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UBC Theses and Dissertations

Steroid hormone receptors and plasminogen activator in human breast cancer : their value in prognosis Humphries, Karin Hartmann 1985

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STEROID HORMONE RECEPTORS AND PLASMINOGEN ACTIVATOR IN HUMAN BREAST CANCER: THEIR VALUE IN PROGNOSIS By KARIN HARTMANN HUMPHRIES B . G . S . , Simon F r a s e r U n i v e r s i t y , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Depar tment of P a t h o l o g y ) We a c c ep t t h i s t h e s i s as c on f o rm i ng t o the r e q u i r e d s t a n d a r d THE © UNIVERSITY OF BRITISH COLUMBIA DECEMBER 1985 K a r i n Hartmann Humphr i es ,1985 f>8 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT S t e r o i d hormone r e c e p t o r a n a l y s i s has emerged as an i m p o r t a n t a i d t o p r o g n o s i s i n human b r e a s t c a n c e r . The 125 a v a i l a b i l i t y o f I - l a b e l e d e s t r a d i o l l e d t o the deve lopment o f a d u a l - l a b e l assay f o r the s imu l t a n eou s d e t e r m i n a t i o n o f e s t r o g e n and p r o g e s t e r o n e r e c e p t o r c o n t e n t . There a re i n d i c a t i o n s t h a t p l a sm inogen a c t i v a t o r a c t i v i t y may a l s o s e r v e as a p r o g n o s t i c i n d i c a t o r i n b r e a s t c a n c e r . Two methods, one f l u o r o m e t r i c and the o t h e r s p e c t r o p h o t o m e t r i c were examined f o r t h e i r s u i t a b i l i t y i n a s s a y i n g f o r p l a sm inogen a c t i v a t o r a c t i v i t y . Bo th a s s a y s were found to be e q u a l l y s u i t a b l e f o r the d e t e r m i n a t i o n o f t h i s enzyme i n b r e a s t tumour c y t o s o l s . C y t o s o l f rom tumour samples s t o r e d a t -70°C f o r 18 - 52 months was as sayed f o r s t e r o i d r e c e p t o r c o n t e n t and p l a sm inogen . a c t i v a t o r a c t i v i t y . E s t r o g e n r e c e p t o r (ER) v a l u e s de t e rm ined a t the t ime of the o r i g i n a l assay were compared w i t h the r e - a s s a y e d e s t r o g e n r e c e p t o r c o n t e n t . The r e s u l t s showed t h a t t h i s s t e r o i d r e c e p t o r i s r ema rkab l y s t a b l e to s t o r a ge a t - 7 0 °C . Compar i sons between the two s t e r o i d r e c e p t o r s and p l a sm inogen a c t i v a t o r (PA) a c t i v i t y showed s i g n i f i c a n t c o r r e l a t i o n s between e s t r o g e n r e c e p t o r and p r oge s t e r one r e c e p t o r (PgR) c on t e n t and between p r o g e s t e r o n e r e c e p t o r and p l a sm inogen a c t i v a t o r a c t i v i t y . T h e r e was no s i g n i f i c a n t c o r r e l a t i o n between ER and PA a c t i v i t y . i i S u r v i v a l c u r v e s were c o n s t r u c t e d to examine the e f f e c t of s t e r o i d r e c e p t o r c o n t e n t and p l a sm inogen a c t i v a t o r a c t i v i t y on the s u r v i v a l o f b r e a s t c a r c i noma p a t i e n t s . Due to the l i m i t e d number of p a t i e n t s a v a i l a b l e , s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n s u r v i v a l were not e v i d e n t . However, t h e r e was an i n d i c a t i o n t h a t e s t r o g e n r e c e p t o r p o s i t i v e p a t i e n t s s u r v i v e d l o n g e r than t h e i r e s t r o g e n r e c e p t o r n e g a t i v e c o u n t e r p a r t s . PA a c t i v i t y and p r o g e s t e r o n e r e c e p t o r c o n t e n t d i d not appear t o i n f l u e n c e s u r v i v a l . i i i TABLE OF CONTENTS ABSTRACT i i L IST OF TABLES v i L IST OF FIGURES v i i ACKNOWLEDGMENTS i x ABBREVIATIONS x I INTRODUCTION i ) Mechanisms of S t e r o i d Hormone A c t i o n 1 i i ) Methods f o r S t e r o i d A n a l y s i s 9 i i i ) C l i n i c a l A p p l i c a t i o n s of Re cep t o r Theory 23 a . R e c e p t o r s & H i s t o l o g i c a l Pa r ame te r s 25 b. Re cep t o r vs T h y m i d i n e - l a b e l i n g Index 26 c . R e c e p t o r s & E p i d e m i o l o g i c a l Data 27 d . Tumour S t a g i n g 28 e . R e c e p t o r s & Serum L e v e l s o f E s t r o g e n & P r o g e s t e r o n e 28 f . P r o g n o s i s & M o l e c u l a r P r o p e r t i e s of Re cep t o r s 29 g . Re cep t o r S t a t u s & Response t o Chemotherapy 30 i v ) P l a sm inogen A c t i v a t o r A c t i v i t y & B r e a s t Cancer 32 v) Methods f o r P l a sm inogen A c t i v a t o r A n a l y s i s 40 v i ) Resea r ch Aims & I n v e s t i g a t i v e Scope 44 I I MATERIALS AND METHODS 48 i ) . R e c ep t o r Assay : M a t e r i a l s 48 i i ) . R e c ep t o r Assay : Method 49 i i i ) . I s o t o p e S e p a r a t i o n 53 i v ) . P r o t e i n and A lbumin D e t e r m i n a t i o n 55 i v v) . PAA As say s : M a t e r i a l s 57 v i ) . PAA Assays : Method 57 I I I RESULTS & DISCUSSION 63 IV CONCLUSION 109 VI APPENDIX 1 2 3 V REFERENCES ^ 2 v LIST OF TABLES NUMBER TITLE PAGE I B u f f e r Compar i son 64 I I I n c u b a t i o n Time f o r PgR 69 I I I E f f e c t o f P r o t e i n on PgR 71 IV ER D e t e r m i n a t i o n s Us i ng 3 H - E 2 & 1 2 5 I - E 2 78 V D u a l - L a b e l and S i n g l e - L a b e l Compar i son 79 VI ER and PgR i n I n d i v i d u a l Tumour Samples 82 V I I Day - t o -Day V a r i a t i o n i n F l u o r o m e t r i c Assay 89 V I I I E f f e c t o f P r o t e i n on the F l u o r o m e t r i c Assay 90 IX E f f e c t o f L y o p h i l i z a t i o n 96 X D i l u t i o n Study 97 XI PAA i n B r e a s t Tumour Samples 99 X I I P o l y t r o n vs D ismembrator 101 v i LIST OF FIGURES NUMBER TITLE PAGE 1 O r i g i n a l S t e r o i d Hormone Mode l 2 2 R e v i s e d Mode l o f S t e r o i d Hormone A c t i o n 8 3a Sample Wool f P l o t 18 3b Sample S c a t c h a r d P l o t 19 4 C o n v e r s i o n o f P l a sm inogen t o P l a s m i n 34 5 I n c u b a t i o n Time f o r PgR Assay 68 6 Time Course o f B i n d i n g f o r ER Assay 70 7 P r o t e i n E f f e c t on PgR Assay 72 8 P r o t e i n E f f e c t and the P r o g e s t e r o n e Assay 73 9 Energy P r o f i l e s o f 3 H and 1 2 5 I 76 3 125 10 Compar i son of H and I E s t r a d i o l 77 11a Dua l and S i n g l e L a b e l ER Assay 80 l i b Dua l and S i n g l e L a b e l PgR Assay 81 12a Re - a s sayed vs O r i g i n a l ER Con ten t 85 12b P r o g e s t e r o n e vs E s t r o g en Re cep t o r Conten t 86 13 P ro t am ine S u l p h a t e S t anda rd Curve 88 14 S t a b i l i t y o f the F l u o r e s c e n t End -P roduc t 91 15 U r o k i n a s e S t anda r d Curve 93 16 S t r e p t o k i n a s e S t anda rd - Curve 94 17 PAA vs P r o g e s t e r o n e Re cep t o r Con ten t 103 v i i NUMBER TITLE PAGE 18 PAA vs E s t r o g e n Re cep t o r Con ten t 104 19 PAA and S u r v i v a l 1 ° 6 20 PgR and S u r v i v a l 1 ° 7 21 ER and S u r v i v a l 108 v i i i ACKNOWLEDGEMENTS I t i s w i t h s i n c e r e a p p r e c i a t i o n and g r a t i t u d e t h a t I e x p r e s s my t hank s t o D r . W i l l i a m G o d o l p h i n , my s u p e r v i s o r , who o f f e r e d c o n t i n u e d encouragement and h e l p f u l s u g g e s t i o n s as w e l l as c o n s t r u c t i v e c r i t i c i s m th roughou t the c ou r s e o f t h i s p r o j e c t . I w i sh to e x t end my a p p r e c i a t i o n to Ms. B e r y l J a c ob son , and the E s t r o g e n Re cep t o r L a b o r a t o r y t e c h n o l o g i s t s , L u l u G a r c i a and Mary McLennan, whose suppo r t gave me the i n c e n t i v e t o comp le te t h i s p r o j e c t . Fo r f i n a n c i a l a s s i s t a n c e r e c e i v e d f rom the N a t i o n a l Cancer I n s t i t u t e o f Canada, I e x p r e s s my g r a t i t u d e . A l s o , I w i sh t o ex tend my t hanks to D r s . Pudek and Seccombe, who gave me t ime o f f f rom t h e i r r e s e a r c h p r o j e c t , t o enab l e me to comp l e t e the w r i t i n g o f t h i s t h e s i s . i x ABBREVIATIONS S Svedberg Units SK Streptokinase UK Urokinase IU International Units -15 fmol femtomoles = 1x10 moles U.V. Ultraviolet Kd dissociation constant Km Michaelis constant - substrate concentration at which one-half of maximum velocity is attained. PMN Polymorphonuclear leukocytes M.W. Molecular Weight BI Binding Index CP Cytosol Protein TP Total Protein x I . INTRODUCTION i . MECHANISMS OF STEROID HORMONE ACTION I t has been a lmos t a c e n t u r y s i n c e the o b s e r v a t i o n was made t h a t human b r e a s t c a n c e r responded t o a b l a t i v e e n d o c r i n e t r e a t m e n t s such as oophorec tomy, (1) hypophysectomy and ad rena l e c t omy ( 2 , 3 ) . The enormous p r o g r e s s i n t he u n d e r s t a n d i n g of hormona l i n t e r a c t i o n w i t h s p e c i f i c t a r g e t t i s s u e s p roceeded f rom the s y n t h e s i s i n the 1 960 ' s of h i g h s p e c i f i c a c t i v i t y r a d i o l a b e l e d e s t r o g e n s . A knowledge of s t e r o i d r e c e p t o r t h eo r y i s f undamen ta l t o the u n d e r s t a n d i n g of the m o l e c u l a r p a t h o l o g y of ho rmona l l y r e s p o n s i v e neop lasms such as b r e a s t c a n c e r . S t e r o i d hormone a c t i o n depends on and i s r e s t r i c t e d t o c e l l s wh i ch c o n t a i n the a p p r o p r i a t e i n t r a c e l l u l a r r e c e p t o r p r o t e i n s c a pab l e o f t r a n s l o c a t i o n i n t o