with enhanced specificity for rapidly growing cells [70]. The phenotype of OZ- and OKA-treated cells is consistent with inhibition of a mitotic motor. Aside from any possible therapeutic benefit, identification of targets of OZ and OKA could prove useful for the biochemical study of the DNA damage checkpoint pathway and early events of mitosis. There are two general approaches one can try for identifying molecular targets. Based on current knowledge of the cell cycle, the phenotype of treated cells may suggest targets to test the inhibitors against. This was the rationale behind the experiments in Chapter 3. A second approach to this challenge is developing labelled analogues of checkpoint inhibitors. A labelled compound is an extremely useful tool for purifying protein targets. For example, a label can track targets during protein chromatography. If an affinity label is used, it has the added advantage of being able to purify targets from complex mixtures, greatly increasing purity in a single step. Our initial attempts at chemical modification were aimed at OZ. Unfortunately, it was resistant to most modifications. We were able to radiolabel the closely related analogue 15-oxozoapatlin with [ 3H]methyl, which allowed for labelling of several proteins (Chapter 2). However, this did not advance our efforts towards identification of protein targets. In this chapter, I describe our attachment of a biotin group to OKA (abbreviated B-OKA). 92 Several careful experiments were performed to ascertain that biotinylation had not disrupted the biochemical activities of OKA. Streptavidin reagents were used in two applications. Firstly, fluorescent streptavidin labelled the distribution of B-OKA within the cell. Secondly, streptavidin agarose enabled purification of proteins that bound to B-OKA. We identified B-OKA-interacting proteins by mass spectrometry, resulting in a short list of candidate targets for B-OKA. One of the candidate targets identified was the Ran-binding protein RanBP2. Ran is a member of the Ras family of GTPases, and is involved in diverse cellular processes including nucleocytoplasmic transport and reassembly of the nuclear envelope after mitosis (reviewed in [183, 184]) . Ran GTPase activity is regulated by a guanine exchange factor (RCCl) and by the GTPase activator RanGAPl and its co-activator RanBPl . Several actions of Ran suggest that interference with its activity through RanBP2 could account for the checkpoint and mitotic effects exerted by OZ and OKA. Ran influences mitotic spindle dynamics [68, 185] , and is essential for the correct positioning of chromosomes during metaphase [ 1 8 6 ] . Furthermore, Ran regulates the action of mitotic motors found on the spindle: in X. laevistqq extracts, Ran-GTP increases the movement of the Eg5 motor protein to the plus ends of microtubules [ 1 8 5 ] . RanBP2 is a large (358 kDa) protein containing four Ran-binding domains, a leucine zipper at its amino terminus, eight zinc fingers, a carboxy terminal cyclophilin domain, and 26 nuclear pore motif repeats [187, 188] . Electron microscopy has localized RanBP2 to the cytoplasmic f ibri ls emanating from nuclear pore (NPC) [187-189 ] . In HeLa cells, RanBP2 associates with mitotic spindles, kinetochores and centrosomes [ 1 9 0 ] . Several functions are proposed for RanBP2: a docking site for complexes of transport cargo and the a and (3 93 importins at the NPC [187 ] , a facil i tator of Ran GTPase activation by RanGAPl [191 ] , and a SUMO E3 ligase [192-195 ] . To begin to explore possible new roles for RanBP2 in the G 2 DNA damage checkpoint and mitosis, we tested whether B-OKA binds to RanBP2. 4.3. EXPERIMENTAL PROCEDURES 4.3.1. Streptavidin-Alexa 594 Affinity Labelling for Fluorescence Microscopy MCF-7 mp53 or HCT116-/- cells were seeded onto cover slips at a density of 100,0007ml (150,000 cells total) and irradiated the following day (0, 10 or 12 Gy). 16 h after irradiation, biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 U.M) was applied to the cells for 8 h. Cells were fixed in 3.7 % formaldehyde solution in TBS, washed twice in TBS, and blocked in TBS containing 1 % BSA and 0.1 % Tx-100 for 30 min. P-tubulin was labelled by incubation with E-7 antibody (diluted 1:20 in TBS containing 1 % BSA) for 1 h. After three washes in TBS, cells were incubated for 30 min with goat anti-mouse secondary antibody conjugated to Alexa-488 (Molecular Probes, diluted 1:500 in TBS containing 1 % BSA) and biotin was simultaneously labelled by streptavidin conjugated to Alexa-594 (Molecular Probes, diluted 1:40,000). After three washes in TBS, DNA was stained with Hoechst 33258 (20 ng/ml in TBS) for 7 min. After a final wash in TBS, slides were mounted in Fluormount and images collected by confocal microscopy. 4.3.2. Streptavidin Affinity Purification of Proteins Interacting with B-OKA Proteins that bound to B-OKA were purified by streptavidin affinity precipitation according to the method of Zimmer eta/. [ 196 ] . HCT116-/- cells (5 x 10 6 ) were seeded onto 10 cm 2 94 dishes and the following day were treated with nocodazole (300 ng/ml) or ionizing radiation (12 Gy from a 6 0 Co source). After 16 h, cells were given biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 |JM) for 8 h. Cells were collected by trypsinization, washed once in PBS containing 0.2 mM PMSF, resuspended in 20 mM Tris, pH 8.0 and finally lysed by addition of an equal volume of 2X RIPA lysis buffer (20 mM Tris, pH 8.0, 0.2 % SDS, 2 % NP-40, 2 % sodium deoxycholate, 300 mM NaCl, 0.04 % sodium azide, 0.2 mM PMSF, and 10 pg/ml each of leupeptin, aprotinin, antipain and pepstatin). Lysates were passed several times through a syringe fitted with a 2 i y 2 gauge needle and incubated on ice for 1 h. Supernatants were collected after centrifugation at 12,600 gior 30 min. The protein concentration of each sample was measured by BCA protein assay and equalized with I X RIPA buffer. Lysates (approximately 3-7 mg of protein) were loaded onto streptavidin agarose beads (Molecular Probes, 100 (Jl of a 50 % slurry) that had been pre-washed three times in RIPA buffer, and nutated overnight at 4 °C. The beads were washed three times in RIPA buffer, followed by one wash in 20 mM Tris-HCI, pH 6.8. Proteins were eluted by boiling in SDS-PAG E sample loading buffer for 10 min with agitation. For experiments requiring mass spectrometric analysis, the following changes were made to the purification protocol to increase the yield of B-OKA-binding proteins: six 10 cm 2 dishes per condition were seeded with HCT116-/- cells, lysate supernatants were collected after a 1 h centrifugation (177,000 g, 4 °C) and typically yielded 20 mg of protein, and the volume of streptavidin agarose beads and elution buffer were both increased to 300 (Jl. Eluted proteins (approximately 50 pg in B-OKA samples) were precipitated in acetone and finally resuspended in SDS-PAG E sample buffer (40 p i ) . 95 4.3.3. Detection of Biotinylated Proteins by Streptavidin-HRP Overlay Proteins purified by streptavidin affinity precipitation were separated by SDS-PAG E in 8 % acrylamide gels. Gels were equilibrated in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 10 % methanol) and electrotransferred to PVDF membrane (Mill ipore) using a semi-dry apparatus at a constant current density of 1 mA/cm 2 for 2 h. After a quick wash in PBS, blots were blocked overnight at 4 °C in PBS containing 5 % BSA and 0.02 % sodium azide. Biotinylated proteins were labelled by incubation with H RP-conjugated streptavidin (Molecular Probes; diluted 1:200,000 in PBS-T containing 1 % BSA) for 1 h at room temperature. After washing the blots three times in PBS-T, labelled bands were detected by incubation with chemiluminescent substrate (Pierce) and exposed to f i lm. For direct detection of biotinylated proteins on the blot, the method was similar except that the streptavidin-HRP dilution was reduced to 1:50,000 and bands were detected by incubation in 3,3,5,5-tetramethylbenzidine (TMB, Pierce) chromogenic substrate for several minutes. 4.3.4. Silver Staining of Electrophoresed Proteins After SDS-PAGE (8 % gels), proteins were fixed by incubation in methanokacetic acid (40 %:10 %, (v/v)) for 30-45 min and treated with a solution of potassium ferricyanide and sodium thiosulfate (dry powders in a ratio of 5:8 (w:w)) for 10 min. After washing in water, the gel was incubated in 120 mM silver nitrate solution for 30-45 min, and incubated in carbonate buffer (2.9 %) containing formaldehyde (0.037 %) until protein bands were visible. Development was stopped by incubation with 5 % acetic acid. Gels were washed in several changes of distilled water and then dried. 96 4.3.5. Analysis of B-OKA-Binding Proteins by Mass Spectrometry Proteins that bound to B-OKA were identified using mass spectrometry by Dr. M. Flory (Laboratory of Dr. R. Aebersold, Institute for Systems Biology, Seattle, WA). Proteins isolated by B-OKA affinity precipitation were diluted in 20 mM Tris pH 8.3, 5 mM EDTA and digested with 20 ng/pl trypsin overnight at 37 °C. The resulting peptides were purified via electrostatic charge by off-line strong cation exchange-liquid chromatography using ICAT™ cation exchange syringe cartridges (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. Peptides were desalted and further purified using C18 ZipTip pipette tips (Mil l ipore, Bedford, MA) according to the manufacturer's recommendations. Purified peptides were subsequently fractionated by on-line reversed-phase liquid chromatography using a 100 min gradient of 5-35 % acetonitrile to elute peptides from a 10 cm microcapillary column packed with 200 A Magic C18 resin (Michrom Bioresources, Auburn, CA). Tandem mass spectra for peptides were acquired by on-line electrospray ionization tandem mass spectrometry using an LCQ ion trap mass spectrometer (Thermofinnigan, San Jose, CA) operating in data-dependent mode to select first-, second-and third-most intense ions from mass spectral survey scans for collision-induced dissociation. Peptide sequences, and the identities of corresponding proteins, were determined using S EQU EST software to search acquired tandem mass spectra against human sequence databases ( [197 ] , (http://fields.scripps.edu/sequest/index.html)). For each data set, potential protein identifications were analyzed and ranked using INTERACT and ProteinProphet software tools [198, 199] . 97 4.3.6. Precipitation and Blotting Methods for Testing an Interaction Between B-OKA and RanBP2 An association between RanBP2 and B-OKA was investigated by testing if biotinylated RanBP2 could be isolated from X. /aev/segq extracts treated with B-OKA. Extracts (1 mg protein) were incubated with biotinylated 1-methyl-l-cyclohexane carboxylic acid (as a negative control) or B-OKA (both at 10 UM) in XB buffer (10 mM Hepes, pH 7.7, 100 mM KCI, 1 mM MgCI 2 / 0.1 mM CaCI 2 / 50 mM sucrose, 1 mM PMSF, and chymostatin, aprotinin, leupeptin, and pepstatin each at 10 u.g/ml) for 8 h at 4 °C. Proteins were then precipitated using either of the two following methods. In the f irst method, RanBP2 was immunoaffinity precipitated by incubation with guinea pig serum containing polyclonal antibody against X. laevis RanBP2 (2 U.I, overnight) followed by protein A agarose (20 U.I, 2 h). In the second method, biotinylated proteins were affinity precipitated by incubation with streptavidin agarose (50 U.I, overnight). After precipitation using either method, beads were washed three times in XB buffer and once in 20 mM Tris-HCl, pH 6.8 containing protease inhibitors at the same concentrations as for XB. Bound proteins were eluted by boiling in SDS-PAG E sample loading buffer for 10 min with agitation. Proteins purified by RanBP2 immunoaffinity precipitation or by streptavidin agarose affinity precipitation were separated by SDS-PAG E (4-20 % gradient gels, Invitrogen) and detected by western blotting and streptavidin-HRP overlay. For western blotting, blots were blocked in nonfat milk protein (5 % in PBS, overnight) and incubated with RanBP2 antibody for 1 h (diluted 1:3,000 in PBS-T containing 1 % milk). After three washes in PBS-T, blots were incubated with H R P-conjugated goat anti-guinea pig antibody (diluted 1:10,000 in PBS-T containing 1 % milk) for 30 min and washed a final three times in PBS-T. For streptavidin overlay, blots were blocked in BSA (5 % in PBS, overnight) and incubated with 98 H RP-conjugated streptavidin (diluted 1:20,000 in PBS-T) for 1 h. After three washes in PBS-T, blots were incubated with chemiluminescent substrate (Pierce) and exposed to f i lm. In later experiments, the procedure was modified to reduce proteolysis of RanBP2. Treatments with the negative control compound and B-OKA were reduced to 3 h. Incubations with guinea pig serum, protein A agarose, and streptavidin agarose were also reduced to 1 h, 1 h, and 2 h, respectively. Complete protease inhibitor cocktail (Roche) was included in all solutions, according to the manufacturer's protocol. X. laeviseqq extracts and RanBP2 antibody were the generous gifts of Dr. M. Dasso (National Institutes of Health, Bethesda, MD). 4.4. RESULTS 4.4.1. Biotinylation Does Not Inhibit the Checkpoint and Antimitotic Activities of OKA The carboxyl group of OKA suggested a potential reactivity towards chemical modification. OKA was modified by reaction in basic refluxing acetone with /V-iodoacetyl-/V-biotinylhexylenediamine (Pierce), a cell membrane-permeable reagent with an extended spacer region. This was attached to OKA through an ester linkage at C18, a position that is far removed from the a,B-unsaturated carbonyl that is necessary for activity in OZ (Chapter 2). The structure of the reaction product was confirmed by NMR and mass spectrometry (Figure 4.1). The chemical reaction and structural analysis were performed by J . Nelson, laboratory of Dr. R. J . Andersen, Departments of Chemistry and Oceanography-Earth Ocean Sciences. 1-methyl-l-cyclohexane carboxylic acid is a hexane ring with methyl and carboxylic acid groups attached to a single carbon, a structure that is identical to the region of OKA that 99 A/-iodoacetyl-A/-biotinylhexylenediamine OKA o o biotinylated OKA (B-OKA) Figure 4.1 Chemical modification of OKA with A/-iodoacetyl-A/-biotinylhexylenediamine. O K A w a s m o d i f i e d by r e a c t i o n w i t h N - i o d o a c e t y l - N - b i o t i n y l h e x y l e n e d i a m i n e in b a s i c r e f l u x i n g a c e t o n e t o g e n e r a t e B - O K A . 100 reacts with the biotinylation reagent (Appendix I ) . This compound was also modified by reaction with /V-iodoacetyl-/l/-biotinylhexylenediamine. We anticipate that any proteins that interact with biotin, the linker, or with the first hexane ring (C1-C5) in B-OKA wil l also interact wi th biotinylated i-methyl-l-cyclohexane carboxylic acid. This was included as a negative control in my experiments to identify any non-specific protein interactions with B-OKA. One of the major pitfalls in developing labelled analogues is an inhibitory effect of the labelling on the activity of the compound. I f irst tested whether the biotinylation reaction interfered with checkpoint activity. 16 h after irradiation (12 Gy), HCT116-/- cells were treated with nocodazole (300 ng/ml) and a range of concentrations of biotinylated 1-methyl-1-cyclohexane carboxylic acid, OKA and B-OKA for 8 h. Entry into mitosis after irradiation was evaluated in mitotic spreads. B-OKA released up to 40 % of cells from arrest in G 2 (Fig. 4.2). A peak of activity was observed at 5 UM for B-OKA. Similar results were obtained for MCF-7 mp53 cells (not shown). A t concentrations above 10 u M , B-OKA-treated cells began to round up and detach. A concentration of 5 UM B-OKA was used for all subsequent experiments. Next, I examined in immunofluorescence microscopy whether the mitotic effects of OKA had been altered by biotinylation. In agreement with results for OZ and OKA, labelling of (3-tubulin and DNA revealed that B-OKA-treated cells arrested in prometaphase, with condensed chromosomes and normal mitotic spindles (not shown). Furthermore, B-OKA treatment caused a re-localization of CENP-E and hBubRl to the centrosomes (not shown). Finally, B-OKA did not affect Y-tubulin localization (not shown). In all respects, the effects of B-OKA on mitosis were indistinguishable from those of OKA and OZ. 1 0 1 0 1 10 100 [compound] (u,M) Figure 4.2 Biotinylation does not prevent checkpoint inhibition by B-OKA. 1 6 h after i r rad iat ion (12 Gy) , H C T I 1 6-1- cells were t reated with nocodazo le (300 ng /m l ) a n d biot inylated 1 -methyl-1 -cyc lohexane carboxyl ic ac id (white circles), O K A (black squares) o r B -OKA (black circles). Cells were col lected after 8 h a n d mitot ic spreads were p repared (Experimental Procedures, C h a p t e r 5 ) . M i to t i c f igures were coun ted under the microscope and are expressed as percentages of the to ta l n u m b e r of cells. Results are means ± S.D. f r o m two independent exper iments. 1 0 2 These results demonstrated that B-OKA retained OKA's two activities of interest: B-OKA is a checkpoint inhibitor and causes arrest in prometaphase. This is the f i rst instance of successful attachment of an affinity ligand, without loss of activity, to one of the checkpoint inhibitors discovered by our laboratory. B-OKA is therefore a useful tool for biochemical studies of the interactions of OKA within the cell. 4.4.2. Intracellular Localization of B-OKA I performed affinity labelling experiments to follow the uptake and distribution of B-OKA within treated cells. I f a specific subcellular localization were observed, it may suggest possible targets for B-OKA. A t the least, determination of a membrane, nuclear or cytoplasmic localization of this compound would be useful for purification of B-OKA targets. The intracellular localization of B-OKA was detected by fluorescent affinity labelling. MCF-7 mp53 cells were irradiated and treated with biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 |JM). After 6 h, cells were fixed and labelled with Alexa 594-conjugated streptavidin, and simultaneously immunostained with B-tubulin and Alexa 488-conjugated secondary antibodies. B-OKA treatment resulted in bright labelling of interphase and mitotic cells. Streptavidin staining was observed throughout the cell (Fig. 4.3). A very weak background signal was observed in the negative controls. Similar patterns were observed for both MCF-7 mp53 and HCTI 16-/- cells, with or without prior irradiation. Although these results are a clear confirmation that B-OKA is taken up by the cell, they unfortunately did not suggest protein targets to pursue. 103 DNA p-tubulin Biotin Merge Control B-OKA V — Figure 4 .3 B-OKA distributes freely throughout the cell. 16 h after irradiation, MCF-7 mp53 cells were treated with biotinylated 1-methyl-l-cyclohexane (control, 5 uM) or B-OKA (5 uM) for 8 h. Cells were immunostained with p-tubulin and Alexa 488-conjugated goat anti-mouse secondary antibodies, affinity labelled with Alexa 594-conjugated streptavidin, and chromosomes were stained with Hoechst 33258 (Experimental Procedures). Fluorescences associated with DNA (blue), microtubules (green) and biotin (red) were measured in confocal microscopy. Bar, 10 um. 104 4.4.3. Purification of B-OKA in Complexes with Protein Targets To begin to isolate targets of B-OKA, I f irst tested whether this compound binds covalently to proteins, as we had hypothesized for OZ (Chapter 2). After treatment with biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA for 6 to 8 h, proteins were extracted, separated by SDS-PAGE and electrotransferred to membranes. HRP-conjugated streptavidin detected multiple protein bands, in a manner dependent on B-OKA concentration (not shown). No signal was detected in the absence of B-OKA. The persistent binding of B-OKA to proteins after SDS-PAG E, which involves boiling in 1 % SDS and 100 mM DTT, is consistent wi th covalent binding of B-OKA to its targets. Next, B-OKA complexes were purified from treated cells by streptavidin agarose affinity precipitation. I f i rst investigated whether the synthesis of B-OKA-binding proteins was regulated by the cell cycle. HCTI 16-/- cells were irradiated (12 Gy) or treated with nocodazole (300 ng/ml). After 16 h, these cells and no-treatment controls were given biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 |JM) for 8 h. Biotinylated proteins were purified from cell extracts by streptavidin agarose affinity precipitation, and detected by streptavidin overlay (Experimental Procedures). Biotinylated proteins were detected only in the samples treated with B-OKA and not in the biotinylated 1-methyl-l-cyclohexane carboxylic acid controls (Fig. 4.4). Numerous biotinylated proteins were detected both in the biotinylated cell extracts and in the purified samples. Most, but not al l , prominent bands in the cell lysate samples were recovered in the purification. The most strongly labelled proteins in the purified samples had apparent relative molecular masses of approximately 35, 45, 52, 60, 6 1 , 95 and 116 (x 10 3 ) . I then tested whether additional proteins had co-purified with those directly bound to B-105 cell lysate streptavidin bound control B-OKA control B-OKA C Y N C Y N C Y N C y N M f Figure 4.4 Streptavidin agarose affinity purification of B-OKA-binding proteins. H C T I 1 6-1- c e l l s w e r e g i v e n n o t r e a t m e n t (C), i r r a d i a t e d (1 2 G y , (y)), o r c u l t u r e d in t h e p r e s e n c e o f n o c o d a z o l e ( 3 0 0 n g / m l , ( N ) ) . A f t e r 1 6 h , ce l l s w e r e i n c u b a t e d w i t h b i o t i n y l a t e d 1 - m e t h y l - 1 - c y c l o h e x a n e c a r b o x y l i c a c i d (control) o r B - O K A (5 U.M) f o r 8 h. B i o t i n y l a t e d p r o t e i n s w e r e p u r i f i e d b y s t r e p t a v i d i n a g a r o s e a f f i n i t y p r e c i p i t a t i o n , s e p a r a t e d by S D S - P A G E a n d e l e c t r o t r a n s f e r r e d t o m e m b r a n e s ( E x p e r i m e n t a l P r o c e d u r e s ) . B i o t i n y l a t e d p r o t e i n s w e r e d e t e c t e d by s t r e p t a v i d i n - H R P o v e r l a y a n d e x p o s e d t o f i l m . T h e resul ts a r e t y p i c a l o f six i n d e p e n d e n t e x p e r i m e n t s . M r , r e l a t i v e m o l e c u l a r m a s s (x 1 0 3 ) . 1 0 6 OKA. Streptavidin affinity precipitation was performed on HCT116-/- cells treated with biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 uM) (Experimental Procedures). Proteins eluted from the streptavidin beads were separated by SDS-PAG E and detected by silver staining (Fig. 4.5). Very few proteins were detected in the negative control samples (none in the figure shown here, and faint bands migrating at approximately 45 and 100 (x 10 3 ) in other experiments). Prominent protein bands migrated at approximately 40, 45, 49, 50, 54, 55, 57, 59, 60, 70, 85, 92, and 200 (x 10 3 ) . A greater number of protein bands was detected by silver staining than by streptavidin-HRP overlay, suggesting that some of the direct targets of B-OKA are in complexes with other proteins. In several independent experiments with both MCF-7 mp53 and HCT116-/- cells, I observed no difference in the proteins purified from cycling, irradiated, or nocodazole-treated cells. I concluded that the expression of B-OKA targets does not appear to be regulated by the cell cycle. 