the nu c l e u s and subsequent i n t e r a c t i o n w i t h s p e c i f i c n u c l e o p r o t e i n s of the c h r oma t i n . The o r i g i n a l model f o r hormone a c t i o n was p roposed i n the e a r l y 1 9 6 0 ' s . Wh i l e some a s p e c t s o f t h i s model have r e c e n t l y been changed , many o f t he f undamen ta l p r i n c i p l e s rema in the same. F i g u r e one summar izes the major s t e p s i n t h i s model f o r s t e r o i d hormone a c t i o n . The e n t r y o f s t e r o i d s i n t o the c e l l appears t o be n o n - s p e c i f i c , and i s c u r r e n t l y p o o r l y u n d e r s t o o d . P a s s i v e d i f f u s i o n i s b e l i e v e d t o be the most p r o b a b l e means o f e n t r y , s i n c e s t e r o i d s a r e n o n - i o n i c and l i p o p h i l i c i n n a t u r e , and thus 1 T i g . l The o r i g i n a l model o f e s t r o g e n a c t i o n i n a t a r g e t c e l l . E s t r o g e n (E) d i s s o c i a t e s from t h e plasma p r o t e i n s (P) and d i f f u s e s i n t o t h e c y t o p l a s m . The l i g a n d b i n d s w i t h t h e c y t o p l a s m i c r e c e p t o r (R) and becomes a c t i v a t e d (ER*) . F o l l o w i n g t r a n s l o c a t i o n i n t o t h e n u c l e u s and i n t e r a c t i o n w i t h n u c l e a r a c c e p t o r s , t h e a c t i v a t i o n o f RNA polymerase and DNA p o l y m e r a s e r e s u l t s i n p r o t e i n s y n t h e s i s . The n u c l e a r r e c e p t o r i s e i t h e r r e c y c l e d o r d e s t r o y e d and new c y t o p l a s m i c r e c e p t o r i s s y n t h e s i z e d . 2 should experience no d i f f i c u l t y i n c r o s s i n g the c e l l membrane. F a c i l i t a t e d t r a n s p o r t cannot be discounted however. On the b a s i s of s t e r o i d entry vs s t e r o i d c o n c e n t r a t i o n , both s a t u r a b i l i t y and s p e c i f i c i t y have been demonstrated ( 4 ) . The demonstration that c e r t a i n s u l p h y d r y l b l o c k i n g agents i n h i b i t s t e r o i d e n t r y , lends f u r t h e r support f o r f a c i l i t a t e d t r a n s p o r t as a p o s s i b l e entry mechanism ( 4 ) . A compromise e x p l a n a t i o n i s the p o s t u l a t i o n that s m a l l d i f f e r e n c e s i n s t e r o i d s t r u c t u r e may be r e s p o n s i b l e f o r the e f f e c t s on plasma membrane p e r m e a b i l i t y . The c o n c e n t r a t i o n ( e x t r a c e l l u l a r ) of s t e r o i d s a v a i l a b l e f o r — 18 entry i n t o t a r g e t c e l l s i s i n the range 10 x 10" M to 10 x 10~ 1 0M. E x t r a c e l l u l a r s t e r o i d s are 90% protein-bound. Albumin, due to i t s high c o n c e n t r a t i o n and i n s p i t e of i t s low s p e c i f i c i t y and low binding a f f i n i t y i s the major candidate f o r e x t r a c e l l u l a r hormone b i n d i n g . That there i s some s e l e c t i v i t y , i s evident from the o b s e r v a t i o n s that the a f f i n i t y of albumin f o r a p a r t i c u l a r s t e r o i d v a r i e s i n v e r s e l y with the number of p o l a r groups on the s t e r o i d . The major high a f f i n i t y t r a n s p o r t p r o t e i n i s sex-hormone bindin g g l o b u l i n (SHBG). The other high a f f i n i t y e x t r a c e l l u l a r p r o t e i n s are e i t h e r a or g g l y c o p r o t e i n s , such as t r a n s c o r t i n , a - f e t o p r o t e i n and progesterone-binding plasma p r o t e i n s (PBG). B i n d i n g of s t e r o i d s to these p r o t e i n s i s b e l i e v e d to be non-covalent. The s t e r o i d e n t e r s the cytoplasm of a t a r g e t c e l l i t forms an a c t i v e complex with i t s r e c e p t o r p r o t e i n . T h i s 3 b i n d i n g i s c h a r a c t e r i z e d by h i gh a f f i n i t y and s p e c i f i c i t y but a g a i n appears n o n - c o v a l e n t i n n a t u r e . T r a n s i e n t a s s o c i a t i o n i n the form of weak b i n d i n g i s a l s o p o s t u l a t e d t o o c cu r w i t h endop l a sm i c r e t i c u l u m and /o r components o f the i n n e r p lasma membrane. A f t e r b i n d i n g , the s t e r o i d - r e c e p t o r complex undergoes a t empe r a t u r e dependent c o n f o r m a t i o n a l change and i s then t r a n s l o c a t e d i n t o the nu c l e u s w i t h subsequent b i n d i n g t o DNA and s p e c i f i c c h r o m a t i n p r o t e i n s ( 4 , 5 , 6 ) . The s t e r o i d r e c e p t o r demons t r a t e s s p e c i f i c b i n d i n g o f a f i n i t e number o f b i n d i n g s i t e s . T h i s i s r e p o r t e d t o range f rom 5 -16 ,000 per c e l l , w i t h a Kd o f 2 x 1 0 " 1 0 M ( 4 ) . Some o t h e r i n t r a c e l l u l a r n o n - s p e c i f i c b i n d i n g p r o t e i n s have a l s o been demons t r a t ed , (Kd l x l 0 ~ 6 M ) , but u n l i k e the s p e c i f i c r e c e p t o r s a re c o n s i d e r e d t o be n o n - s a t u r a b l e . The n o n - s p e c i f i c s t e r o i d b i n d i n g p r o t e i n s can be d i s t i n g u i s h e d f rom the s a t u r a b l e s p e c i f i c s t e r o i d r e c e p t o r s bo th by s e d i m e n t a t i o n p r o p e r t i e s and by c o m p e t i t i v e b i n d i n g w i t h compounds o f s i m i l a r a f f i n i t e s and s p e c i f i c i t i e s . Low s a l t ( l e s s t han 0.15M KC1) s u c r o s e d e n s i t y g r a d i e n t work has i d e n t i f i e d the agg rega t e form of the r e c e p t o r p r o t e i n w i t h an appa ren t m o l e c u l a r we igh t o f g r e a t e r t han 200,000 d a l t o n s and a s e d i m e n t a t i o n c o e f f i c i e n t of 8S. H igh s a l t ( g r e a t e r t han 0.4M KC1) r e s u l t s i n d i s s o c i a t i o n t o a d imer wh ich sed imen t s a t a p p r o x i m a t e l y 4S w i t h a m o l e c u l a r we igh t o f a p p r o x i m a t e l y 130 ,000 4 d a l t o n s . The d imer when exposed t o 5M u rea d i s s o c i a t e s i n t o two n o n - i d e n t i c a l s u b u n i t s A and B. S e d i m e n t a t i o n m o l e c u l a r we i gh t s a re g i v e n as a p p r o x i m a t e l y 65 ,000 d a l t o n s , w h i l e SDS - a c r y l am i d e g e l e l e c t r o p h o r e s i s g i v e s m o l e c u l a r we i gh t s o f 110 ,000 d a l t o n s f o r s u b u n i t A and 117 ,000 d a l t o n s f o r s u b u n i t B. Each d imer b i n d s two s t e r o i d m o l e c u l e s . T h e a g g r e g a t i o n t o a t e t r a m e r seen i n low s a l t i s c h a r a c t e r i s t i c o f the s p e c i f i c r e c e p t o r p r o t e i n s and i s r e v e r s i b l e by a l t e r a t i o n o f the i o n i c s t r e n g t h . H i g he r agg rega t e s ( g r e a t e r t han 8S) have not been o b s e r v e d . Recent work w i t h e l e c t r o n m i c r o s copy i n d i c a t e t h a t f o r e s t r o g e n , p r o g e s t e r o n e and d i h y d r o t e s t o s t e r o n e the d imers a re o b l a t e e l l i p s o i d a l s t r u c t u r e s ( 4 ) . T r a n s l o c a t i o n changes the 8S t o a 5.4S m o i e t y . W i t h i n t he n u c l e u s the 5.4S complex must r e c o g n i z e a s p e c i f i c a c c e p t o r s i t e i n o r d e r to f u l f i l l i t s r o l e i n t r a n s c r i p t i o n a l c o n t r o l . R e c o g n i t i o n i n v o l v e s bo th DNA and p r o t e i n w i t h the p r o g e s t e r o n e r e c e p t o r s u b u n i t A i n t e r a c t i n g w i t h the f o rmer and the B s u b u n i t d e m o n s t r a t i n g h i g h s p e c i f i c i t y f o r a c i d i c chromosomal p r o t e i n s ( 4 ) . The s t e r o i d - r e c e p t o r complex p r e f e r e n t i a l l y b i n d s t o e u ch r oma t i n not h e t e r o c h r o m a t i n . T h i o l b l o c k i n g agen t s i n h i b i t e d e s t r a d i o l b i n d i n g t o r e c e p t o r s i n mammary t i s s u e , t hus s u g g e s t i n g the impo r t an ce of s u l p h y d r y l g roups i n l i g a n d r e c e p t o r i n t e r a c t i o n s ( 5 ) . Indeed the a d d i t i o n o f s u l p h y d r y l p r o t e c t i n g agen t s such as d i t h i o t h r e i t o l o r m o n o t h i o g l y c e r o l i n c r e a s e d the s p e c i f i c 5 e s t r o g e n b i n d i n g o f mammary t i s s u e s ( 7 ) . W i th r e ga r d to the n a t u r e o f the l i g a n d - r e c e p t o r i n t e r a c t i o n s bo th the 8-9S and the 4-5S s u b u n i t s can b i n d e s t r a d i o l - 1 7 3. P r o t e a s e s have been shown t o c o m p l e t e l y d e s t r o y s p e c i f i c e s t r o g e n b i n d i n g , w h i l e d e o x y r i b o n u c l e a s e s and r i b o n u c l e a s e s do n o t . S p e c i f i c b i n d i n g s i t e s appear t o be p r i n c i p a l l y p e p t i d e i n n a t u r e , though t h e r e i s some s u g g e s t i o n t h a t they may c o n t a i n phosphorous and c a r b o h y d r a t e m o i e t i e s ( 5 ) . I t has been no ted t h a t any s u b s t i t u t i o n o r s t e r i c h i n d r a n c e o f the C 3 - h y d r o x y l group d r a s t i c a l l y l owe r s o r even a b o l i s h e s the a f f i n i t y of the l i g a n d f o r the b i n d i n g s i t e . The C-17 3 h y d r o x y l p o s i t i o n i s l e s s c r i t i c a l t o b i n d i n g s i t e r e c o g n i t i o n , but a change f rom to i n t h i s p o s i t i o n r e s u l t s i n a 25 f o l d r e d u c t i o n i n b i n d i n g a f f i n i t y ( 6 ) . The degree o f s a t u r a t i o n o f the A r i n g a l s o appea r s t o be c r i t i c a l . I f o n l y one doub l e bond i s p r e s e n t such as i n 5 ( 1 0 ) e s t r a n - 3 < * , 1 7 B d i o l , e f f e c t i v e n e s s of b i n d i n g i s r educed t o 1% o f t h a t o f e s t r a d i o l . S t u d i e s unde r t a ken t o i n v e s t i g a t e the t h e r m o s t a b i l i t y o f e s t r o g e n b i n d i n g p r o t e i n s show t h a t o c c u p i e d e s t r o g e n p r o t e i n s a re more s t a b l e t han t ho se wh i ch a re unoc cup i ed and f u r t h e r t h a t the p re sence of s u l p h y d r y l p r o t e c t i n g agen t s have no e f f e c t on t empe ra t u r e s e n s i t i v i t y o f the b i n d i n g component . The h a l f l i f e o f o c c up i e d r e c e p t o r s i t e s at 25°C i s g i v e n as 2 1 0 m i nu t e s , whereas unoccup i ed s i t e s a t the same t empe ra t u r e have a h a l f l i f e o f 95 m inu te s ( 6 ) . Recent work ( 8 - 1 2 ) s u gge s t s a p o s s i b l e m o d i f i c a t i o n o f the 6 c l a s s i c two s t e p model j u s t d i s c u s s e d . There i s mount ing e v i d e n c e t h a t t h e r e may be e x c e p t i o n s t o the dogma t h a t c y t o p l a s m i c r e c e p t o r s w i l l o n l y e n t e r the nu c l e u s when bound to hormone. F i g u r e two i l l u s t r a t e s t h i s second mode l , wh ich p r opose s t h a t e s t r o g e n d i f f u s e s d i r e c t l y i n t o the nu c l e u s where i t b i n d s w i t h unoc cup i ed r e c e p t o r , becomes a c t i v a t e d and i n t e r a c t s w i t h a c c e p t o r s to i n i t i a t e b i o l o g i c a l r e spon se s such as c e l l p r o l i f e r a t i o n and p r o t e i n s y n t h e s i s . Unbound r e c e p t o r i s i n e q u i l i b r i u m , p a r t i t i o n e d between c y t o p l a sm and n u c l e u s a c c o r d i n g t o t h e i r r e s p e c t i v e f r e e wate r c o n t e n t . The r e c e p t o r s a re a c t i v a t e d i n s i t u and the n u c l e a r f r a c t i o n o f s t e r o i d - r e c e p t o r comp l exes , b i n d s t o the c h r o m a t i n and thus p r e c i p i t a t e s out o f s o l u t i o n . The e f f e c t o f t h i s b i n d i n g i s such t h a t the n u c l e a r volume " a p p e a r s " d e vo i d o f r e c e p t o r s . The c y t o p l a s m i c r e c e p t o r s move i n t o the n u c l e u s ( t r a n s l o c a t i o n ) t o r e - e s t a b l i s h the o r i g i n a l e q u i l i b r i u m . A u t o r a d i o g r a p h i c l o c a l i z a t i o n o f the hormone b i n d i n g t o t he nu c l e u s i n n o n - t r a n s l o c a t i o n c o n d i t i o n s (11) s uppo r t s t h i s mode l . The o r i g i n a l e v i d e n c e f o r t he t w o - s t e p model was t h a t on homogen i z a t i o n i n b u f f e r most of the b i n d i n g appeared i n the c y t o s o l . T h i s may s i m p l y be a consequence o f the change i n the f r e e water c o n t e n t o f the c y t o p l a s m i c compar tment . Upon homogen i z a t i o n o f t i s s u e i n b u f f e r , the wate r c on t en t o f t he c y t o p l a s m i c compartment would i n c r e a s e ove r t h a t i n the n u c l e a r compar tment . To r e - e s t a b l i s h the o r i g i n a l e q u i l i b r i u m , the 7 F i g . 2 The r e v i s e d m o d e l o f e s t r o g e n a c t i o n i n t a r g e t c e l l s . P l a s m a e s t r o g e n (E) d i f f u s e s d i r e c t l y i n t o t h e n u c l e u s w h e r e i t b i n d s w i t h t h e n u c l e a r e s t r o g e n r e c e p t o r s (R) a n d i n i t i a t e s p r o t e i n s y n t h e s i s a n d c e l l p r o l i f e r a t i o n . 8 n u c l e a r r e c e p t o r would e n t e r the c y t o s o l . Bo th a u t o r a d i o g r a p h i c and immunochemica l e v i d e n c e sugges t t h a t the r e v i s e d model ( f i g u r e 2) i s a more a c c u r a t e i n t e r p r e t a t i o n of s t e r o i d hormone a c t i o n ( 9 - 1 1 ) . i i . METHODS FOR STEROID HORMONE ANALYSIS F o l l o w i n g F o l c a ' s o b s e r v a t i o n t h a t c e r t a i n tumours c o u l d r e t a i n s p e c i f i c s t e r o i d s ( 1 3 ) , T o f t and G o r s k i used an i n v i t r o method t o demons t r a t e ER p r o t e i n i n u t e r i n e t i s s u e , w i t h t he subsequent d e m o n s t r a t i o n of c y t o p l a s m i c ER i n human b r e a s t t i s s u e by Jensen ( 1 4 ) . S i n c e then t h e r e has been a p r o l i f e r a t i o n of c y t o p l a s m i c and n u c l e a r ER assay methods and r e p o r t s o f c o r r e l a t i o n s between r e c e p t o r c on t en t and r e sponse t o e n d o c r i n e t h e r a p y , as w e l l as p r o g n o s i s . P r o g e s t e r o n e r e c e p t o r , an end - p r odu c t of e s t r o g e n s t i m u l a t i o n and t hus a measure o f hormone r e s p o n s i v e n e s s , has a l s o been assayed i n an a t t empt to improve the a b i l i t y t o p r e d i c t the hormona l r e s p o n s i v e n e s s o f a g i v e n b r e a s t tumour . A s u i t a b l e assay f o r s t e r o i d r e c e p t o r s must measure bound s t e r o i d and d i f f e r e n t i a t e between s e v e r a l s p e c i e s of r e c e p t o r o f wh ich f r e e c y t o p l a s m i c , o c c up i e d c y t o p l a s m i c and n u c l e a r r e c e p t o r s a re t he majo r fo rms f o r c o n s i d e r a t i o n . The d i f f e r e n t i a t i o n o f f i l l e d and u n f i l l e d r e c e p t o r s i t e s i s based on the t empe ra t u r e dependence of the r a t e o f s t e r o i d d i s s o c i a t i o n ( 1 5 ) . At 0-4°C o c c u p i e d s t e r o i d r e c e p t o r s d i s s o c i a t e ve r y 9 s l o w l y w i t h a t ^ 2 o f 20-30 hou r s , whereas u n f i l l e d s i t e s r e a d i l y b i nd t he 3 H - l a b e l e d l i g a n d . At e l e v a t e d t e m p e r a t u r e s , i n a d d i t i o n t o the s a t u r a t i o n o f r e c e p t o r s i t e s , p r e v i o u s l y f i l l e d r e c e p t o r s i t e s r e a d i l y d i s s o c i a t e and b i nd added 3 3 H - l a b e l e d s t e r o i d . H - l a b e l e d s t e r o i d s were the f i r s t 125 l i g a n d s u s ed . In 1979 Hochberg (16) i n t r o d u c e d I - l a b e l e d 125 e s t r a d i o l . Wh i l e I i s a gamma e m i t t e r , i t can be coun ted as a b e t a e m i t t e r i n a l i q u i d s c i n t i l l a t i o n sytem by v i r t u e o f t he f a c t t h a t i t s s e conda ry e l e c t r o n s (Auger o r c o n v e r s i o n ) a re c a p a b l e of e x c i t i n g the c o c k t a i l components t o p roduce p h o t o n s . L i q u i d s c i n t i l l a t i o n c o u n t i n g i n v o l v e s the c o n v e r s i o n o f the k i n e t i c energy o f a r a d i o a c t i v e decay p a r t i c l e i n t o p h o t o n s . In t h i s s y s t em, the decay p a r t i c l e ( p a r t i c l e ) i n t e r a c t s w i t h a mo l e c u l e ( f l u o r ) t o e x c i t e i t above i t s ground s t a t e e n e r g y . When the f l u o r r e t u r n s t o t he ground s t a t e i t em i t s v i s i b l e l i g h t . Because the p e n e t r a t i o n o f a be t a p a r t i c l e i s so s h o r t , w i t h a h a l f - t h i c k n e s s 0.008mm, the f l u o r o r s c i n t i l l a t o r must be mixed w i t h the samp le . Thus the sample i s d i s s o l v e d i n a s o l u t i o n ( t o l u e n e ) wh i ch c o n t a i n s the s c i n t i l l a t o r . The most w i d e l y used f l u o r i s 2 , 5 - d i p h e n y l o x a z o l e (PPO) . The number o f pho tons e m i t t e d i s p r o p o r t i o n a l t o the energy o f the e l e c t r o n wh i ch i n t e r a c t e d w i t h f l u o r . These pho tons a re then t r a n s m i t t e d t h r ough the c o c k t a i l , t h r ough the w a l l s o f the v i a l and a re c o l l e c t e d by a s p e c i a l l i g h t d e t e c t i n g sytem onto the f a c e s o f two p h o t o m u l t i p l i e r t u b e s . E l e c t r o n i c components c o n v e r t t h i s 10 energy i n t o v o l t a g e p u l s e s whose amp l i t u d e i s p r o p o r t i o n a l t o the number o f pho tons d e t e c t e d . Coun t i ng c h a n n e l s a re used t o s e p a r a t e t he se v o l t a g e s on the b a s i s o f t h e i r h e i g h t , such t h a t o n l y p u l s e s h i g h e r t han the c h a n n e l ' s l owe r l i m i t and l owe r t han the upper l i m i t a re r e g i s t e r e d as a " c o u n t " . An impo r t a n t f a c t o r i n l i q u i d s c i n t i l l a t i o n c o u n t i n g i s quen ch i ng , wh ich i s d e f i n e d as "any p r o c e s s which r educes the number o f pho tons ob se r ved f o r a g i v e n amount o f i n p u t t o the l i q u i d s c i n t i l l a t i o n s o l u t i o n " ( 1 7 ) . Three t ype s o f quench ing a re o b s e r v e d . Chem i c a l quench ing o c c u r s when c o c k t a i l components a re p r e s e n t wh i ch impede the t r a n s f e r o f energy f rom the s o l v e n t t o the f l u o r . C o l o u r quench ing r e f e r s t o the p re sence o f components wh ich abso rb the l i g h t pho tons e m i t t e d by the p r ima r y and secondary f l u o r and f i n a l l y o p t i c a l quench ing wh ich i s a b s o r p t i o n o f e m i t t e d pho tons by i m p u r i t i e s such as c o n d e n s a t i o n o r r e s i d u e on the e x t e r n a l s u r f a c e o f the c o u n t i n g v i