4.4.4. Mass Spectrometric Identification of B-OKA-Binding Proteins In the next investigation, we set out to identify some of the targets that interact with B-OKA. Proteins were purified using two different strategies, outlined below, and identified by mass spectrometry (Dr. Mark Flory, laboratory of Dr. R. Aebersold, Institute for Systems Biology, Seattle, WA). In the f irst approach (batch purification), I performed large-scale streptavidin affinity precipitations on cycling HCT116-/- cells treated with biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 UM) (Experimental Procedures). Purified proteins were digested with trypsin, purified by cation exchange and C18 chromatography, and fractionated 107 cell lysate streptavidin bound control B-OKA control B-OKA M r C Y N C Y N C Y N C Y N Figure 4.5 SDS-PAGE analysis of proteins co-purified with B-OKA. H C T I 167-ce l l s w e r e i r r a d i a t e d ( 1 2 G y , (y)), c u l t u r e d in t h e p r e s e n c e o f n o c o d a z o l e ( 3 0 0 n g / m l , (N)), o r g i v e n n o t r e a t m e n t ( Q . A f t e r 1 6 h , ce l l s w e r e i n c u b a t e d w i t h b i o t i n y l a t e d 1 - m e t h y l - 1 - c y c l o h e x a n e c a r b o x y l i c a c i d [control) o r B - O K A f o r 8 h (5 uM) f o r 8 h . P r o t e i n s w e r e p u r i f i e d b y s t r e p t a v i d i n a g a r o s e a f f i n i t y p r e c i p i t a t i o n , s e p a r a t e d by S D S - P A G E a n d d e t e c t e d b y s i l v e r s t a i n i n g ( E x p e r i m e n t a l P r o c e d u r e s ) . T h e resul ts a r e t y p i c a l o f f o u r i n d e p e n d e n t e x p e r i m e n t s . M r , r e l a t i v e m o l e c u l a r m a s s (x 1 0 3 ) . 108 by on-line reversed phase chromatography. Mass spectra and sequence data for eluted peptides were collected by tandem mass spectrometry [200 ] . Proteins were identified by comparison of the spectra and sequence data to the S EQU EST databank. This purification and analysis was performed three-separate times. In the second approach (band excision), I set out to identify the proteins that bind to B-OKA directly. HCT116-/- cells were treated with biotinylated 1-methyl-l-cyclohexane carboxylic acid or B-OKA (both at 5 UM) for 8 h, and B-OKA binding proteins purified by streptavidin agarose affinity precipitation. Protein samples were separated by SDS-PAG E, electrotransferred to membranes, and overlayed with H R P-conjugated streptavidin. Biotinylated proteins were detected directly on the blot by incubation with the chromogenic substrate 3,3,5,5 tetramethylbenzidine (TMB) (Fig. 4.6). Labelled bands were excised and identified by mass spectrometry as described above. The combined dataset from the batch purification and band excision experiments resulted in a list of numerous potential targets for B-OKA. To narrow this list down to reproducibly identified proteins, I applied the following criteria: candidates must have been identified in both purification approaches (batch purification and band excision), and they must not have been detected in the biotinylated 1-methyl-l-cyclohexane carboxylic acid control samples. Six proteins met these criteria (Table 4.1). Based on their repeated identification in several independent experiments using two different methods, I consider these proteins to be plausible candidate targets for B-OKA. Interestingly, three of the six candidates have potential E3 ligase activity in common. 109 B-OKA control M r Figure 4.6 Chromogenic labelling of B-OKA-binding proteins. H C T 1 1 6-1 - ce l l s w e r e t r e a t e d w i t h b i o t i n y l a t e d 1 - m e t h y l - 1 -c y c l o h e x a n e c a r b o x y l i c a c i d (con t ro l ) o r B - O K A (5 uM) f o r 8 h. B i o t i n y l a t e d p r o t e i n s w e r e p u r i f i e d by s t r e p t a v i d i n a g a r o s e a f f i n i t y p r e c i p i t a t i o n , s e p a r a t e d by S D S - P A G E a n d e l e c t r o t r a n s f e r r e d t o m e m b r a n e s . B i o t i n y l a t e d p r o t e i n s w e r e l a b e l l e d by s t r e p t a v i d i n -HRP o v e r l a y a n d i n c u b a t e d w i t h c h r o m o g e n i c s u b s t r a t e ( E x p e r i m e n t a l P r o c e d u r e s ) . Arrows i n d i c a t e p o s i t i o n s o f p r o t e i n b a n d s t h a t w e r e e x c i s e d a n d a n a l y z e d by m a s s s p e c t r o m e t r y . T h e resu l ts a r e r e p r e s e n t a t i v e o f t h r e e s e p a r a t e e x p e r i m e n t s . M r , r e l a t i v e m o l e c u l a r m a s s (x 1 0 3 ) . 110 Table 4.1 Candidate targets for inhibition by B-OKA. P r o t e i n s t h a t b i n d t o B - O K A w e r e p u r i f i e d by s t r e p t a v i d i n a g a r o s e a f f i n i t y p r e c i p i t a t i o n a n d i d e n t i f i e d by m a s s s p e c t r o m e t r y . T h e l is ted p r o t e i n s w e r e r e p r o d u c i b l y i d e n t i f i e d by b o t h b a t c h p u r i f i c a t i o n a n d b a n d e x c i s i o n o f B - O K A -b i n d i n g p r o t e i n s ( E x p e r i m e n t a l P r o c e d u r e s ) . W h e r e p o s s i b l e , p o t e n t i a l f u n c t i o n s a r e i n d i c a t e d . Protein: Predicted Function 1. FKBP-rapamycin associated protein protein kinase 2. similar to PHD finger protein E3 ubiquitin ligase 3. phosphate carrier protein, mitochondrial precursor phosphate transport 4. similar to nasopharyngeal carcinoma susceptibility protein unknown 5. similar to putative E3 ligase E3 ubiquitin ligase 6. Ran-binding protein 2 nuclear transport, SUMO E3 ligase 111 4.4.5. Testing an Interaction Between B-OKA and the Candidate Target RanBP2 I next investigated whether one of the candidate targets identified by mass spectrometry could be confirmed using a different biochemical approach. I focussed my investigation upon Ran binding protein 2 (RanBP2), a large protein that associates with the cytoplasmic face of the nuclear pore complex (NPC) during interphase [187-189] . RanBP2 has been localized to kinetochores, spindles and centrosomes during mitosis, suggesting a new mitotic role for this protein [190 ] . RanBP2 has SUMO E3 ligase activity [192, 194, 195] , and SUMOylation could affect interactions or localization of proteins that play a role in the G 2 checkpoint and/or chromosome congression. Furthermore, disruption of the Ran system by RNAi in C. elegans embryos prevents chromosome congression [186 ] . We had proposed earlier that the effects of OZ and OKA on the DNA damage checkpoint and on chromosome congression were caused by action on a single protein. Because the phenotypes exerted by OZ and OKA are consistent with the function of RanBP2, and reagents for studying RanBP2were available, I tested an interaction between OKA and this candidate. I f irst tested whether OZ and OKA treatment inhibited nucleocytoplasmic transport. Cyclin B l is transported back and forth between the nucleus and cytoplasm through the NPC. MCF-7 mp53 cells were irradiated (0 or 10 Gy) and after 16 h were treated with the nuclear export inhibitor leptomycin B (100 ng/ml), OZ (10 UM) or OKA (20 U.M) for 1, 2, or 4 h, and cyclin B l localization was examined by immunofluorescence microscopy. Leptomycin B treatment resulted in a strong nuclear accumulation of cyclin B l (not shown). In contrast, OZ- or OKA-treated cells were indistinguishable from controls in all conditions and time points (not shown), indicating that these compounds do not affect nuclear import or export. Next, I tested whether an interaction between RanBP2 and B-OKA could be confirmed 112 using a technique other than mass spectrometry. These experiments used X. laevis egg extracts, which are a better source of soluble RanBP2 protein than mammalian cells. After treatment with B-OKA or biotinylated 1-methyl-l-cyclohexane carboxylic acid for 8 h, RanBP2 was immunoaffinity precipitated overnight wi th guinea pig serum containing RanBP2 polyclonal antibody and protein A agarose. Eluted proteins were separated by SDS PAGE, electrotransferred to membranes, and probed by streptavidin-HRP overlay. Several biotinylated protein bands were detected in extracts treated with B-OKA but not in the negative controls (Fig. 4.7 A, supernatant). Three prominent biotinylated bands were precipitated by RanBP2 antibody. Their interaction with B-OKA appears to be specific, because RanBP2 precipitated from 1-methyl-l-cyclohexane carboxylic acid-treated extracts was not biotinylated (Fig. 4.7 A, bound). I then performed the converse experiment. X. laevis extracts were treated with B-OKA or biotinylated 1-methyl-l-cyclohexane carboxylic acid as described above. Biotinylated proteins were affinity precipitated with streptavidin agarose and probed by RanBP2 western blotting. RanBP2 antibody recognized faint protein bands in streptavidin precipitates from the B-OKA treated extracts but not in the controls (Fig. 4.7 B). Taken together, these two approaches yielded agreeing results and suggest that B-OKA-labelled RanBP2 was recovered In both the reciprocal precipitation approaches described above, the biotinylated RanBP2 signal was much smaller than expected for the full-length protein (358 kDa, e.g. Fig 4.7. B, lane E). RanBP2 is extremely sensitive to proteolysis ([1913, and personal communication from the laboratory of Dr. M. Dasso), which could account for its observed reduction in M r Therefore, I tested whether the experimental conditions could be altered to recover ful l -length RanBP2. Al l incubation times were greatly reduced, and a broader range of protease 113 / / B & M. E M B M r M B M B 173 111 80 61 49 36 25 19 13 9 173 — 1 111 80 — 61 — 49 — 36 — 25 — 19 — 13 — 9 — Figure 4.7 Reciprocal precipitation of biotinylated RanBP2. X. / aev i s e g g ex t rac ts (1 m g ) w e r e t e s t e d f o r a n i n t e r a c t i o n b e t w e e n R a n B P 2 a n d B-OKA. Ext racts (£) w e r e i n c u b a t e d w i t h b i o t i n y l a t e d 1 - m e t h y l - l - c y c l o h e x a n e c a r b o x y l i c a c i d (M, 5 uM) o r B -OKA (8, 5 uM) f o r 8 h. A j , R a n B P 2 w a s i m m u n o a f f i n i t y p r e c i p i t a t e d by i n c u b a t i o n w i t h g u i n e a p i g s e r u m c o n t a i n i n g p o l y c l o n a l R a n B P 2 a n t i b o d y a n d p r o t e i n A a g a r o s e . T h e s u p e r n a t a n t a n d b o u n d f r a c t i o n s w e r e p r o b e d f o r b i o t i n by H R P - c o n j u g a t e d s t r e p t a v i d i n o v e r l a y ( E x p e r i m e n t a l P r o c e d u r e s ) . 8), In t h e c o n v e r s e a p p r o a c h , b i o t i n y l a t e d p r o t e i n s w e r e p r e c i p i t a t e d by i n c u b a t i o n w i t h s t r e p t a v i d i n a g a r o s e , a n d R a n B P 2 d e t e c t e d by w e s t e r n b l o t t i n g . M r , r e la t i ve m o l e c u l a r m a s s (x 1 0 3 ) . 114 inhibitors was included in all steps. After incubation with biotinylated 1-methyl- l-cyclohexane carboxylic acid or B-OKA (both at 5 MM) for 3 h, RanBP2 was immunoaffinity precipitated with guinea pig polyclonal antibody, and biotinylated proteins were precipitated with streptavidin agarose (Fig. 4.8). Proteins were separated by SDS-PAG E and electrotransferred to the same membrane. One half of the membrane, containing the streptavidin precipitates, was probed with streptavidin-HRP (lanes M a n d B). A RanBP2 western blot was performed on the other half of the blot, containing egg extract proteins and immunoaffinity precipitated RanBP2 (lanes £ a n d I). In contrast to earlier experiments, ful l -length RanBP2 was detected in both the egg extracts and in RanBP2 antibody precipitates (Fig 4.8, £ a n d D. This strongly suggests that the reduced M r o f RanBP2 in the initial experiments was caused by proteolysis. No biotinylated proteins were precipitated from extracts treated with 1-methyl-l-cyclohexane carboxylic acid (Fig 4.8 lane M). In contrast, several biotinylated proteins were isolated from B-OKA-treated extracts. Only one prominent band was detected above the 111,000 Mr standard (Fig. 4.8 B). This biotinylated protein co-migrated with full-length RanBP2. However, I was unable to recover this signal in reciprocal precipitations, perhaps due to the inefficiency of immunoaffinity precipitation for full-length RanBP2. 4.5. DISCUSSION Here I have presented experiments designed to identify the target(s) of OKA. Because the structure of OKA is very similar to OZ, and OKA faithfully reproduces every aspect of the biological activities of OZ, i t is likely that by identifying the target(s) for OKA we wil l also 115 M M B M E I r r Figure 4.8 Co-migration of RanBP2 and a B-OKA-labelled protein. X. laevis e g g e x t r a c t s (E, 1 m g ) w e r e i n c u b a t e d w i t h b i o t i n y l a t e d 1 - m e t h y l - l -c y c l o h e x a n e (A/I), o r B - O K A (8) f o r 3 h b e f o r e i n c u b a t i o n w i t h g u i n e a p i g s e r u m c o n t a i n i n g p o l y c l o n a l R a n B P 2 a n t i b o d y ( l a n e /) o r s t r e p t a v i d i n a g a r o s e ( l a n e s M a n d 8 ) ( E x p e r i m e n t a l P r o c e d u r e s ) . P r o t e i n s w e r e s e p a r a t e d by S D S - P A G E a n d e l e c t r o t r a n s f e r r e d t o t h e s a m e m e m b r a n e . L a n e s A/I a n d 8, s t r e p t a v i d i n p r e c i p i t a t e s p r o b e d f o r b i o t i n w i t h HRP-c o n j u g a t e d s t r e p t a v i d i n . L a n e s E a n d / , e g g e x t r a c t p r o t e i n s a n d R a n B P 2 i m m u n o a f f i n i t y p r e c i p i t a t e s , r e s p e c t i v e l y , l a b e l l e d in a R a n B P 2 w e s t e r n b l o t . Asterix, R a n B P 2 . M r , r e l a t i v e m o l e c u l a r m a s s (x 1 0 3 ) . 116 find the target(s) for OZ. Modification of OKA by biotinylation did not reduce its activity. Although it is not direct proof, this fact may provide another clue to structure-activity relationships in OZ and OKA. Both the checkpoint and antimitotic activities of OKA are preserved after attachment of a bulky linker and biotin group to the carboxylic acid at position C18. By analogy with observations for OZ, one would anticipate that modification of the a,(3-unsaturated carbonyl would inhibit checkpoint activity in OKA. This implies that the region around C l l through C17, and the attached groups, are more important for interactions with checkpoint and mitotic targets than is the first hexane ring (C1-C5) and the carboxylic acid. Probing B-OKA-treated cells with fluorescently labelled streptavidin allowed tracking of the compound's distribution throughout the cell. Unfortunately, the widespread distribution of B-OKA did not suggest targets, as we had hoped. It is possible that B-OKA disperses its targets from their native cellular localization. An alternative, and perhaps more likely, explanation refers to the weak specificity of B-OKA. As shown in the streptavidin overlay experiments (Fig. 4.4), this compound interacts with a number of proteins, and its diffuse intracellular labelling could result from the superimposition of the distributions of all B-OKA targets. Attachment of a biotin group to OKA, and the covalent nature of the interaction between OKA and its targets, permitted observation of the M , of proteins that interact with B-OKA. The most striking result was that B-OKA binds to numerous proteins. This is perhaps not surprising, as most small molecule inhibitors do not interact with only a single protein. As well, both OZ and OKA are cytotoxic, which suggests interactions with other cellular targets. In these experiments, biotinylated RanBP2 has been detected by streptavidin batch affinity 117 purification, by excision of biotinylated proteins from blots, by RanBP2 immunoaffinity precipitation, and by RanBP2 western blotting. The M,. of the labelled bands was much lower than would be expected for full-length RanBP2 (358,000). In the band excision experiments, RanBP2 was identified in three separate bands in one experiment, and in one band in a subsequent experiment. Al l of these bands migrated well below the M,of RanBP2. The high degree of sensitivity of RanBP2 to proteolysis was noted during its original purification [191 ] , and has been a l imit ing factor in its expression and purification (personal communication, Dr. J . Jamon, laboratory of Dr. M. Dasso, National Institutes of Health, Bethesda, MD). By adjusting the experimental procedures to minimize the risk of proteolysis, I was able to recover a small amount of full-length RanBP2, which co-migrated with a biotinylated band in B-OKA treated extracts. RanBP2 is unusually large, and there was only one protein with molecular mass above 111 kDa that reacted with B-OKA. This suggests that the co-migration of RanBP2 with the biotinylated band is not simply coincidental. Taken together, these combined results strongly support a direct interaction between RanBP2 and B-OKA. 4.6. ACKNOWLEDGEMENTS I would like to thank J im Nelson for preparation of B-OKA, Dr. Mark Flory for mass spectrometric analysis, and Dr. Mary Dasso for providing RanBP2 antibody and X. laevis extracts. 118 Chapter 5 INVESTIGATION OF A G 2 DNA DAMAGE CHECKPOINT ROLE FOR GSK-3 p KINASE 5.1. SUMMARY In this chapter, a potential role for GSK-3 Bin the Gz DNA damage checkpoint was tested. This kinase was over-expressed in MCF-7 mp53 cells by transient or stable transfection, and a clonal cell line (called 3G3) that stably over-expressed the kinase zvas created. Immunofluorescence microscopy demonstrated that cells expressing large amounts of GSK-3 (3 were less likely than controls to enter mitosis after DNA damage. Similarly, mitotic spreads showed that fewer 3G3 cells were in mitosis after irradiation. I developed a method for triply labelling cells for flow cytometry, to allow simultaneous measurement of cell cycle markers and GSK-3 (3 expression in transfected cells. In these experiments, over-expression of GSK-3 j3 increased the population of cells in G1 and reduced entry into mitosis after irradiation. The effects of didemnimide A, an analogue of the GSK-3 (3 inhibitor isogranulatimide, were also examined. Didemnimide A lacked checkpoint inhibiting activity. Because in vitro kinase assays indicated that didemnimide A is a potent inhibitor of GSK-3 (3,1 conclude that GSK-3 B does not function in the G2 DNA damage checkpoint pathway. 5.2. INTRODUCTION In 1998, the Roberge and Andersen laboratories undertook the f i rst high throughput cell-based screen for G 2 checkpoint inhibitors and tested a collection of 1,300 extracts from marine organisms [ 1 5 5 ] . This yielded several new checkpoint inhibitors including 119 debromohymenialdisine (DBH) and isogranulatimide (IGR) ( IC 5 0 values 8 (JM and 2 u M , respectively, in ELICA checkpoint inhibition assays) [155, 1563. IGR was chosen for further development as a potential therapeutic compound based on its abil ity to synergistically increase cell killing by DNA damage in a p53-selective manner, and its relatively low level of toxicity towards non-irradiated cells [155 ] . Kinetech, Inc. licensed IGR for development and assayed its effects on 14 different recombinant kinases in vitro. In these assays, IGR significantly inhibited only glycogen synthase kinase P (GSK-3 P, I C 5 0 0.5 (JM). Similar assays showed that DBH also inhibited GSK-3 (3. The structures of IGR and DBH are quite different (Fig. 5.1 A, B), yet both compounds are inhibitors of GSK-3 (3. I set out to determine whether this kinase has a function in the G 2 checkpoint pathway. GSK-3 (3 was originally identified as one of the kinases that phosphorylate glycogen synthase in rabbit skeletal muscle [ 2 0 1 , 202] . I t is a dual-specificity kinase that is well conserved among species [203 ] . I t is activated by phosphorylation on Tyr216 [204] and inactivated by phosphorylation on Ser9 [203, 205] . Kinases that inactivate GSK-3 pi include cAMP-dependent protein kinase, p90 r s k , protein kinase B and integrin-linked kinase [206-208] . The name of this kinase does not adequately describe the diversity of its targets. GSK-3 P phosphorylates the microtubule-associated protein tau, several transcription factors including Jun and Myc, P-catenin, cyclin D l , the RII regulatory subunit of cAM P-dependent protein kinase, and the protein translation initiation factor eIF2B (reviewed in [209-212] . GSK-3 P is part of the Wnt and APC/P-catenin pathway that determines cell fate in developing embryos and is oncogenic (reviewed in [213, 214]) . GSK-3 P also regulates TCF/LEF1, NF-KB and p53, transcription factors that play a role in the cell cycle [215-218] . In S. cerevisiae, genetic screens have identified the GSK-3 P homologue M c k l in a 120 Figure 5.1 Structural formulae for debromohymenialdesine (A), isogranulatimide (6), and didemnimide A (C). 121 pathway that inhibits the Cdc28-Clb complex [ 2 1 9 1 No involvement of GSK-3 p in the DNA damage checkpoint has been reported, but since its targets are so diverse, we reasoned that there may be other functions of G S K-3 P yet to be discovered. Because two different inhibitors of GSK-3 P kinase activity are also checkpoint inhibitors, I tested a G 2 checkpoint function for GSK-3 p. After transfection, the effects of transient or stable over-expression of this kinase on responses to ionizing radiation were tested in ELICA checkpoint assays, immunofluorescence microscopy, mitotic spreads, and flow cytometry. I also explored whether didemnimide A, an analogue of the GSK-3 P inhibitor IGR, exerts effects on the checkpoint response. 5.3. EXPERIMENTAL PROCEDURES 5.3.1. Cell Transfection and Generation of Pooled or Clonal Stable Cell Lines Cells were transfected with hemagglutinin (HA) epitope tagged wild-type GSK-3 P or a kinase-dead mutant (K85A) in pcDNA3 vector [220 ] (gifts of Dr. S. Dedhar), and simultaneously co-transfected with pCMVLacI repressor vector encoding hygromycin resistance (gift of Dr. C. Roskelley). MCF-7 mp53 cells were seeded into 35 mm 2 tissue culture dishes and grown to 80 % confluence. A mixture of 3 p i lipofectamine (Gibco), 1.8 ug GSK-3 P DNA, and 0.2 Ug pCMVLacI DNA was prepared in 1 ml of transfection medium (DMEM containing no antibiotics or serum). After washing with three changes of transfection medium, cells were incubated with the DNA and lipofectamine mixture at 37 °C. After 6 h incubation, 1 ml of media containing 20 % FBS was added to each well. To generate stable cell lines, transfected cells were cultured in media containing 300 ug/ml hygromycin B (Sigma). For the cells referred to as pooled stable cell lines, no further 122 separation was performed. Clones were obtained from pooled stable cell lines by limiting dilution plating in 96-well plates. Wells that contained a single cell were selected for amplification and the expression of GSK-3 P was tested by western blotting. 5.3.2. Western Blotting and Immunoaffinity Precipitation MCF-7 mp53 cells were washed twice in ice-cold PBS containing 0.2 mM PMSF and lysed by incubation with RIPA (10 mM Tris, pH 8.0, 0.1 % SDS, 1 % NP-40, 1 % sodium deoxycholate, 150 mM NaCl, 0.02 % sodium azide) on ice for 1 h. Lysis supernatants were collected after centrifugation at 13,000 gfor 1 h, separated by SDS-PAGE (8 % gels) and transferred to Immobilon membrane (Mil l ipore). After blocking the membrane in 5 % nonfat milk protein in PBS containing 0.1 % Tween-20 (PBS-T), blots were incubated with PBS-T containing 1 % milk and HA antibody (12CA5 ascites diluted 1:5,000) or GSK-3 P antibody (Transduction Laboratories, diluted 1:2,500) for 1 h. After washing in three changes of PBS-T, blots were incubated with H RP-conjugated goat anti-mouse antibody (Sigma, diluted 1:3,000 in PBS-T) for 30 min. After a final three washes in PBS-T, HRP was detected by incubation with chemiluminescent substrate (Pierce) and exposure of blots to f i lm. GSK-3 P immunoprecipitations were performed according to the method of Kinetech, Inc. The volumes of cell lysates (250-500 pg protein) were brought to 1 ml with isotonic homogenization buffer (100 mM Tris, pH 8.0, 0.5 % Tx-100, 150 mM NaCl, 0.2 mM PMSF, 1 mM sodium orthovanadate, 1 Ug/ml leupeptin, 1 Ug/ml aprotinin), mixed with GSK-3 P or HA antibodies (1:500 and 1:2,000 dilution, respectively), and nutated for 2 h a t 4 °C. Samples were then incubated with protein A Sepharose (50 ul, for GSK-3 P) or a 1:1 mixture of protein A and protein G Sepharose (50 ul total volume, for HA) for 45 min at 4 123 °C. Samples were washed in 4 changes of 3 % NETF (3 % Nonidet P-40 (v/v), 100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCI, pH 7.4, 50 mM NaF). Proteins were eluted by boiling the beads in 1 vol SDS-PAG E sample buffer, and analyzed by western blotting. 5.3.3. Mitotic Spreads Cells were collected by trypsinization and incubated in 1 ml 75 mM KC I for 10 min, and fixed by incubation in freshly prepared ice-cold Carnoy's fixative (1:3 acetic acid:methanol (v:v), 10 min). Cells were then resuspended in 50 ul of the same solution, spotted onto glass slides from a height of 1 m and allowed to dry. Chromosomes were stained by incubation with Hoechst 33258 (1 ug/ml in PBS) for 7 min. After draining this solution away, slides were mounted in Fluormount and sealed with nail polish. Mitotic figures were counted under the microscope and calculated as percentages of the total number of cells. 