a l . C o r r e c t i o n f o r t h i s e f f e c t i s ve ry i m p o r t a n t s i n c e the r e l a t i v e s e n s i t i v i t y o f the d e t e c t i o n may be s i g n i f i c a n t l y r e du ced , such t h a t the v a l u e s are no l o n g e r s t a t i s t i c a l l y s i g n i f i c a n t . In the case o f d u a l l a b e l c o u n t i n g one e n c o u n t e r s t he added p rob lem of " s p i l l o v e r " . T h i s phenomenon r e f e r s t o the s h i f t o f the h i g h e r energy i s o t o p e c oun t s i n t o the l owe r energy i s o t o p e ' s c h anne l as a r e s u l t o f q u e n c h i n g . S e v e r a l t e c h n i q u e s have been deve l oped t o c o r r e c t f o r t h i s e f f e c t . The i n t e r n a l s t a n d a r d t e c h n i q u e i n v o l v e s c o u n t i n g of the 11 sample b e f o r e and a f t e r a d d i t i o n o f a s m a l l amount o f r a d i o a c t i v e s t a n d a r d , t hus e n a b l i n g c a l c u l a t i o n s o f e f f i c i e n c y f rom the change i n the coun t r a t e . The sample c h a n n e l r a t i o t e c h n i q u e r e l i e s on the f a c t t h a t quench ing w i l l s h i f t the p u l s e h e i g h t s t o s m a l l e r p u l s e h e i g h t s ( e n e r g i e s ) . A s h i f t o f t h i s t ype w i l l be r e f l e c t e d i n a change i n the r a t i o between two c h anne l s s e t so t h a t the t o t a l spec t rum i s coun ted i n one c h anne l w h i l e the o t h e r c o un t s on l y a f i x e d f r a c t i o n ( i e . the upper t h i r d ) . In e x t e r n a l s t a n d a r d i z a t i o n methods , a gamma ray sou r ce i s p l a c e d near the samp le . Some of the gamma r a y s i n t e r a c t w i t h the l i q u i d s c i n t i l l a t i o n c o c k t a i l c a u s i n g the p r o d u c t i o n o f Compton e l e c t r o n s t h a t p roduce f l u o r e s c e n c e wh ich v a r i e s w i t h the degree of q uen ch i n g . A p l o t o f the number o f p u l s e s vs the p u l s e h e i g h t y i e l d s a Compton p u l s e h e i g h t d i s t r i b u t i o n . A measure o f a p o r t i o n of t h i s d i s t r i b u t i o n ( u s u a l l y the h i g h e s t p u l s e s ) i s r e c o r d e d ove r a p e r i o d o f t ime and e xp r e s s ed as the e x t e r n a l s t a n d a r d count ESC. Thus the more h i g h l y quenched a sample i s the fewer the number o f c oun t s i n the h i g h e r p u l s e amp l i t u d e p o r t i o n o f the d i s t r i b u t i o n and the l owe r the ESC number. The e x t e r n a l s t a n d a r d c h a n n e l r a t i o n ESCR t e c h n i q u e goes one s t ep f u r t h e r by m o n i t o r i n g two p o r t i o n s o f the Compton d i s t r i b u t i o n and e x p r e s s i n g i t as a r a t i o . In my work two t e c h n i q u e s were used t o c o r r e c t f o r q u e n c h i n g . H# and au t oma t i c quench c o r r e c t i o n AQC, bo th f e a t u r e s o f the Beckman LS-9000 l i q u i d s c i n t i l l a t i o n c o u n t e r . H# - T h i s 12 t e c h n i q u e i s based on the i n s t r u m e n t a l d e t e r m i n a t i o n o f the c h a n n e l s e t t i n g o f the Compton edge f rom an e x t e r n a l gamma s o u r c e . Measurements o f s p e c i f i c p u l s e h e i g h t s i n the Compton edge r e g i o n do not depend on the c o u n t i n g r a t e o f the samp le , or t h e a c t i v i t y o f t h e e x t e r n a l s o u r c e . F u r t h e r t h i s d e t e r m i n a t i o n i s u n a f f e c t e d by the p o s i t i o n of the v i a l and e x t e r n a l gamma s o u r c e . In the Beckman l i q u i d s c i n t i l l a t i o n c o u n t e r u sed , the Compton edge i s based on ^"'57Cs . Fo r each sample the Compton p u l s e h e i g h t d i s t r i b u t i o n i s measured and a c o m p u t e r - d i r e c t e d s can i s i n i t i a t e d t o l o c a t e the Compton edge and the i n f l e c t i o n p o i n t o f the Compton edge i s d e t e r m i n e d . I n a quenched sample t he Compton edge i n f l e c t i o n p o i n t w i l l s h i f t t o a l owe r c h anne l s e t t i n g . The d i f f e r e n c e i n Compton edge i n f l e c t i o n p o i n t s between a s e a l e d unquenched s t a n d a r d and the sample i s the H#. RELATIVE PULSE HEIGHTS 13 Au toma t i c quench c o r r e c t i o n i s based on the H#. I t i n v o l v e s t he au t oma t i c ad j u s tmen t of the c o u n t i n g c h a n n e l s , i n p r o p o r t i o n to t he H# of the samp le . As a r e s u l t the i n s t r u m e n t i s a b l e to t r a c k the r a d i o i s o t o p e ' s p u l s e h e i g h t d i s t r i b u t i o n and t hus a d j u s t f o r the " s p i l l i n g o v e r " e f f e c t wh ich can g r e a t l y d i s t o r t c a l c u l a t i o n when two i s o t o p e s a re be i ng s i m u l t a n e o u s l y m o n i t o r e d . Nugent and Mayes (5) i n i t i a l l y deve l oped a method f o r measu r i ng c o r t i c o s t e r o i d s i n p lasma u s i n g c o r t i c o s t e r o i d b i n d i n g g l o b u l i n (CBG) and d e x t r a n coa t ed c h a r c o a l (DCC) . Korenman i n 1968, used the p r o cedu r e t o demons t r a t e s p e c i f i c e s t r o g e n b i n d i n g components i n c y t o s o l p r epa r ed f rom u t e r i o f p r egnan t r a b b i t s . Korenman and Dukes t hen went on t o measure s p e c i f i c e s t r o g e n - b i n d i n g c a p a c i t y i n human b r e a s t c an ce r ( 1 8 ) . M o d i f i c a t i o n s o f t he o r i g i n a l p r o cedu r e i n v o l v i n g the i n c u b a t i o n of DCC a t bo th a s p e c i f i e d t ime and t empe r a t u r e w i t h the "^H-labeled s t e r o i d r e c e p t o r complexes were i n t r o d u c e d by F e h e r t y e t a l . ( 1 9 ) . T h i s i n c u b a t i o n p r o cedu r e was found to e l i m i n a t e b i n d i n g t o low a f f i n i t y and n o n - s p e c i f i c s i t e s , t hu s r e s u l t i n g i n improved a c cu r a c y and s e n s i t i v i t y . A s i m i l a r m o d i f i c a t i o n was adopted by the EORTC B r e a s t Cancer C o o p e r a t i v e Group (1973) ( 2 0 ) . B r aunsbe rg (21) sugges t ed t h a t i n c u b a t i o n w i t h DCC r e s u l t e d i n i n a c c u r a c i e s when s t e r o i d b i n d i n g c a p a c i t i e s were e s t i m a t e d i n t i s s u e e x t r a c t s w i t h a p p r e c i a b l e c o n c e n t r a t i o n s o f b i n d i n g components w i t h low a f f i n i t y s i t e s . L a t e r s t u d i e s have shown IA t h a t the i n c l u s i o n o f an u n l a b e l e d c o m p e t i t i t v e i n h i b i t o r o f 3 H - l a b e l e d e s t r a d i o l - 1 7 B such as n a f o x i d i n e o r d i e t h y l s t i l b e s t e r o l (DES) c o u l d ensu re a c c u r a t e e s t i m a t i o n o f n o n - s p e c i f i c b i n d i n g components . There a re a number o f assay methods a v a i l a b l e f o r s t e r o i d r e c e p t o r assessment and a r ev i ew o f the g e n e r a l a s p e c t s o f t he se methods f o l l o w s , w i t h emphas i s on t he d e x t r a n - c o a t e d c h a r c o a l (DCC) method wh ich was employed i n t h i s p r o j e c t . Most e x i s t i n g methods a re based on the measurement of bound r a d i o l a b e l e d e s t r a d i o l t o the s o l u b l e p r o t e i n s of the c y t o p l a s m i c f r a c t i o n o f a t i s s u e homogenate. The methods d i f f e r f rom one ano the r i n two key a r e a s : 1) the p r o c edu r e used t o s e p a r a t e bound hormone f rom t h a t wh i ch rema ins unbound 2) the a b i l i t y o f the method t o d i s c r i m i n a t e between t r u e r e c e p t o r b i n d i n g and n o n - s p e c i f i c b i n d i n g t o o t h e r p r o t e i n s i n c l u d i n g a l bum in and sex hormone b i n d i n g g l o b u l i n (SHBG). The methods f o r s e p a r a t i n g bound f rom f r e e l i g a n d a re n o n - e q u i l i b r i u m i n n a t u r e - i n the sense t h a t the hormone must rema in bound t o i t s r e c e p t o r i n the absence of any f r e e hormone f o r t he b r i e f p e r i o d d u r i n g wh ich t he s e p e r a t i o n i s c a r r i e d o u t . True e q u i l i b r i u m methods , such as e q u i l i b r i u m d i a l y s i s have not been emp loyab l e i n r e c e p t o r a n a l y s i s s i n c e the s m a l l q u a n t i t i e s o f r e c e p t o r do not s h i f t the e q u i l i b r i u m s u f f i c i e n t l y t o p e rm i t 15 accurate measurement. Besides g r e a t e r s e n s i t i v i t y , n o n - e q u i l i b r i u m methods a l s o take advantage of the d i f f e r e n c e s i n a f f i n i t y of the l i g a n d f o r s p e c i f i c r e c e p t o r and non-receptor p r o t e i n s . Most of the non-receptor p r o t e i n s bind the s t e r o i d s with low a f f i n i t y , such that the d i s s o c i a t i o n i s r a p i d , while the s p e c i f i c r e c e p t o r - l i g a n d complex b a r e l y d i s s o c i a t e s at a l l . The d i s s o c i a t i o n constant i s f r e q u e n t l y used to d i s t i n g u i s h s p e c i f i c from n o n - s p e c i f i c b i n d i n g , s i n c e r e c e p t o r s have a higher a f f i n i t y f o r t h e i r l i g a n d than even the high a f f i n i t y plasma bi n d i n g g l o b u l i n s . The amount of s t e r o i d bound at approximate e q u i l i b r i u m with three or more f r e e s t e r o i d c o n c e n t r a t i o n s i