5.3.4. Immunofluorescence Examination of GSK-3 Q and HA Epitope Expression in Transiently Transfected Cells MCF-7 mp53 cells were plated onto cover slips (800,000 cells/ 2 ml media) in 35 mm tissue culture wells. The following day, cells were transfected with pcDNA3.1 vector, HA-tagged wild-type or (K85A) GSK-3 P (Section 5.3.1). 12 h after transfection began, cells were re-plated into 10 cm 2 dishes. 24 h after transfection began, a set of controls was collected. The remaining cells were irradiated (3.3 Gy), treated with nocodazole (100 ng/ml), and collected at 36 and 48 h post-transfection. The following method was used to label cells for immunofluorescence. After a wash in PBS, cells were fixed by incubation in formaldehyde solution (3.7 % in PBS) for 15 min at 4 °C 124 and washed twice in KB (10 mM Tris HCI, pH 7.5, 0.1 % BSA, 0.1 % Tx-100, 150 mM NaCl) . Cells were immunostained by incubation with GSK-3 (3 (Transduction Laboratories) or HA (12CA5 ascites fluid) antibodies (diluted 1;200 or 1:1,000, respectively, in KB) for 30 min. After washing twice in KB, cells were incubated with Cy3-conjugated goat anti-mouse antibody (diluted 1:500 in KB) for 30 min. After a further two washes in KB, chromosomes were labelled by incubation with Hoechst 33258 (100 ng/ml in KB) for 7 min. After a final wash in KB, slides were mounted in Fluormount and sealed with nail polish. Cells that stained brightly for GSK-3 P or the HA epitope were counted and classified as interphasic or mitotic based on their chromosome condensation, and mitotic cells expressed as a percentage of the total number of transfected cells. 5.3.5. Preparation of the Biotinylated GF-7 Antibody and its Application in Flow Cytometry The GF-7 mitosis-specific antibody was biotinylated according to the method of M. Roederer (http://www.drmr.com/abcon). Partially purified GF-7 from hybridoma tissue culture supernatant (gift of Dr. P. Davies) was separated by gel f i l t rat ion on Sephadex (Pharmacia) and biotinylated by incubation with N-hydroxysuccinimido-biotin (Pierce, 80 ug biotin/mg antibody in 100 mM carbonate, pH 8.4) for 4 h in darkness. A second gel f i l tration step exchanged the biotinylated GF-7 antibody (B-GF-7) into storage buffer (10 mM Tris, pH 8.2, 150 mM NaCl, 500 ng/ml pentachlorophenol). For flow cytometry labelling, MCF-7 mp53 cells were collected by trypsinization, washed once in ice-cold PBS containing PMSF (0.2 mM), fixed in ice-cold ethanol (70 %) and stored overnight at 4 °C. After one wash in standard azide buffer containing Tween-20 (Tw-SAB, PBS containing 0.5 % Tween-20, 1 % fetal calf serum and 0.5 % sodium azide), cells were 125 permeabilized by incubation in the same buffer for 30 min on ice. Mitotic epitopes were labelled by incubating 1 x 10 6 cells with B-GF-7 in 100 (Jl labelling buffer (diluted 1:50, Tw-SAB containing 6 % fetal calf serum) for 1 h on ice. After two washes in Tw-SAB, cells were labelled with PE-conjugated avidin (Molecular Probes, 10 ug/ml in 200 (Jl Tw-SAB) for 1 h on ice. After two washes in Tw-SAB, cells were treated with RNAse A (Roche, 500 units/ml) in sodium citrate buffer (4 m M , pH 8.4) for 30 min at 37 °C. DNA was stained by adding an equal volume of 7-AAD (Molecular Probes, 10 ug/ml final concentration) and incubating on ice for 20 min. Cells were stored at 4 °C until the following day. 5.3.6. Flow Cytometric Evaluation of the Effects of Over-Expressing GSK-3 P on the Cell Cycle MCF-7 mp53 cells (5 x 10 6 ) were seeded into 10 cm 2 tissue culture dishes and grown to 80 % confluency. Cells were transfected with pcDNA3.1 vector or with HA-tagged wild-type GSK-3 P (50 ug DNA and 75 p i lipofectin) (Section 5.3.1) for 6 h and re-plated into several 10 cm dishes. 24 h after transfection, cells were irradiated and treated with nocodazole (100 ng/ml). 16 h after irradiation, cells were labelled for indirect immunofluorescence as described for the B-G F-7 antibody (Section 5.3.5), with modifications in the primary and secondary labels that were applied. To label HA and mitotic epitopes, 2 x 1 0 6 cells were incubated with rat HA (Roche) and mouse GF-7 antibodies simultaneously (both diluted 1:100 in 200 pi labelling buffer) for 1 h on ice. These were detected by Alexa 488-conjugated goat anti-rat and Alexa 633-conjugated goat anti-mouse secondary antibodies (Molecular Probes, 10 pg/ml in 200 pi labelling buffer) for 30 min on ice. After three washes in Tw-SAB, DNA was stained with PI as previously described (Section 2.3.2.). 126 5.4. RESULTS 5.4.1. Effects of Stable Over-expression of GSK-3 3 on G2 Arrest in Pooled Stable Cell Lines To investigate whether GSK-3 3 plays a role in the DNA damage checkpoint, I over-expressed the kinase and tested for an effect on G 2 arrest after irradiation. MCF-7 mp53 cells were co-transfected with hygromycin B resistance vectors and either wild-type or kinase-dead (K85A) GSK-3 p. In these constructs, a hemagglutinin (HA) epitope was fused to GSK-3 3 as an expression marker. Stably transfected cells were selected by culture for at least two weeks in the presence of hygromycin B (300 Ug/ml). Entry into mitosis was evaluated by ELICA assay 16 h after irradiation [155 ] . As the radiation exposure increased, fewer MCF-7 mp53 cells transfected with the hygromycin resistance vector entered mitosis, with an approximate I C 5 0 of 5 Gy (Fig. 5.2). There was no significant difference between these controls and the wild-type and K85A GSK-3 3 transfectants. There were two factors that made the interpretation of these results difficult, however: there was significant variation between experiments, and the level of expression of the transfected gene was also variable. 5.4.2. Generation of a Stable Clonal Cell Line Over-expressing GSK-3 3 To overcome the problem of variable expression levels in the pooled transfected cells described above, I set out to clone cells over-expressing wild-type HA-tagged GSK-3 3-Transfected cells were plated to limiting dilution in 96-well plates. Seven hygromycin-resistant clones were screened in western blots for G S K-3 3- A positive clone was identified and named 3G3. In GSK-3 3 western blots, this clone contained endogenous GSK-3 3 and a slower-migrating band corresponding to the HA-tagged form, in approximately equal 127 0 i I i 1 1 1 1 1 1 1 1 r 0 3 6 9 12 Radia t ion D o s e (Gy) Figure 5.2 Stable over-expression of wild-type or K85A GSK-3 6 does not affect entry into mitosis after irradiation. M C F - 7 m p 5 3 cel ls w e r e c o -t r a n s f e c t e d w i t h w i l d - t y p e o r K 8 5 A G S K - 3 P a n d a h y g r o m y c i n res is tance v e c t o r . H y g r o m y c i n resistant cel ls w e r e t r e a t e d w i t h t h e i n d i c a t e d a m o u n t s o f i o n i z i n g r a d i a t i o n a n d g i ven n o c o d a z o l e ( 1 0 0 n g / m l ) a t t h e t i m e o f r a d i a t i o n . A f te r 1 6 h, entry in to mi tos is w a s e v a l u a t e d by ELICA assay [ 1 5 5 ] . Results a r e expressed as p e r c e n t a g e s o f t h e O G y c o n t r o l s f o r e a c h cel l l i ne , a n d a r e a v e r a g e va lues f o r t w o i n d e p e n d e n t e x p e r i m e n t s 128 abundance (Fig. 5.3). I performed GSK-3 (3 immunoaffinity precipitation-kinase assays and found that 3G3 contained approximately twice the kinase activity of non-transfected cells, and this was inhibited by IG R (not shown). To confirm that the 3G3 clone truly contained HA-tagged GSK-3 P, I performed reciprocal immunoaffinity precipitations (Fig. 5.4). The 12CA5 antibody, directed against the HA epitope, precipitated GSK-3 P from 3G3 and not from non-transfected cells (Fig. 5.4 A). Similarly, the GSK-3 p antibody brought down a protein bearing the HA epitope only in the 3G3 clone (Fig 5.4 B). From these data, I concluded that 3G3 was a clonal cell line that stably over-expressed active, wild-type HA-GSK-3 p. I used the 3G3 cell line to study the effects of over-expressing GSK-3 P on G 2 arrest after a range of radiation exposures. The experiments began with MCF-7 mp53 and 3G3 cells plated at the same concentration. 16 h after irradiation and nocodazole treatment, mitotic spreads were prepared and cells counted. The percentage of mitotic cells was approximately 75 % for both cell lines when they were treated with nocodazole alone (Fig. 5.5). This number dropped steeply as radiation exposures increased, and was reduced by 50 % after 2.5 Gy. After exposures of 1 to 4 Gy, there was a small but consistent reduction in the number of mitotic 3G3 cells. After exposures of 6 Gy or greater, the values for MCF-7 mp53 and 3G3 cells were indistinguishable. These results suggested that 3G3 cells were slightly less likely than controls to enter mitosis after lower radiation exposures, perhaps reflecting enhanced arrest in G 2 by GSK-3 p. 5.4.3. Effects of Transient Transfection of GSK-3 P on the Radiation Response Analysis of the 3G3 clone had suggested that over-expression of GSK-3 P could enhance 129 A B C D Figure 5.3 Identification of the 3G3 clonal cell line over-expressing wild-type GSK-3 B. M C F - 7 m p 5 3 ce l l s w e r e c o - t r a n s f e c t e d w i t h H A - t a g g e d w i l d t y p e G S K - 3 p a n d a v e c t o r e n c o d i n g r e s i s t a n c e t o h y g r o m y c i n B. A f t e r c u l t u r e f o r s e v e r a l w e e k s in t h e p r e s e n c e o f h y g r o m y c i n B ( 3 0 0 u g / m l ) , s t a b l e t r a n s f e c t a n t s w e r e c l o n e d by l i m i t i n g d i l u t i o n p l a t i n g a n d s c r e e n e d by w e s t e r n b l o t t i n g w i t h G S K - 3 P a n t i b o d y . A, n o n - t r a n s f e c t e d M C F - 7 m p 5 3 c e l l s ; B, ce l l s t r a n s f e c t e d w i t h h y g r o m y c i n v e c t o r o n l y ; C , p o o l e d t r a n s f e c t e d c e l l s ; D, 3 G 3 c l o n a l ce l l l i n e . Asterix i n d i c a t e s e n d o g e n o u s G S K - 3 P, arrow d e n o t e s H A - t a g g e d G S K - 3 p. Resul ts a r e r e p r e s e n t a t i v e o f s e v e r a l e x p e r i m e n t s . 130 MCF-7 mp53 3G3 B MCF-7 mp53 3G3 Figure 5.4 The 3G3 clone expresses HA-tagged GSK-3 (3. T h e e x p r e s s i o n o f G S K - 3 P a n d t h e H A e p i t o p e w a s c o m p a r e d in M C F - 7 mp53 ce l l s (left) a n d in t h e 3G3 c l o n e (right). A n t i b o d i e s a g a i n s t G S K - 3 P a n d t h e H A e p i t o p e w e r e i n c u b a t e d w i t h p r o t e i n s e x t r a c t e d f r o m e a c h ce l l l i n e , p r e c i p i t a t e d by i n c u b a t i o n w i t h p r o t e i n A s e p h a r o s e , a n d a n a l y z e d by w e s t e r n b l o t t i n g ( E x p e r i m e n t a l P r o c e d u r e s ) . A, H A a n t i b o d y p r e c i p i t a t e s , p r o b e d w i t h G S K - 3 P a n t i b o d y ; 8, G S K - 3 P a n t i b o d y p r e c i p i t a t e s , d e t e c t e d w i t h H A a n t i b o d y . 131 100 Radiation Dose (Gy) Figure 5.5 The 3G3 clone is sensitized to low doses of ionizing radiation. T h e r e s p o n s e s o f n o n - t r a n s f e c t e d ce l l s a n d ce l l s o v e r -e x p r e s s i n g G S K - 3 B w e r e c o m p a r e d o v e r a r a n g e o f r a d i a t i o n e x p o s u r e s . M C F - 7 m p 5 3 (white circles) a n d 3 G 3 ce l l s (black circles) w e r e i r r a d i a t e d as i n d i c a t e d a n d t r e a t e d w i t h n o c o d a z o l e (100 n g / m l ) i m m e d i a t e l y . 16 h a f t e r i r r a d i a t i o n , m i t o t i c s p r e a d s w e r e p r e p a r e d a n d m i t o t i c f i g u r e s w e r e c o u n t e d u n d e r t h e m i c r o s c o p e ( E x p e r i m e n t a l P r o c e d u r e s ) . M i t o s e s a r e e x p r e s s e d as p e r c e n t a g e s o f t h e t o t a l n u m b e r o f ce l l s . Resul ts a r e m e a n s ± S. D. f r o m t h r e e i n d e p e n d e n t e x p e r i m e n t s . 1 3 2 the G 2 checkpoint, however, this effect was too slight to be convincing. This could be due to the fact that GSK-3 P levels were only doubled in the 3G3 line. I therefore performed transient transfections of GSK-3 P, in the hope that I would be able to achieve higher levels of GSK-3 P expression. I performed immunofluorescence microscopy to examine entry into mitosis exclusively in transfected cells. Immunolabelling with either GSK-3 P or HA antibodies clearly identified a population of brightly stained cells after transient transfection, indicating that strong expressers could be selected by microscopy. Time-course studies indicated that the peak of HA-GSK-3 P expression occurred 24 h after transfection had begun (not shown). With either antibody, the transfection efficiency was approximately 10 % for wild-type GSK-3 P and 1 % for the K85A mutant (not shown). 24 h after transfection with wild-type or K85A GSK-3 P, a set of control samples were collected for immunofluorescence labelling. At this time, other samples were treated with nocodazole and a low dose of radiation (3 Gy) and collected 12 and 24 h after the controls (36 and 48 h post-transfection). Mitotic figures were counted under the microscope in transfected cells (Fig 5.6). All samples collected before radiation and nocodazole treatment had very few mitotic cells. After 12 and 24 h, the number of mitoses in no-vector transfection controls increased to 15 and 22 •%. This increase was not observed in expressers of wild-type HA-GSK-3 P, in which the frequency of mitoses was approximately 4 % at both time points. In cells expressing K85A GSK-3 P, mitoses were at least as frequent as in the controls. Taken together, these results suggested that strong expressers of wild-type GSK-3 P repressed mitosis after DNA damage. The inhibitory effects of GSK-3 P also appeared to depend upon kinase activity, as they were not observed in expressers of the K85A mutant. There were technical problems with this approach that 133 40 30 H 0 12 24 Time Elapsed after Irradiation and NOC (h) Figure 5.6 Transient transfection of wild-type but not K85A GSK-3 p inhibits entry into mitosis after DNA damage. M C F - 7 m p 5 3 ce l l s w e r e t r a n s i e n t l y t r a n s f e c t e d w i t h l i p o f e c t a m i n e a l o n e (white), w i l d - t y p e H A - G S K - 3 P (black), o r K 8 5 A G S K -3 P (diagonal stripes). 2 4 h a f t e r t r a n s f e c t i o n b e g a n , ce l l s w e r e e i t h e r c o l l e c t e d i m m e d i a t e l y (0 h , left) o r i r r a d i a t e d (3 G y ) a n d t r e a t e d w i t h n o c o d a z o l e ( N O C , 1 0 0 n g / m l ) f o r 1 2 (middle) o r 2 4 h (right). C e l l s w e r e l a b e l l e d w i t h G S K - 3 P a n t i b o d y a n d c h r o m o s o m e s w e r e s t a i n e d w i t h H o e c h s t 3 3 2 5 8 ( E x p e r i m e n t a l P r o c e d u r e s ) . M i t o t i c f i g u r e s w e r e c o u n t e d in b r i g h t l y f l u o r e s c e n t t r a n s f e c t e d ce l l s a n d a r e e x p r e s s e d as p e r c e n t a g e s o f t h e t o t a l n u m b e r s o f t r a n s f e c t e d ce l l s . T h e resu l ts a r e t y p i c a l o f t w o i n d e p e n d e n t e x p e r i m e n t s . Asterix, n o t d e t e r m i n e d . 134 limited the significance of these experiments, however. Firstly, the visual scoring of transfected cells (over 250 cells per data point) was laborious and tedious. Secondly, selecting strong expressers under the microscope is a subjective process. For these reasons, I began to seek methods for examining large numbers of transfected cells using an unbiased approach. 5.4.4. Development of Flow Cytometric Methods for Cell Cycle Analysis in Transfected Cells I applied flow cytometry to the exploration of GSK-3 P's role in the checkpoint pathway. Two properties of flow cytometry were particularly attractive: fluorescence values are quantitative, and analysis of large samples is possible (10-20,000 cells in a typical sample). My goal was to label transiently transfected cells for three different markers: HA expression, DNA content, and mitotic epitopes. These three labels would allow me to assess the level of GSK-3 3 expression on a cell-by-cell basis, and to determine the cell cycle phase within expressing cells. I tried two different triple labelling approaches for flow cytometry. In the f irst labelling scheme I attempted, both the HA tag and mitotic epitopes were detected with mouse primary antibodies (Fig 5.7). The HA antibody was conjugated directly to fluorescein isothiocyanate (FITC). A biotinylated mitosis-specific antibody was required, and would be detected by avidin conjugated to phycoerythrin (PE). DNA content was measured by staining with 7-aminoactinomycin D (7-AAD). To begin, I selected a mitosis-specific antibody suitable for flow cytometry. The TG-3 mitosis-specific antibody has been used in flow cytometry and in other applications by our laboratory [221 ] , but as this belongs to the IgM class it would be diff icult to biotinylate. Four mitosis-specific antibodies (GF-1, GF-7, GF-25 and GF-31, gifts of Dr. Peter Davies) 135 Fig 5.7 A scheme for assessing the effects of HA-GSK-3 p expression on the cell cycle, using biotinylated GF-7 antibody. Th is d i a g r a m o u t l i n e s t h e a p p l i c a t i o n o f t h r e e f l u o r o c h r o m e s t o ce l l s t r a n s f e c t e d w i t h H A - G S K - 3 P (diagonal stripes). H A - G S K - 3 P e x p r e s s i o n is d e t e c t e d w i t h a F I T C - c o n j u g a t e d a n t i b o d y d i r e c t e d a g a i n s t t h e h e m a g g l u t i n i n (HA) e p i t o p e . C e l l s a r e c l a s s i f i e d as b e i n g in G 1 ; S, o r G 2 / M p h a s e by m e a s u r i n g D N A c o n t e n t w i t h t h e D N A d y e 7 - a c t i n o m y c i n D (7 -AAD). M p h a s e ce l l s a r e d i s t i n g u i s h e d f r o m t h o s e in G 2 by l a b e l l i n g w i t h a b i o t i n y l a t e d a n d m i t o s i s - s p e c i f i c a n t i b o d y G F - 7 (B-GF -7 ) , a n d a r e d e t e c t e d by a v i d i n c o n j u g a t e d t o p h y c o e r y t h r i n ( a v i d i n - P E , stippled). T h e in tens i t i es o f 7-A A D , PE a n d F I T C f l u o r e s c e n c e a r e m e a s u r e d o n a c e l l - b y - c e l l bas is in f l o w c y t o m e t r y . 136 were tested in western blotting. GF-7 labelled several mitotic and no interphasic proteins (not shown). Immunofluorescence microscopy confirmed that GF-7 antibody labelled mitotic cells specifically (not shown). In flow cytometry, G F-7 yielded bright fluorescence that was well-resolved from background (not shown). I prepared a biotinylated form of the GF-7 antibody (B-GF-7) by reaction with NHS-Biotin (Experimental Procedures). Far western blots with HRP-conjugated streptavidin confirmed biotinylation of the GF-7 antibody (not shown). B-GF-7 and GF-7 antibodies labelled the same percentages of mitotic cells in flow cytometry (not shown). In immunofluorescence microscopy, mitotic cells were brightly labelled by B-GF-7 when detected with avidin conjugated to either FITC or PE (not shown). Repeated flow cytometric titrations indicated that the brightest PE signal was obtained when B-GF-7 and avidin-PE were applied at 1:25 dilution and 20 Ug/ml, respectively. A t this concentration of B-GF-7, mitotic cells were detected equally frequently by avidin conjugated to PE or FITC (Fig. 5.8 B, C) or fluorescent secondary antibody (Fig. 5.8 D) (51 % vs. 52 % and 55 %, respectively). Unfortunately, the PE fluorescence was poorly resolved from the background (Fig. 5.8 B). Furthermore, the FITC-conjugated HA antibody (Boehringer Mannheim), intended to mark HA-GSK-3 3 expression, was of very poor quality. For these reasons, I developed a different combination of labels for f low cytometry. In the second approach, I took advantage of species-specific secondary antibodies to allow differential fluorescence of HA and mitotic markers (Fig 5.9). In immunofluorescence microscopy, rat HA antibody labelled HA-expressing cells brightly and specifically (not shown). In flow cytometry, cells transfected with HA-GSK-3 3 were detected with equal frequency by GSK-3 3 and fat HA antibodies (not shown). To label mitotic cells, I titrated 137 00% LU 0. o 19.0% 0.4 % 0.4% DNA 8o.es B 0.2% 52 n 0 4% 54.5% O I— I 0 15.6% DNA 52.1 % 1023 32.0'X 00 CO «t ro x c mi 0 15.2% DNA 54.5 % 1023 29.9% Fig. 5.8 Biot inylated GF-7 ant ibody (B-GF-7) al lows count ing of mitot ic cells in f low cytometry. MCF-7 mp53 cells were treated with nocodazole (100 ng/ml) for 8 h before fixation and labelling for flow cytometry (Experimental Procedures). Mitotic epitopes were labelled with B-GF-7 and DNA was stained with 7-AAD. PE, FITC, and Alexa 488 fluorescence was detected in flow cytometry and plotted against DNA content. In the density plots shown here, low frequency events are marked in blue and high frequency events indicated in red. (A), 7-AAD only. B-GF-7 antibody was detected with PE-conjugated avidin (B), FITC-conjugated avidin (C), or goat anti-mouse antibody conjugated to Alexa 488 (D). Mitotic cells were counted and are indicated as percentages of the total number of cells. 138 5.9 A strategy for labelling HA-GSK-3 p-expressing cells for cell cycle analysis using rat and mouse primary antibodies. In th is a p p r o a c h , d i f f e r e n t i a l l a b e l l i n g o f H A - G S K - 3 P a n d m i t o t i c e p i t o p e s is a c h i e v e d u s i n g s p e c i e s -s p e c i f i c a n t i b o d i e s . T h e H A e p i t o p e is i m m u n o l a b e l l e d w i t h a r a t a n t i b o d y ( a - H A (rat)) a n d d e t e c t e d w i t h A l e x a 4 8 8 - c o n j u g a t e d g o a t a n t i - r a t a n t i b o d y . D N A c o n t e n t is m e a s u r e d a f t e r p r o p i d i u m i o d i d e (PI) s t a i n i n g , a n d c lass i f i es c e l l s a s b e i n g in Gv S, o r G 2 / M p h a s e . M i t o t i c c e l l s a r e s e l e c t e d by l a b e l l i n g w i t h m o u s e G F - 7 a n d A l e x a 6 3 3 - c o n j u g a t e d g o a t a n t i - m o u s e a n t i b o d i e s . F l u o r e s c e n c e s o f t h e s e t h r e e l a b e l s a r e m e a s u r e d in f l o w c y t o m e t r y ( E x p e r i m e n t a l P r o c e d u r e s ) . 139 GF-7 primary and Alexa 633-conjugated goat-anti mouse secondary antibodies, and found that dilutions of 1:100 and 1:200 (10 ug/ml), respectively, gave the best signal in flow cytometry. A scheme for cell cycle data analysis of transfected cells, using the second labelling strategy described above, is outlined in Fig. 5.10. Transfected cells contained a sub-population that was strongly labelled by the HA antibody, corresponding to HA-GSK-3 P expressers, and indicated by bright Alexa 488 fluorescence (Fig. 5.10 A, B, left. During analysis, the cell cycle data from this expressing population were considered separately, and compared to the non-expressing controls. Histograms of the propidium iodide (PI) fluorescence gave the profiles of DNA content that typically represent the cell cycle distribution. These were further refined by examining Alexa 633 fluorescence that was associated with the GF-7 antibody, which allowed the counting of mitotic cells. Using this strategy, it was possible to count the percentages of mitoses in only those cells that were expressing the HA epitope, and to compare these values with those determined for the vector controls. 5.4.5. Effects of Over-expressing HA-GSK-3 P on the Cell Cycle After DNA damage I began by examining entry into mitosis after DNA damage. MCF-7 mp53 cells were transfected with wild-type HA-tagged GSK-3 P or with empty pcDNA3.1 vector as a control. At the peak of HA-GSK-3 P expression (24 h), cells were irradiated and treated with nocodazole (100 ng/ml). 16 h after irradiation, cells were fixed and labelled for flow cytometry with rat HA and mouse GF-7 antibodies (Experimental Procedures). Mitotic cells were counted and expressed as percentages of the total number of cells in the HA-expressing 140 Fig 5.10 Analysis of flow cytometry data for measuring the effects of ectopic gene expression on the cell cycle. T h e c e l l c y c l e e f f e c t s o f e x p r e s s i n g a g e n e o f i n t e r e s t c a n b e s t u d i e d in f l o w c y t o m e t r y by a c o m b i n a t i o n o f e x p r e s s i o n - a n d m i t o s i s - s p e c i f i c a n t i b o d i e s . In th is e x a m p l e , ce l ls w e r e t r a n s f e c t e d w i t h e m p t y p c D N A 3 . 