s p l o t t e d and e x t r a p o l a t e d to s a t u r a t i o n to determine the number of r e c e p t o r s i t e s present (Bmax). This i s accomplished using e i t h e r the Scatchard or the Woolf p l o t (22). Since i n many i n s t a n c e s , s a t u r a t i o n of the s p e c i f i c b i n d i n g s i t e s i s not accomplished, the data i s e x t r a p o l a t e d to s a t u r a t i o n , thus a l l o w i n g the d e t e r m i n a t i o n of the number of bin d i n g s i t e s . The Scatchard p l o t and/or the Woolf p l o t are s u i t a b l e g r a p h i c a l methods f o r the c a l c u l a t i o n of b i n d i n g parameters from known values f o r bound (B) and unbounded or f r e e (F) l i g a n d . The Michaelis-Menton equation d e s c r i b e s the r e l a t i o n s h i p between B and F as f o l l o w s : B=(B F)/Kd + F max 16 T h i s i s t he e q u a t i o n of a h y p e r b o l a . C a l c u l a t i o n o f Kd ( d i s s o c i a t i o n c o n s t a n t ) and B m g x (number o f s p e c i f i c r e c e p t o r s i t e s ) i s c o n s i d e r a b l y e a s i e r w i t h s t r a i g h t l i n e f i t t i n g and f o r t h i s r eason the above e q u a t i o n has been l i n e a r i z e d . In the case o f the S c a t c h a r d p l o t , the e q u a t i o n becomes B/F=(B /Kd) + ( - l / K d ) B max The Woolf e q u a t i o n i s F/B=Kd/B + (1/B )F max max Thus f o r the S c a t c h a r d p l o t , the i n t e r c e p t o f the e x t r a p o l a t e d l i n e w i t h the a b s c i s s a g i v e s the v a l u e f o r B . w h i l e t he a max' s l o p e o f the l i n e ( - 1 /Kd ) g i v e s the v a l u e f o r Kd . I n the case o f t he Wool f p l o t , B i s de t e rm ined f rom the s l o p e and Kd f rom K ' max K the i n t e r c e p t o f the e x t r a p o l a t e d l i n e w i t h the o r d i n a t e such t h a t K d = y - i n t e r c e p t x B m a x ' ( s e e f i g u r e 3) The most commonly used p l o t i s the S c a t c h a r d , w i t h l i n e f i t t i n g a c c omp l i s h ed by the l e a s t squa re s r e g r e s s i o n a n a l y s i s . Much of i t s p o p u l a r i t y a r i s e s f rom the f a c t t h a t the i m p o r t a n t v a l u e B can be read d i r e c t l y f rom the g r a p h . There a re max however some drawbacks t o t h i s p l o t wh ich a re l a r g e l y overcome by u s i n g the Wool f p l o t . Fo r l e a s t squa re s r e g r e s s i o n a n a l y s i s to be a p p l i e d , the i ndependen t v a r i a b l e s hou l d be r e l a t i v e l y e r r o r f r e e . In the case o f the S c a t c h a r d p l o t the i ndependen t v a r i a b l e i s B, wh ich i s p rone t o c o n s i d e r a b l y g r e a t e r e r r o r t han F wh i ch i s the i ndependen t v a r i a b l e i n the Wool f p l o t . Ano the r r e qu i r emen t o f l e a s t squa re s r e g r e s s i o n a n a l y s i s i s t h a t the 17 SAMPLE WOOLF PLOT F R E E S T E R O I D F i g u r e 3A S a m p l e W o o l f P l o t . F r e e / s p e c i f i c b o u n d i s p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n o f f r e e s t e r o i d . Bmax i s c a l c u l a t e d f r o m 1 / s l o p e a n d t h e d i s s o c i a t i o n c o n s t a n t (Kd) i s d e t e r m i n e d f r o m t h e p r o d u c t o f t h e y - i n t e r c e p t a n d Bmax. 18 SAMPLE SCATCHARD PLOT r c e p t BOUND S T E R O I D F i g u r e 3B S a m p l e S c a t c h a r d P l o t . The c o n c e n t r a t i o n o f s p e c i f i c b o u n d / f r e e i s p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n o f s p e c i f i c b o u n d . Bmax i s d e t e r m i n e d f r o m t h e x - i n t e r c e p t , w h i l e t h e d i s s o c i a t i o n c o n s t a n t i s d e t e r m i n e d f r o m - 1 / s l o p e . 19 e r r o r i n the dependent v a r i a b l e be homogeneous. Fo r the S c a t c h a r d p l o t , the e r r o r i n B/F i s d i s t r i b u t e d r a d i a l l y such t h a t the e r r o r i s g r e a t e s t a t low i n c u b a t i n g c o n c e n t r a t i o n s o f l i g a n d and l owe s t a t h i g h i n c u b a t i n g c o n c e n t r a t i o n s . T h i s may w e l l be because the l e v e l o f a c t i v i t y i n the p o s t - c h a r c o a l s u p e r n a t a n t s i s l owe s t when the i n c u b a t i n g c o n c e n t r a t i o n o f l i g a n d i s l owe s t and t h e r e f o r e i s s u b j e c t t o the g r e a t e s t e r r o r ( 2 2 ) . In c o n t r a s t , t he e r r o r d i s t r i b u t i o n i n the Wool f p l o t ( F /B ) i s r e l a t i v e l y c o n s t a n t and i ndependen t of the i n c u b a t i n g c o n c e n t r a t i o n o f l i g a n d . F i n a l l y , i n the p re sence o f o u t l i e r s , a f r e q u e n t o c cu rance i n s t e r o i d a s s a y s , the Woolf p l o t pe r f o rms b e t t e r ( 2 2 , 2 3 ) . The s p e c i f i c i t y o f the assay i s enhanced by t a k i n g advantage o f t he f a c t t h a t s p e c i f i c r e c e p t o r s i t e s s a t u r a t e a t r e l a t i v e l y low s t e r i o d c o n c e n t r a t i o n s . Thus by add ing a 1 0 0 - f o l d e x ce s s o f n o n - r a d i o a c t i v e l i g a n d , 99% of the r e c e p t o r s i t e s w i l l be f i l l e d w i t h t h i s l i g a n d . S i n c e the low a f f i n i t y n o n - s p e c i f i c b i n d i n g p r o t e i n s w i l l be nowhere near s a t u r a t i o n , the subsequent a d d i t i o n of l a b e l l e d l i g a n d w i l l a l l o w q u a n t i t a t i o n of the n o n - s p e c i f i c b i n d i n g s i n c e the p r e sence o f u n l a b e l e d l i g a n d w i l l have l i t t l e e f f e c t on t he b i n d i n g of the l a b e l e d l i g a n d t o t he se p r o t e i n s . The n o n - s p e c i f i c b i n d i n g (NSB) measured i n t h i s way i s t hen s u b t r a c t e d f rom the t o t a l b i n d i n g to y i e l d s p e c i f i c b i n d i n g . S p e c i f i c i t y can be f u r t h e r enhanced by a p ruden t c h o i c e o f r a d i o l i g a n d and u n l a b e l e d c o m p e t i t o r . L a b e l e d e s t r a d i o l b i n d s to 20 t he e s t r a d i o l r e c e p t o r w i t h h i g h a f f i n i t y , but a l s o u n f o r t u n a t e l y shows c o n s i d e r a b l e a f f i n i t y f o r SHBG. T h i s s i t u a t i o n can be c o r r e c t e d f o r by u s i n g u n l a b e l e d d i e t h y l s t i l b e s t e r o l (DES) r a t h e r t han c o l d e s t r a d i o l as the c o m p e t i t o r . DES has an a f f i n i t y f o r ER e q u a l to t h a t o f e s t r a d i o l , but i t s a f f i n i t y f o r SHBG i s f a r l o w e r . The a n t i - e s t r o g e n n a f o x i d i n e has a l s o been used f o r bo th the same r ea son and the same pu rpo se , but the p o s s i b i l i t y t h a t t h i s compound a c t s a l l o s t e r i c a l l y a t a secondary s i t e on the r e c e p t o r has been f o rwa rded ( 1 4 ) . An a l t e r n a t i v e approach i s t o 3 3 r e p l a c e H - e s t r a d i o l w i t h the s y n t h e t i c e s t r o g e n H-R2858 wh i ch app rox ima t e s e s t r a d i o l i n i t s a f f i n i t y f o r the r e c e p t o r , but has a ve ry low a f f i n i t y f o r SHBG and o t h e r i n t e r f e r i n g p r o t e i n s . F i n a l l y , some methods p h y s i c a l l y s e p a r a t e the bound r e c e p t o r f rom the n o n - s p e c i f i c b i n d i n g p r o t e i n s . D e n s i t y g r a d i e n t s e d i m e n t a t i o n , a d s o r p t i o n on h y d r o x y a p a t i t e o r DEAE f i l t e r s a re examples of such methods ( 5 , 1 5 ) . T i s s u e h a n d l i n g and p r e p a r a t i o n a re ve ry s i m i l a r among the v a r i o u s a s s a y s . I t i s now w e l l a c c ep t ed t h a t t i s s u e t o be as sayed f o r r e c e p t o r c on t en t must be c h i l l e d immed i a t e l y upon remova l and as sayed w i t h i n hours o r f r o z e n i n l i q u i d n i t r o g e n . Tumour s t o r a g e shou l d be a t - 70° C o r c o l d e r . B u f f e r s f o r the r e c e p t o r assay a re a l s o ve ry s i m i l a r . The pH u s u a l l y chosen i s 7 . 4 , w i t h s t a b i l i t y of the r e c e p t o r r e p o r t e d o ve r the range pH 7-9. The most common b u f f e r i n g agent i s TRIS 21 (trisChydroxymethyl] aminoethane) a t 10-50 mM. EDTA (1 o r 2 mM) i s f r e q u e n t l y added t o p r e ven t r e c e p t o r a g g r e g a t i o n . 