1 v e c t o r (A) o r w i t h H A - t a g g e d w i l d - t y p e G S K - 3 (3 (8). Le f t , s i m u l t a n e o u s l y l a b e l l i n g ce l l s w i t h r a t H A a n d A l e x a 4 8 8 - c o n j u g a t e d g o a t a n t i - r a t a n t i b o d i e s a l l o w s s e l e c t i o n o f H A - e x p r e s s i n g a n d n o n - e x p r e s s i n g c e l l p o p u l a t i o n s . Right, t h e ce l l c y c l e is a n a l y z e d in H A - e x p r e s s i n g a n d n o n - e x p r e s s i n g p o p u l a t i o n s a n d c o m p a r e d t o assess a n y e f fec t s o f o v e r - e x p r e s s i n g H A - G S K - 3 (3. By l a b e l l i n g c e l l s w i t h t h e D N A d y e p r o p i d i u m i o d i d e (PI), t h e m i t o s i s - s p e c i f i c m o u s e G F - 7 a n d A l e x a 6 3 3 - c o n j u g a t e d g o a t a n t i -m o u s e a n t i b o d i e s , t h e d i s t r i b u t i o n o f ce l l s t h r o u g h e a c h p h a s e o f t h e c e l l c y c l e c a n b e p r e c i s e l y c o u n t e d . In t h i s e x a m p l e , e x p r e s s i o n o f w i l d - t y p e H A - G S K - 3 |3 h a s r e s u l t e d in a n i n c r e a s e in G , a n d a d e c r e a s e in t h e n u m b e r o f m i t o t i c ce l l s . 141 Selection of Transfected Cells Cell Cycle Analysis 142 population and in the vector controls (Fig. 5.11). In the vector controls, approximately 80 % of cells were arrested in mitosis after treatment with nocodazole alone. As the exposure to radiation was increased, fewer cells entered mitosis as a consequence of G 2 arrest. In three independent experiments, HA-GSK-3 P-expressing cells had consistently fewer mitotic cells after treatment with nocodazole alone or with radiation (Fig 5.11 and data not shown). Examination of the cell cycle profiles in HA-GSK-3 P-expressing cells revealed that there is a corresponding increase in the number of cells in G x {e.g. compare Fig. 5.10 A and B, right). The main effect of over-expressing GSK-3 P is an increase in the G x and not the G 2 population, which argues against a role for this kinase in the G 2 DNA damage checkpoint pathway. 5.4.6. Checkpoint Effects of Didemnimide A In a separate approach to answering the question of whether GSK-3 P is a checkpoint protein, I investigated didemnimide A, a compound that is very closely related to IGR. Didemnimide A was originally isolated from the ascidian Didemnum conchy Iiatum, which grows on the seagrass Thalassia testudinum [ 222 ] . The ascidian helps to repel predation by carnivorous fish. Didemnimide A is identical to IGR in its structure, except that it is lacking a bond that joins the indole and imidazole rings (Fig. 5.1). I tested whether didemnimide A is a checkpoint inhibitor in f low cytometry. 16 h after irradiation, cells were treated with nocodazole and a range of didemnimide A concentrations. After 8 h, cells were collected and labelled with GF-7 and PI for flow cytometry (Chapter 2, Experimental Procedures). No checkpoint inhibition was found at any concentration tested (not shown). 143 Radiation Dose (Gy) Fig. 5.11 Expression of wild-type HA-GSK-3 p reduces entry into mitosis after DNA damage. M C F - 7 m p 5 3 ce l l s w e r e t r a n s i e n t l y t r a n s f e c t e d w i t h w i l d - t y p e H A - G S K - 3 P (black circles) o r w i t h p c D N A 3 . 1 v e c t o r a l o n e (white circles). A t t h e p e a k o f H A - G S K - 3 P e x p r e s s i o n ( 2 4 h ) , ce l l s w e r e i r r a d i a t e d a n d t r e a t e d w i t h n o c o d a z o l e (1 0 0 n g / m l ) . 1 6 h a f t e r i r r a d i a t i o n , ce l l s w e r e f i x e d , i m m u n o s t a i n e d w i t h H A a n d G F - 7 p r i m a r y a n t i b o d i e s , a n d A l e x a 4 8 8 a n d 6 3 3 - 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 ( E x p e r i m e n t a l P r o c e d u r e s ) . D N A w a s l a b e l l e d w i t h p r o p i d i u m i o d i d e (PI). M i t o t i c ce l l s in t h e v e c t o r c o n t r o l a n d H A -e x p r e s s i n g p o p u l a t i o n s w e r e c o u n t e d in f l o w c y t o m e t r y , a n d a r e e x p r e s s e d as p e r c e n t a g e s o f t h e t o t a l n u m b e r o f ce l l s . T h e resu l ts a r e t y p i c a l f o r t w o i n d e p e n d e n t e x p e r i m e n t s . 1 4 4 In a separate experiment, the effects of didemnimide A were tested on immunoaffinity precipitated GSK-3 P in in vitro kinase assays. Didemnimide A potently inhibited GSK-3 P kinase activity, reducing activity by 95 % at 50 UM (Dr. Y. J . Wang, personal communication). To confirm that didemnimide A is reaching intracellular targets, and therefore could access GSK-3 P in vivo, I tested the effects of this compound on cell proliferation. MCF-7 mp53 cells were treated with didemnimide A for 24 h, the compound was washed away, and cells cultured for 3 d before measuring their proliferation in the MTT assay (Fig. 5.12) [155, 223] . Didemnimide A exerted a modest inhibitory effect, reducing the growth of cells by 50 % at 100 UM. While not a direct proof, these results suggest that didemnimide A inhibits proliferation because i t enters the cell. Taken together, the results show that didemnimide A inhibits GSK-3 P and yet is not a checkpoint inhibitor, indicating that GSK-3 P activity is not necessary for arrest in G 2 . 5.5. DISCUSSION In this work, a role for GSK-3 P in cell cycle arrest in G 2 after DNA damage was investigated. This was initiated because the checkpoint inhibitors DBH and IGR are different in structure yet both inhibit the in vitro activity of this kinase. The rationale behind my studies was that if GSK-3 P were a checkpoint kinase, over-expressing the protein could enhance the checkpoint and would reduce the number of mitoses after irradiation. Several different strategies for over-expression were attempted. I used transiently transfected cells to examine high levels of GSK-3 P expression, and created a clonal cell line in order to study cells expressing a consistent amount of GSK-3 p. In immunofluorescence 145 0 10 100 [compound] (u.M) 1000 Figure 5.12 Effects of didemnimide Aon cell proliferation. M C F - 7 m p 5 3 ce l l s w e r e c u l t u r e d in t h e p r e s e n c e o f t h e i n d i c a t e d c o n c e n t r a t i o n o f d i d e m n i m i d e A (black circles) o r t h e c o r r e s p o n d i n g a m o u n t o f D M S O (white circles) f o r 24 h. A f t e r w a s h i n g a w a y t h e c o m p o u n d , ce l l s w e r e c u l t u r e d f o r 3 d a n d i n h i b i t i o n o f g r o w t h e v a l u a t e d by M T T a s s a y [ 1 5 5 ] . Resul ts a r e e x p r e s s e d as p e r c e n t a g e s o f t h e v a l u e s o b s e r v e d in n o - t r e a t m e n t c o n t r o l s , a n d a r e m e a n s ± S. D. f o r t h r e e i n d e p e n d e n t e x p e r i m e n t s . 146 microscopy of transiently transfected cells and mitotic spreads of 3G3, a reduction in the number of mitotic cells after DNA damage was detected. A decrease in the frequency of mitoses was also observed in flow cytometric studies. Examination of the cell cycle profiles revealed that in both non-irradiated and irradiated cells over-expressing the kinase, the reduction in mitoses correlated with an increase in G : phase. One would expect an increase in G 2 phase after over-expressing a G 2 checkpoint protein. GSK-3 (3 phosphorylates cyclin D l , targeting it for destruction by the proteasome [224 ] . I t is likely that in cells over-expressing GSK-3 P, cyclin D l levels were reduced and prevented exit from G r While this research was underway, the GSK-3 P gene was disrupted in mice [215 ] . No defect in the response to ionizing radiation was observed in the knockout mice. Didemnimide A closely mimics the structure of IG R, except that its lack of one bond makes it less rigid than IG R and potentially alters interactions with target proteins. I found no evidence for checkpoint inhibition by didemnimide A. This is in agreement with previous ELICA results obtained by our lab [ 1 5 5 ] . Didemnimide A inhibits GSK-3 P kinase activity (Dr. Y.-J. Wang, personal communication). GSK-3 P inhibition has also been shown recently by a small number of aryl and anilino maleimide derivatives that are closely related to IGR and didemnimide A in structure [225, 226] . Didemnimide A inhibits GSK-3 P yet does not have any effects on the checkpoint response. Taken together, these results indicate that GSK-3 P activity is not needed for the checkpoint pathway. As this investigation was underway, our laboratory, together with collaborators, found that in addition to GSK-3 P, IGR and DBH inhibit Chkl and Chl<2 kinases (unpublished, and [156] ) . These kinases are effectors of the DNA damage checkpoint pathway, and they are phosphorylated in response to DNA damage by ATM and ATR (reviewed in [19] ) . Inhibition 147 of Chkl and Chl<2 is very likely to account for the checkpoint effects of IG R and DBH. This flow cytometric evaluation of the effects of over-expression of GSK-3 P could be extended to test other candidate checkpoint proteins. In the case where a mouse antibody must be used to measure expression levels, the biotinylated form of G F-7 antibody could label mitotic cells. A possible extension of this technique would make use of one of the fluorescent proteins (e.g. GFP) as an expression tag, reducing the number of labelling steps required. Two aspects of this method should be improved for its application to be practical. First, the efficiency of transfection was very low (2-5 %) and must be improved to take advantage of the quantitative power of flow cytometry. Secondly, the brightness of the Alexa 633 secondary antibody was disappointing. This should be replaced with a different fluorochrome to increase the clarity of the data. 5.6. ACKNOWLEDGEMENTS I am grateful to Geoff Osborne and Andy Johnston for many helpful discussions on flow cytometry, and would like to thank Peter Davies for his continued generosity in giving our lab mitosis-specific antibodies, and Dr. Y.-J. Wang for sharing kinase assay data. 148 Chapter 6 DISCUSSION 6.1. Thesis Overview A primary research focus in our laboratory is the identification of new inhibitors of the G 2 DNA damage checkpoint. In this thesis, the cell cycle effects of three new checkpoint inhibitors (IGR, OZ and OKA) are described, and strategies for identification of their molecular targets are presented. My research was primari ly on the related compounds OZ and OKA. OZ potently inhibited G 2 arrest after DNA damage. Unlike other checkpoint inhibitors, OZ did not inhibit checkpoint protein kinases or protein phosphatases. This suggested that OZ may inhibit the checkpoint pathway by acting against a distinct enzyme activity. I t was also discovered that OZ acts as an antimitotic agent that prevents cells from reaching metaphase. The phenotype induced by OZ in mitotically arrested cells was atypical for an antimitotic agent and unique for a checkpoint inhibitor. During the course of these studies, OKA was identified as a checkpoint inhibitor. Structural analysis revealed OKA to be closely related to OZ. These two compounds were very similar with respect to checkpoint inhibition and effects on localization of cell cycle proteins. Considering that a target of OZ and OKA may be involved in both the DNA damage checkpoint and in the prometaphase stage of mitosis, we became interested in identifying the target(s) of these compounds. 