0.5-5mM d i t h i o t h r e i t o l o r 1-12 mM m o n o t h i o g l y c e r o l a re added t o p r o t e c t t he s u l p h y d r y l g roups o f the r e c e p t o r wh i ch a re b e l i e v e d t o be i m p o r t a n t to the b i n d i n g p r o c e s s . G l y c e r o l a t 10% w h i l e not known t o a f f e c t the ER a s s ay s h e l p s s t a b i l i z e the p r o g e s t e r o n e r e c e p t o r s and i s deemed e s s e n t i a l i n the l a t t e r assay ( 2 4 ) . The f r o z e n tumour can be p u l v e r i z e d w h i l e s t i l l f r o z e n u s i n g a Braun d i smembra to r o r i t can be p a r t i a l l y thawed f o r P o l y t r o n d i s r u p t i o n . P u l v e r i z a t i o n i n t h i s f a s h i o n a c c omp l i s h e s two t h i n g s - i t b r eak s up the f i b r o u s t i s s u e so p r e v a l e n t i n human b r e a s t tumours and a l s o t h o r o u g h l y m ixes the t he tumour samp le . The r e s u l t i n g powder i s then homogenized i n a few volumes of b u f f e r o r mere l y a l l o w e d t o thaw out i n the b u f f e r . S e p a r a t i o n o f the c y t o s o l f rom p a r t i c u l a t e f r a c t i o n s i s e f f e c t e d by c e n t r i f u g a t i o n . There i s c o n s i d e r a b l e v a r i a b i l i t y i n p r e p a r a t i o n a t t h i s s t a g e . Most worke r s use the u l t r a c e n t r i f u g e a t 100,000X g f o r 20 m inu te s t o one hou r , w h i l e o t h e r s have used low speed c e n t r i f u g a t i o n a t 2,000X g ( 2 5 , 1 9 ) . J acobsen (26) de t e rm ined t h a t t he 100 ,000 xg ( c y t o s o l i c ) f r a c t i o n c o n s i s t e n t l y y i e l d e d a l owe r ER c on t e n t than the 40 ,000 xg f r a c t i o n ( s u p e r n a t a n t ) . Re cep to r v a l u e s a re g e n e r a l l y r e p o r t e d per m i l l i g r a m c y t o s o l p r o t e i n , w i t h the Lowry method f o r p r o t e i n d e t e r m i n a t i o n a f r e q u e n t l y used method. T o t a l t i s s u e p r o t e i n can i n c l u d e 22 c o n s i d e r a b l e amounts o f a l bum in ( 2 6 , 2 0 ) . Wh i l e a l bum in has not been shown t o c o n t r i b u t e s i g n i f i c a n t l y t o the n o n - s p e c i f i c b i n d i n g , i t can l e a d t o c o n s i d e r a b l e q u a n t i t a t i v e u n d e r - e s t i m a t i o n o f the s p e c i f i c b i n d i n g . Fo r t h i s r e a s o n , a l bum in a s say s and subsequent c o r r e c t i o n f o r serum p r o t e i n c o n t a m i n a t i o n to y i e l d ER pe r m i l l i g r a m c y t o s o l i c p r o t e i n i s recommended ( 2 6 ) . The a l bum in can be measured by i m m u n o d i f f u s i o n and t hus a c o r r e c t i o n f o r a l bum in c o n t a m i n a t i o n i s p o s s i b l e . i i i . CLINICAL APPLICATIONS OF RECEPTOR THEORY Hormonal m a n i p u l a t i o n has been an i m p o r t a n t component o f the t r e a tmen t of human b r e a s t c ance r s i n c e the o b s e r v a t i o n i n 1896 by Bea t s on of r e m i s s i o n o f the d i s e a s e w i t h oophorectomy ( 1 ) . I n b r e a s t and p r o s t a t i c tumours wh i ch have r e t a i n e d hormone r e s p o n s i v e n e s s , a p p r o p r i a t e hormona l m a n i p u l a t i o n r e s u l t s i n s i g n i f i c a n t l y g r e a t e r p a l l i a t i o n than would c h emo the r apeu t i c o r r a d i a t i o n reg imes ( 4 ) . F u r t h e r m o r e , hormona l m a n i p u l a t i o n i s a s s o c i a t e d w i t h l e s s m o r b i d i t y and l e s s non- tumour c y t o t o x i c s eque l a e than the o t h e r t h e r a p e u t i c m o d a l i t i e s . S i n c e hormona l m a n i p u l a t i o n can i n v o l v e s u r g i c a l p r o c e d u r e s such as o opho r e c t om i e s , a d r e n a l e c t o m i e s and /o r hypophysec tom ies i t i s d e s i r a b l e t o i d e n t i f y as a c c u r a t e l y as p o s s i b l e t ho se p a t i e n t s most l i k e l y to b e n e f i t f rom t he se p r o c e d u r e s . S e l e c t i o n p u r e l y on c l i n i c a l d a t a a l one i s i n adequa t e and may r e s u l t i n 23 unneces sa r y and u n d e s i r a b l e d e l a y s i n t r e a tmen t by o t h e r m o d a l i t i e s . To t h i s end marke rs have been sought f o r hormone r e s p o n s i v e n e s s . F o l c a i d e n t i f i e d j u s t such a marker when he was a b l e t o demons t r a t e t h a t the tumours of p a t i e n t s most r e s p o n s i v e to ad rena l e c t omy f o r t r e a tmen t o f b r e a s t c a n c e r , s e que s t e r e d g r e a t e r 3 q u a n t i t i e s o f i n j e c t e d H - h e x o e s t e r o l t han d i d tumours i n p a t i e n t s u n r e s p o n s i v e t o t h i s form of hormona l m a n i p u l a t i o n ( 1 3 ) . T h i s o b s e r v a t i o n i s i n keep i ng w i t h s t e r o i d r e c e p t o r t h eo r y wh ich h y p o t h e s i z e s t h a t h o rmona l l y r e s p o n s i v e t i s s u e s hou l d c o n t a i n h i g h a f f i n i t y s t e r o i d p r o t e i n s , w h i l e i n the absence of such s p e c i f i c r e c e p t o r s r e sponse would be p r e c l u d e d . F o l c a ' s s u g g e s t i o n t h a t c e r t a i n tumours can r e t a i n and c o n c e n t r a t e s p e c i f i c s t e r o i d s has s i n c e been v e r i f i e d bo th i n v i v o and i n v i t r o ( 2 7 , 2 8 ) . By 1970 many l a b s had begun c l i n i c a l t r i a l s t o c o r r e l a t e tumour r e c e p t o r c on t en t w i t h hormone t he r apy r e s p o n s i v e n e s s . The p r o g n o s t i c v a l u e o f e s t r o g e n r e c e p t o r a n a l y s i s was f i r s t p roposed by Jensen (29) and has s ub s equen t l y been r e p e a t e d l y c o n f i r m e d ( 3 0 - 3 4 ) . A c c o r d i n g to da t a p r e s e n t e d a t the 1974 Wash ing ton Con f e r ence on E s t r o g e n R e c e p t o r s ( 3 4 ) , absence of measu rab l e ER c o r r e l a t e d w i t h e n d o c r i n e t he r apy f a i l u r e i n 95% o f b r e a s t c a n c e r s , w h i l e the p re sence o f ER c o r r e l a t e d w i t h a 55-65% re sponse r a t e . 24 The o v e r a l l a c c u r a c y o f p r e d i c t i o n s based on c y t o p l a s m i c ER i s a p p r o x i m a t e l y 75%, d e r i v e d as f o l l o w s : I n c i d e n c e of Re cep to r P o s i t i v i t y (60%) X P r e d i c t i v e A c cu r a c y (60%) P l u s I n c i d e n c e o f Re cep t o r N e g a t i v i t y (40%) X P r e d i c t i v e A c cu r a c y (95%) The 35-45% of r e c e p t o r p o s i t i v e tumours wh ich f a i l t o respond t o hormona l t h e r apy a re h y p o t h e s i z e d to r e p r e s e n t a f a i l u r e i n one o r more o f the s t e p s subsequent to the c y t o p l a s m i c r e c e p t o r b i n d i n g s t e p . To t h i s end McGu i re (35) i n i t i a t e d the use o f the p r o g e s t e r o n e r e c e p t o r assay f o r p r o g n o s i s and t he r apy s e l e c t i o n i n b r e a s t c a n c e r . The p r o g e s t e r o n e r e c e p t o r c o n c e n t r a t i o n i s an e s t r ogen -dependen t f u n c t i o n and t h e r e f o r e an i n d i c a t o r t h a t s t e p s subsequent t o e s t r o g e n b i n d i n g a re i n t a c t ( 4 , 3 6 ) . Indeed i t has been found t h a t tumours w i t h both ER and PgR have the g r e a t e s t r e sponse r a t e t o e n d o c r i n e t he r apy namely 75-85% ( 3 6 - 4 0 ) . a . RECEPTORS AND HISTOLOGICAL PARAMETERS There a re i n d i c a t i o n s t h a t r e c e p t o r s t a t u s v a r i e s w i t h h i s t o l o g i c a l g rade ( 4 1 , 4 2 , 1 1 6 ) . ER p o s i t i v i t y i s found more 25 f r e q u e n t l y i n low grade i n f i l t r a t i n g duc t t umour s . DNA p l o i d y has a l s o been shown t o c o r r e l a t e i n v e r s l y w i t h ER s t a t u s ( 4 1 , 4 3 ) . Though most wo rke r s r e p o r t no c o r r e l a t i o n between ER s t a t u s and m e t a s t a t i c p a t t e r n s ( 4 , 4 2 ) , a r e c e n t p u b l i c a t i o n (44) i n wh ich 25 s u b j e c t s w i t h m e t a s t a t i c b r e a s t c an ce r were examined a t a u t op s y , ER p o s i t i v e tumours were r e p o r t e d to m e t a s t a s i z e more f r e q u e n t l y to t h y r o i d and / o r p a r a t h y r o i d w h i l e ER n e g a t i v e tumours m e t a s t a s i z e d more f r e q u e n t l y to the l e p t o m e n i n g e s . PgR p o s i t i v e tumours m e t a s t a s i z e d more f r e q u e n t l y t o the myocard ium, s m a l l b owe l , u r o t h e l i a l s t r u c t u r e s as w e l l as t h y r o i d and /o r p a r a t h y r o i d . Recent work i n d i c a t e s t h a t m e t a s t a t i c l e s i o n s do not n e c e s s a r i l y have the same r e c e p t o r p r o f i l e as d i d the p r ima r y tumour ( 4 5 , 4 6 ) , t h o u g h a p o s s i b l e e x p l a n a t i o n f o r t h i s d i f f e r e n c e may be the t r e a tmen t g i v e n to the p a t i e n t d u r i n g the i n t e r v a l between b i o p s i e s . I t i s t hus recommended t h a t the r e c e p t o r s t a t u s o f m e t a s t a t i c l e s i o n s be de t e rm ined whenever t h i s i s f e a s i b l e . b. RECEPTOR vs THYMIDINE-LABELLING INDEX An i n v e r s e r e l a t i o n s h i p between r e c e p t o r c on t en t and l a b e l l i n g - i n d e x has been r e p o r t e d ( 4 7 , 4 8 ) . T h y m i d i n e - l a b e l l i n g Index has been used as a measure o f the growth r a t e o f t umours . A number o f i n v e s t i g a t o r s (49) have r e p o r t e d t h a t low grade tumours have a l owe r t h y m i d i n e - l a b e l l i n g i n d e x . T h i s i s i n agreement w i t h the h y p o t h e s i s t h a t low grade ( h i g h l y d i f f e r e n t i a t e d ) tumours a re l e s s a g g r e s s i v e . 26 S i n c e tumour d i f f e r e n t i a t i o n v a r i e s d i r e c t l y w i t h , and t h y m i d i n e - l a b e l l i n g i n de x v a r i e s i n v e r s e l y w i t h r e c e p t o r c o n t e n t , a number o f wo rke r s began t o s tudy ER i n an a t tempt to d i s c e r n a p o s s i b l e r e l a t i o n s h i p between r e c e p t o r c o n t e n t and d i s e a s e f r e e i n t e r v a l ( 5 0 - 5 2 , 1 1 1 , 1 1 7 ) . c . RECEPTORS AND EPIDEMIOLOGICAL DATA The g e n e r a l consensus i s t h a t t h e r e i s a p o s i t i v e c o r r e l a t i o n between d i s e a s e f r e e i n t e r v a l and r e c e p t o r s t a t u s ( 5 2 , 5 3 ) . There a re o t h e r s t u d i e s however , wh ich f a i l t o c o n f i r m the v a l u e o f e i t h e r ER of PgR i n p r e d i c t i n g e a r l y r e l a p s e i n b r e a s t c an ce r ( 1 1 , 1 1 0 ) . W i th r e g a r d t o o v e r a l l s u r v i v a l , t h e r e i s g e n e r a l agreement t h a t p a t i e n t s w i t h tumours w i t h ER (and pe rhaps PgR) s u r v i v e l o n g e r a f t e r mastectomy than p a t i e n t s whose tumours a re d e v o i d of the r e c e p t o r ( 5 2 - 5 4 ) . What r ema ins u n c l e a r , i s whether t h i s p r o l o nged s u r v i v a l i s a r e s u l t of a l o n g e r d i s e a s e f r e e i n t e r v a l , i n c r e a s e d s u r v i v a l t ime a f t e r r e l a p s e o r a c omb i n a t i o n o f b o t h . The s u g g e s t i o n has been made t h a t the i n c r e a s e d s u r v i v a l a s s o c i a t e d w i t h r e c e p t o r r i c h tumours i s a r e f l e c t i o n o f the h e i gh t ened r e sponse o f t he se tumours t o e ndo c r i n e t he r apy ( 5 4 ) . Thus , the v a l u e o f r e c e p t o r s as p r o g n o s t i c i n d i c a t o r s r ema ins a c o n t e n t i o u s i s s u e . 27 d . TUMOUR STAGING There a re many d i f f e r e n t s t a g i n g s y s t ems , but a lmos t a l l a re based on the TNM c l a s s i f i c a t i o n . S tage I b r e a s t c an ce r i s a tumour c o n f i n e d t o the b r e a s t , l e s s t han 5 cm. i n s i z e and a l l a x i l l a r y lymph nodes a re p a t h o l o g i c a l l y n e g a t i v e f o r tumour . I n S tage I I d i s e a s e the a x i l l a r y nodes a re p o s i t i v e and S tage I I I d i s e a s e i n v o l v e s l a r g e bu l k y tumours a s s o c i a t e d w i t h s k i n changes and w i t h mat ted and f i x e d a x i l l a r y lymph nodes . S tage IV d i s e a s e i s m e t a s t a t i c b r e a s t d i s e a s e . e . RECEPTORS AND SERUM LEVELS OF ESTROGEN AND PROGESTERONE There a re a number o f r e p o r t s i n the l i t e r a t u r e (30 -32 ) o f an i n v e r s e r e l a t i o n s h i p between serum l e v e l s o f e s t r a d i o l and tumour ER c on t e n t ( 5 5 , 5 6 ) . I n v e s t i g a t i o n s o f t h i s t ype were unde r t a k en because i t was a rgued t h a t a c c o r d i n g t o the c u r r e n t model f o r s t e r o i d hormone a c t i o n the i n c r e a s e d serum l e v e l s o f e s t r a d i o l found i n p remenopausa l p a t i e n t s would l e a d t o a) i n c r e a s e d t r a n s l o c a t i o n o f the r e c e p t o r t o the nu c l e u s and b) i n c r e a s e d occupancy o f the a v a i l a b l e c y t o s o l i c r e c e p t o r s i t e s t hus l e a v i n g them u n a v a i l a b l e f o r 3 H - e s t r a d i o l , a n d hence r e s u l t i n g i n a f a i l u r e t o d e t e c t them u s i n g c u r r e n t assay t e c h n i q u e s . I t i s a rgued t h a t the i n c r e a s e i n ER seen w i t h age i s a r e f l e c t i o n of an i n c r e a s e i n unoc cup i ed s i t e s r a t h e r than an a c t u a l t o t a l i n c r e a s e i n r e c e p t o r s . T h i s has been d i s p u t e d by r e c e n t w o r k , i n wh ich i n c r e a s e d l e v e l s o f serum e s t r a d i o l were not found t o 28 c o r r e l a t e w i t h de c r ea sed ER tumour c on t e n t ( 5 7 ) . Saez (58) p o s t u l a t e d t h a t the c y c l i c l e v e l s o f serum p r o g e s t e r o n e seen i n p remenopausa l p a t i e n t s l i m i t s the s y t h e s i s o f c y t o s o l i c e s t r o g e n r e c e p t o r s . V a r i a t i o n s o f ER have been r e p o r t e d w i t h m e n s t r u a l c y c l e i n human b r e a s t c a n c e r s . I n c r e a s e d ER was obse r ved d u r i n g t he e a r l y p r o l i f e r a t i v e phase , w i t h a d e c r e a s i n g ER d u r i n g the l a t e p r o l i f e r a t i v e and s e c r e t o r y phases when e s t r a d i o l i s h i g h as i s p r o g e s t e r o n e ( 5 9 ) . f . PROGNOSIS AND MOLECULAR PROPERTIES OF RECEPTORS The s u c r o s e d e n s i t y c e n t r i f u g a t i o n (SDC) method a s s e s s e s not o n l y the p r e sence o r absence of r e c e p t o r , but a l s o c e r t a i n m o l e c u l a r p r o p e r t i e s o f t h e se p r o t e i n s . By t h i s method, f o u r c a t e g o r i e s based on s e d i m e n t a t i o n p r o f i l e s have been i d e n t i f i e d . Tumours c o n t a i n i n g ER o r PgR may c o n t a i n s p e c i f i c s t e r o i d - b i n d i n g components m i g r a t i n g a t e i t h e r 8S, AS o r bo th 8S and AS. The f o u r t h c a t e g o r y i s f o r t ho se tumours i n wh ich no ER i s d e t e c t a b l e . A p p a r e n t l y the l i g a n d - b i n d i n g s p e c i f i c i t i e s and a f f i n i t i e s o f the 8S and AS s p e c i e s o f ER a re s i m i l a r ( 6 ) . The SDC method c o n f i r m s f i n d i n g s based on the DCC method, namely t h a t 0-5% o b j e c t i v e r e m i s s i o n s a re obse r ved i n p a t i e n t s whose tumours a re ER n e g a t i v e , when t he se p a t i e n t s undergo hormona l t h e r a p y . I n a d d i t i o n , t ho se p a t i e n t s whose tumours c o n t a i n e d on l y the AS component, responded l e s s f r e q u e n t l y t o hormona l m a n i p u l a t i o n than those whose tumours c o n t a i n e d on l y 8S o r bo th 8S and AS. These r e s u l t s i n d i c a t e t h a t bo th the q u a n t i t y 29 ( number o f b i n d i n g s i t e s ) and the q u a l i t y (8S o r 4S) are i m p o r t a n t p r e d i c t i v e i n d i c e s f o r s e l e c t i n g a p p r o p r i a t e t h e r apy ( 6 ) . g . RECEPTOR STATUS AND RESPONSE TO CHEMOTHERAPY Wh i l e the v a l u e o f ER as a p r e d i c t o r o f r e sponse t o hormona l t h e r ap y i s w e l l e s t a b l i s h e d i t s v a l u e i n p r e d i c t i n g r e sponse to c y t o t o x i c t h e r apy r ema ins c o n t r o v e r s i a l ( 3 0 , 6 0 - 6 2 , 5 1 ) . L ippman (62) i n 1978 r e p o r t e d t h a t ER p o s i t i v e tumours responded l e s s o f t e n than ER n e g a t i v e tumours t o c h emo the r apeu t i c r e g i m e s . T h e o r e t i c a l l y , t h i s i s not unexpec t ed , s i n c e ER n e g a t i v e tumours a r e a s s o c i a t e d w i t h an i n c r e a s e d t h y m i d i n e - l a b e l l i n g i ndex and f l o w c y t ome t r y (43) has demons t ra ted t h a t t he se tumours have a g r e a t e r p e r c en t age of c e l l s i n the S -phase of the c e l l c y c l e . R a p i d l y g row ing c e l l s a re thus e xpec t ed t o be more s u s c e p t i b l e to chemothe rapy . A l l e g r a (51) r e p o r t e d r e s u l t s s i m i l a r t o L i p pman ' s f i n d i n g s , but o t h e r g roups ( 30 , 60 ) have found t h a t ER p o s i t i v e tumours a re more r e s p o n s i v e to chemotherapy than ER n e g a t i v e tumours , w h i l e B e r t e l s e n (61) found no d i f f e r e n c e i n r e sponse between ER n e g a t i v e and ER p o s i t i v e t umou r s . To d a t e , t he c o n t r o v e r s y rema ins u n r e s o l v e d . A once f r e q u e n t l y recommended t r e a tmen t f o r pos tmenopausa l women w i t h b r e a s t c a r c i n oma was s u r g i c a l a b l a t i o n o f the a d r e n a l s and /o r p i t u i t a r y g l a n d s . A 50-60% reponse r a t e was r e p o r t e d f o r t h i s t r e a tmen t ( 3 ) . There i s , however , s i g n i f i c a n t m o r b i d i t y and o c c a s i o n a l m o r t a l i t y a s s o c i a t e d w i t h t h i s form of t r e a t m e n t . 30 M e d i c a l t h e r apy was deve l oped as an a l t e r n a t i v e t o s u r g i c a l a b l a t i o n . T h i s t r e a tmen t i s based upon the h y p o t h e s i s t h a t ad rena l e c t omy and hypophysectomy a re e f f e c t i v e by v i r t u e of t h e i r a b i l i t y t o l owe r e s t r o g e n p r o d u c t i o n . M e d i c a l t he r apy i n v o l v e s two s e p a r a t e s t r a t e g i e s . 1) i n t e r f e r e n c e w i t h e x i s t i n g e s t r o g e n a c t i o n t h rough the use o f a n t i e s t r o g e n s and 2) i n h i b i t i o n o f new e s t r o g e n s y n t h e s i s w i t h an enzyme a n t a g o n i s t . Tamox i fen i s an example o f an a n t i e s t r o g e n and a m i n o g l u t e t h i m i d e (AG) i s an example o f a p o t e n t s t e r o i d o g e n e s i s b l o c k e r . S t e r o i d s a re s y n t h e s i z e d f rom c h o l e s t e r o l by a s e r i e s o f en z yma t i c s t e p s i n v o l v i n g c l e a v a g e , h y d r o x y l a t i o n s and the c o n v e r s i o n o f the A r i n g to an a r o m a t i c r i n g . AG b l o c k s a number o f the cy toch rome P-450 med ia ted h y d r o x y l a t i o n s , i n c l u d i n g the c o n v e r s i o n of c h o l e s t e r o l t o p r egneno l one and the c o n v e r s i o n of androgen t o e s t r o g e n s . C l i n i c a l s t u d i e s have shown t h a t AG and r ep l a cemen t g l u c o c o r t i c o i d t h e r apy p roduces o b j e c t i v e r e m i s s i o n as f r e q e u n t l y as s u r g i c a l a b l a t i o n ( 3 ) . Even w i t h da t a on c l i n i c a l s t a t u s , ER and PgR of the p r ima r y tumour and /o r any m e t a s t a s e s , i t i s not p o s s i b l e a t t h i s t ime to a c c u r a t e l y p r e d i c t wh ich t r e a tmen t i s the most b e n e f i c i a l o r wh ich w i l l p r o l o n g s u r v i v a l . To add re s s t h i s s ho r t c om ing many wo rke r s have s ea r ched f o r ano the r marker ; one more l i k e l y t o p r e d i c t the g rowth c h a r a c t e r i s t i c s o f the tumour than s t e r o i d r e c e p t o r s t a t u s . P l a sm inogen a c t i v a t o r has been t o u t e d as j u s t such a ma rke r . 