149 A biotinylated analogue of OKA (B-OKA) was synthesized that retained the principal biological activities of OKA and OZ. B-OKA bound covalently to several proteins. These were purified by streptavidin affinity precipitation and six were identified using mass spectrometry. Among these potential targets was RanBP2, which was chosen for further investigation. Xenopus /aev/s egg extracts were treated with B-OKA and probed for the presence of biotinylated RanBP2. The results strongly suggested that B-OKA binds directly to RanBP2. RanBP2 may be involved in chromosome congression during prometaphase and/or the G 2 DNA damage checkpoint response. Like many other checkpoint inhibitors, IG R inhibits protein kinase activity. IG R selectively inhibited GSK-3 B in vitro, indicating that this kinase may be involved in the G 2 checkpoint pathway. GSK-3 P was over-expressed by transfection and the resulting effects on the response to ionizing radiation were examined. Didemnimide A, a compound closely related to IG R, was also tested as a checkpoint inhibitor and a GSK-3 P inhibitor. Didemnimide A inhibited GSK-3 P but did not block checkpoint arrest. Taken together, these results demonstrate that inhibition of GSK-3 P does not in itself prevent G 2 arrest, and argue against a role for this kinase in the DNA damage checkpoint. 6.2. The Chemical Genetics Approach to Identifying Checkpoint Inhibitors Chemical genetics refers to the perturbation of protein function by small-molecule inhibitors or activators, with the aims of identifying their targets or elucidating protein function (reviewed in [227-229] ) . In many respects, chemical genetics is analogous to traditional forward or reverse genetics. In forward genetics, one mutagenizes an organism, searches for expression of the desired phenotype, and then identifies the causative mutation. 150 In reverse genetics, the activity of a protein of interest is altered by genetic mutation and the resulting effects on the phenotype of the organism are examined. In the chemical genetics approach, small-molecule activators or inhibitors are used in place of genetic mutations. Our laboratory has adopted a forward chemical genetics approach (Fig. 6.1). First, collections of biological extracts are screened for induction of the desired phenotype (i.e. G 2 checkpoint inhibition). Extracts containing inhibitor activity are fractionated and the structures of purified inhibitors are determined. The cellular effects of active compounds are examined and their molecular targets are identified, if possible. Small-molecule inhibitors are very useful biochemical tools for unravelling complex cellular processes. In some instances, these compounds may also have therapeutic potential. In these cases, several stages of animal studies and clinical trials must be completed before a drug can be brought to market. There are two distinct advantages to the forward chemical genetics approach. Firstly, there is the potential to uncover new biological activities, in contrast to developing inhibitors or activators of well-characterized proteins. Secondly, because the end-point of this screen is a cellular effect, one's research is immediately focussed on compounds that succeed in achieving the desired phenotype. In the reverse chemical genetics approach, highly specific inhibitors can be developed. However, these may fail to yield the appropriate cellular effects due to compensatory activities of other enzymes. The drawback of the chemical genetics approach adopted by our laboratory is that it is a diff icult challenge to identify the targets of small-molecule inhibitors. My research fits into an early stage of the chemical genetics approach, in which active compounds are characterized and their molecular targets identified. 151 5 • • • • • • one extract per well 96-well plate containing mammalian cells purify inhibitor identify checkpoint inhibit ing extracts 0 1023 DNA (Pfl structure determination > cellular phenotype target identif ication biological tool biological studies therapeutic tool clinical trials Figure 6.1 Forward chemical genetics approach to identifying checkpoint inhibitors and their targets. N a t u r a l p r o d u c t ex t rac ts a r e t e s t e d in h i g h - t h r o u g h p u t s c r e e n i n g f o r c h e c k p o i n t i n h i b i t i o n in m a m m a l i a n c e l l s . C h e c k p o i n t i n h i b i t o r s a r e p u r i f i e d a n d t h e i r s t r u c t u r e s d e t e r m i n e d by N M R a n d m a s s s p e c t r o m e t r y . T h e b i o l o g i c a l e f fec ts o f p u r i f i e d i n h i b i t o r s a r e c h a r a c t e r i z e d u s i n g t e c h n i q u e s s u c h a s f l o w c y t o m e t r y a n d i m m u n o f l u o r e s c e n c e m i c r o s c o p y . T a r g e t s o f t h e i n h i b i t o r s a r e p o t e n t i a l l y c l a s s i f i e d b a s e d o n t h e c e l l u l a r p h e n o t y p e , o r by s c r e e n i n g a p p r o a c h e s . T h e i d e n t i f i e d i n h i b i t o r s h a v e v a l u e as b i o l o g i c a l t o o l s a n d / o r as l e a d c o m p o u n d s f o r d r u g d i s c o v e r y . If t h e i n h i b i t o r c o n t i n u e s in t h e d r u g d e v e l o p m e n t p r o c e s s , e f f i c a c y a n d tox i c i t y w i l l b e a s s e s s e d in a n i m a l s t u d i e s a n d c l i n i c a l t r i a l s . 6.3. Conclusions and Future Directions IGR I t is my conclusion that GSK-3 P is not a checkpoint enzyme, and further pursuit of a checkpoint role for this kinase is not warranted. Inhibition of the checkpoint kinases Chkl and Chl<2 by IGR is very likely to account for the effects of IGR on the G 2 checkpoint. However, there are several areas of interest related to IGR that are being explored in our laboratory. For example, a crystal structure of Chk l kinase in complex with IGR was obtained and revealed that IGR lies in the ATP-binding pocket of the kinase (M. Robergeand B. B. Xhou, unpublished). This structural information wi l l be applied to the development of more potent and specific analogues of IGR (studies of G. Karjala, in conjunction with the laboratory of Dr. R. J . Andersen). The effectiveness of IGR in vivo\N\\\ also be explored in mouse models, in which checkpoint inhibition wil l be tested on H C T 1 1 6 + / + and HCTI 16-/-tumour implants after irradiation (studies of C. Sturgeon). In this research, the checkpoint role of GSK-3 P was tested by flow cytometric evaluation of the cell cycle in transfected and irradiated cells. This is a generally applicable method, and may be used to detect radiation sensitization or resistance as a consequence of over-expressing a gene of interest. This approach may also be used to test whether a given protein is a target of a checkpoint inhibitor. Over-expression of the protein target should increase resistance to the checkpoint inhibitor, and this may be detected in flow cytometry. OZ and OKA In this work, binding of B-OKA to RanBP2 was detected in several ways: batch affinity purification of B-OKA-binding proteins by streptavidin agarose, band excision of individual 153 biotinylated proteins purified from B-OKA-treated cells, RanBP2 western blots of B-OKA-binding proteins, and by streptavidin-HRP overlay of RanBP2 immunoprecipitates. How might inhibition of RanBP2 affect the DNA damage checkpoint or chromosome congression? There are several alternatives. One possibility explored in this thesis is that OZ and OKA interfere with a nuclear transport function of RanBP2. This protein is found on the cytoplasmic face of the nuclear pore complex (NPC) (Fig 6.2) [187-189 ] . During interphase,' RanBP2 could act as a docking site for cargo proteins before they are imported through the NPC [192, 230] . Because RanBP2 binds Ran and exportins [188, 231] , and stimulates the activity of RanGAPl [ 1 9 1 ] , i t is thought to help dissociate Ran from exportins and transport cargo [230 ] . However, it has recently been shown that depletion of cytoplasmic filaments -in which RanBP2 is a major component- from NPCs in Xenopus /aev/segg extracts apparently has no effects on nuclear import [ 232 ] . Therefore, the actual contribution of RanBP2 to nucleocytoplasmic transport is unclear. My experiments did not demonstrate an effect of OZ or OKA on nuclear transport of cyclin B l . However, the effects of OZ and OKA could be tested more appropriately in an experimental system specifically designed to assay transport (ex. [233 ] ) . RanBP2 is also a SUMO E3 ligase [192, 194, 195] . Attachment of SUMO can affect protein-protein interactions, intracellular localization of proteins, and protein stability (reviewed in [192] ) . OZ and OKA may inhibit SUMOylation, disrupting protein-protein interactions necessary for checkpoint arrest and chromosome congression. For example, RanGAPl was the f irst SUMOylated protein to be identified, and its association with RanBP2 depends upon SUMOylation [190, 234] . 154 INTERPHASE MITOSIS OZ, OKA Figure 6.2 A model for the action of OZ and OKA on RanBP2. The localization and function of RanBP2 (o) is regulated by the cell cycle. In interphase, RanBP2 is found on the cytoplasmic face of the nuclear pore complex (NPC), bound to the GTPase activator RanGAPI (o) . During nuclear transport, RanBP2 and RanBPI dissociate Ran-GTP from exported proteins (not shown). RanBP2 is a SUMO E3 ligase, and may SUMOylate (-S ) proteins immediately before their import through the NPC. OZ and OKA could prevent SUMOylation of checkpoint proteins during their import, altering their localization or function within the nucleus. During mitosis, the RanBP2-RanGAP1 complex is found on kinetochores, centrosomes and the spindle. OZ and OKA may suppress spindle dynamics directly or have an indirect effect through RanGAPI and Ran. 155 At this point, there is no evidence for SUMOylation of the kinetochore motor protein CENP-E. However, CENP-E is modified by ubiquitin, which is closely related to SUMO. Microinjection of cdc34 (an ubiquitin E2 conjugating enzyme) resulted in ubiquitination of CENP-E and its re-distribution from the kinetochores into cytoplasmic aggregates [235 ] . This effect was independent of ubiquitin-mediated proteolysis, suggesting that ubiquitination regulates the intracellular localization of CENP-E through a sorting mechanism or protein-protein interactions. Perhaps SUMOylation also affects the localization of CENP-E, either through direct effects or by antagonizing ubiquitination. SUMO could also affect the DNA damage checkpoint by effects on protein stability. In some instances, SUMOylation prevents ubiquitin-mediated proteolysis. For example, the E3 ubiquitin ligase MDM-2 is a negative regulator of p53 stability and is a substrate for SUMOylation by RanBP2 [195 ] . SUMOylation prevents ubiquitin-mediated proteolysis of MDM-2, which results in de-stabilization of p53. DNA damage results in removal of SUMO from MDM-2, leading to stabilization of p53 and checkpoint-mediated cell cycle arrest. During mitosis, RanBP2 (in complex with the GTPase activator RanGAPl) is found on the centrosomes, kinetochores, and the mitotic spindle [190 ] . Ran is a regulator of spindle dynamics [ 6 8 ] . Furthermore, depletion of Ran by RNAi prevents chromosome congression [186 ] . Taken together, these facts suggest that inhibition of RanBP2 by OZ and OKA may prevent chromosome congression through effects on the Ran system. A t present, there is no direct connection between Ran and CENP-E. However, Ran activity has been shown to direct a similar protein, the mitotic motor protein hEg5, towards the plus ends of microtubules on mitotic spindles in Xenopus laeviszgg extracts [185 ] . 156 There are several ways in which an effect of OZ and OKA on RanBP2 could be explored in future research. The next logical step would be testing whether OZ or OKA can inhibit RanBP2 activity. This protein contains several different domains, and the functions of many of these domains is unknown. Expression of individual domains of RanBP2 is possible in reticulocyte lysates [ 2 3 4 ] . By testing for biotinylation of individual domains, using the methods I have described, one could determine which domain binds to RanBP2. This mapping could be further refined by a mass spectrometric analysis of the B-OKA-labelled domain. After trypsinization, small labelled peptides may be identified by mass spectrometry, and could narrow down the site of interaction with B-OKA to within a few amino acids. This type of mapping analysis could suggest appropriate experimental systems for testing an effect of OZ and OKA on RanBP2. 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