31 i v . PLASMINOGEN ACTIVATOR ACTIVITY AND BREAST CANCER P l a sm inogen a c t i v a t o r s a re a group of enzymes wh ich c o n v e r t t he zymogen p l a sm inogen t o the a c t i v e p r o t e a s e p l a sm i n by c a t a l y z i n g the h y d r o l y s i s o f the Arg 5 6 0 - V a l 561 bond . H i s t o r i c a l l y , i n t e r e s t i n p l a sm i n and p l a sm inogen a c t i v a t o r s a r o s e f rom t h e i r r o l e i n f i b r i n o l y s i s . One of the f i r s t p l a sm inogen a c t i v a t o r s s t u d i e d was s t r e p t o c o c c a l f i b r i n o l y s i n o r s t r e p t o k i n a s e . I n the 1940 ' s i t was shown t h a t p lasma c o n t a i n e d an i n a c t i v e enzyme, p l a sm i nogen , wh ich when c o n v e r t e d by the s t r e p t o c o c c a l f a c t o r y i e l d e d the a c t i v e enzyme p l a s m i n . P l a s m i n i n t u r n was r e s p o n s i b l e f o r the d e g r a d a t i o n o f f i b r i n . A s e a r c h f o r endogenous p l a sm inogen a c t i v a t o r s l e d t o t h e i r d i s c o v e r y i n serum, a lmos t a l l t i s s u e s , v a s c u l a r e ndo t he l i um and many body f l u i d s , i n c l u d i n g u r i n e . Subsequent d i s c o v e r y of f i b r i n o l y t i c a c t i v i t y of c e l l s i n c u l t u r e was shown to be due to the s y n t h e s i s o f p l a sm inogen a c t i v a t o r s . At l e a s t two d i f f e r e n t t y pe s o f p l a sm inogen a c t i v a t o r (PA) have been i d e n t i f i e d . U r o k i n a s e i s the major PA of human u r i n e and some PA i s o l a t e d f rom t i s s u e s r e semb le s u r o k i n a s e i n i t s p r o p e r t i e s w h i l e t he o t h e r major group i s c l a s s i f i e d as n o n - u r o k i n a s e o r t i s s u e p l a sm inogen a c t i v a t o r . Immuno l og i c a l and m o l e c u l a r we igh t d i f f e r e n c e s have been no ted ( 6 3 - 6 5 ) . The m o l e c u l a r we igh t o f u r o k i n a s e i s g i v e n as 45 ,000 - 60 ,000 d a l t o n s and t i s s u e p l a sm inogen a c t i v a t o r i s 55 ,000 - 70 ,000 d a l t o n s . 32 D i f f e r e n c e s i n k i n e t i c d a t a between t he se two p l a sm inogen a c t i v a t o r s has a l s o been r e p o r t e d (66 -68 ) as w e l l as d i f f e r e n c e s i n s p e c i f i c t y f o r s u b s t r a t e s and i n h i b i t o r s . Undegraded human p l a sm inogen has a m o l e c u l a r we igh t o f a p p r o x i m a t e l y 90 ,000 d a l t o n s and i s composed o f a s i n g l e p e p t i d e c h a i n w i t h g l u t a m i n e at the amino t e rm i nu s and a s p a r a g i n e at the c a r b o x y l end . P l a s m i n , wh ich i s g ene r a t ed by two p r o t e o l y t i c c l e a v a g e s , c o n s i s t s o f 2 p e p t i d e c h a i n s , l i n k e d by a d i s u l p h i d e bond ( f i g u r e 4 ) . From k i n e t i c s t u d i e s i t appears as though the most l i k e l y sequence f o r p l a sm inogen a c t i v a t i o n by u r o k i n a s e i s as f o l l o w s : I . 1) . G l u - p l a s m i n o g e n > L y s - p l a s m i n o g e n + a c t i v a t i o n p e p t i d e 92 ,000 86 ,000 7 ,000 2) . L y s - p l a s m i n o g e n > L y s - p l a s m i n 86 ,000 H 63 ,000 - L 25 ,000 An a l t e r n a t e pathway, g i v e n below was d i s c o v e r e d t h r ough the use o f T r a s y l o l , wh i ch i s a t r y p s i n i n h i b i t o r wh ich i n h i b i t s p l a s m i n , but not u r o k i n a s e . In the p r e sence o f t h i s compound, o n l y r e a c t i o n 3 o c c u r s , t hus d e m o n s t r a t i n g t h a t u r o k i n a s e can c l e a v e p l a sm inogen w i t h o u t f i r s t remov ing the 7,000 d a l t o n a c t i v a t i o n p e p t i d e . 33 © [p NH2'Glu'Pro'Leu'Asp-Asp—^.S-SyL.—Glu-Asn/Arg-Lys' Ser* S e r ACTIVATION PEPTIDE u , | / 2 ^ M e t -Lys Cys Pro 6 ly A r g ® ^'^'Val-GlyGlyCys UROKINASE HEAVY CHAIN S S - s - s Leu'Tyr-Val'LyS'Lys PLASMIN -Asn-COOH LIGHT CHAIN • Bond Cleavage,converts Plasminogen to Plasmin d j > Bond Cleavage,gives Lys-plasmin(ogen) Other points of Cleavage which may release'Activation Peptide" F i g . 4 C o n v e r s i o n o f p l a s m i n o g e n t o p l a s m i n . The v a r i o u s s i t e s o f c l e a v a g e by b o t h u r o k i n a s e and p l a s m i n a r e i l l u s t r a t e d . " 34 I I . 3 ) . G l u - p l a s m i n o g e n G l u - p l a s m i n 92 ,000 H 70 ,000 L 25 ,000 4 ) . G l u - p l a s m i n L y s - p l a s m i n + a c t i v a t i o n p e p t i d e H 70 , 000 - L 25 ,000 H 63 , 000 - L 25 ,000 + 7 ,000 I t has been p o s t u l a t e d t h a t the remova l o f the a c t i v a t i o n p e p t i d e and the r e s u l t a n t c o n f o r m a t i o n a l change c o n v e r t s p l a sm inogen i n t o a more r e a d i l y a c t i v a t e d f o rm . The f a c t t h a t g l u - p l a s m i n i s neve r found i n d e t e c t a b l e amounts, would tend t o argue i n f a v o u r o f pathway I . Once p l a s m i n i s formed i t c a t a l y z e s the r emova l of the a c t i v a t i o n p e p t i d e , t hus g r e a t l y i n c r e a s i n g the r a t e o f p l a s m i n f o r m a t i o n . When a c t i v a t i o n i s a c c omp l i s h ed w i t h e q u i m o l a r amounts of s t r e p t o k i n a s e , the heavy and l i g h t c h a i n s o f p l a sm inogen formed appear i n d e n t i c a l w i t h t hose formed f rom l y s - p l a s m i n o g e n by u r o k i n a s e . The f i r s t s t e p i n t h i s a c t i v a t i o n s e r i e s i s the f o r m a t i o n o f a s t o i c h i o m e t r i c complex between s t r e p t o k i n a s e and p l a s m i n o g e n . T h i s r e s u l t s i n a c o n f o r m a t i o n a l change i n t he p l a sm inogen m o l e c u l e , e x po s i n g a p r o t e o l y t i c s i t e . Bo th the p l a sm inogen and the s t r e p t o k i n a s e a re then r a p i d l y c l e a v e d t o y i e l d p l a s m i n and s t r e p t o k i n a s e f r a g m e n t s . I t i s b e l i e v e d t h a t p l a sm inogen a c t i v a t o r f rom t r a n s f o rmed c e l l s a c t i v a t e s p l a sm inogen i n a manner s i m i l a r t o t h a t o u t l i n e d f o r u r o k i n a s e . P l a sm inogen a c t i v a t o r a c t i v i t y was a l s o found i n a s s o c i a t i o n 35 w i t h v a s c u l a r i z a t i o n d u r i n g wound h e a l i n g , i n f l a m m a t i o n and tumour i n f i l t r a t i o n . W i t h such a u b i q u i t o u s d i s t r i b u t i o n , t he h y p o t h e s i s o f i t s c r u c i a l impo r t ance i n many a s p e c t s of c e l l u l a r ma in tenance was f o r w a r d e d . P l a s m i n , u n l i k e the o t h e r p r o t e o l y i c enzymes found i n p l a sma , has a very b road s p e c i f i c i t y . Among i t s many s u b s t r a t e s a re c a s e i n , p r o t a m i n e , c e l l membrane p r o t e i n s , i m m u n o g l o b u l i n s , ACTH, s o m a t o t r o p i n , g l u c a g o n , s e v e r a l components o f complement as w e l l as F a c t o r s V , V I I 1 and X l l a o f the c l o t t i n g s y s t em . P l a s m i n can thus a f f e c t the f u n c t i o n o f a l l t he major p lasma p r o t e i n e f f e c t o r sys tems ( 6 9 ) . Some o f the e a r l i e s t o b s e r v a t i o n s , l i n k i n g PA w i t h n e o p l a s i a were made by U n k e l e s s and Ossowsk i (70) i n c e l l c u l t u r e s . Nagy e t a l (71) showed PA l e v e l s to be e l e v a t e d i n human n e o p l a s t i c t i s s u e s w h i l e the c o r r e s p o n d i n g no rma l t i s s u e s showed l i t t l e a c t i v i t y . Among the t i s s u e s examined were c e r v i c a l , mammary, p r o s t a t i c and o v a r i a n c a n c e r s , as w e l l as l ung c a r c i n o m a s , melanomas and b a s a l i o m a s . A number o f wo rke r s were a b l e to demons t r a t e hormona l c o n t r o l o f PA i n mur ine and r oden t mammary t i s s u e ( 7 2 , 7 3 ) . B u t l e r e t a l (74) was a b l e t o demons t r a t e i n d u c t i o n o f PA by e s t r o g e n i n the human b r e a s t c an ce r c e l l l i n e MCF-7. P l a sm inogen a c t i v a t o r p r o d u c t i o n has been a s s o c i a t e d w i t h s e v e r a l r e p r o d u c t i v e p r o c e s s e s such as o v u l a t i o n , t r o p h o b l a s t imp lan ta t i on ,mammary g l a nd i n v o l u t i o n and u t e r i n e g rowth ( 7 3 , 7 5 , 7 6 ) . A l l t h e se p r o c e s s e s sha re two common f e a t u r e s : 36 1) hormona l c o n t r o l o r modu l a t i o n and 2) c e l l m i g r a t i o n and /o r t i s s u e r e m o d e l l i n g . On the b a s i s o f t h e se f i n d i n g s i t was a rgued t h a t p l a sm inogen a c t i v a t o r a c t i v i t y i n tumours may r e f l e c t not so much the t u m o u r i g e n i c i t y o f the c e l l s , but r a t h e r the e x t e n t to wh ich t h e s e c e l l s r ema in under hormona l c o n t r o l , a f u n c t i o n i n d i c a t i v e of the p re sence of some degree of d i f f e r e n t i a t i o n ( 1 0 5 ) . The no rma l p h y s i o l o g i c a l r o l e f o r p l a sm inogen a c t i v a t o r s i s i n h a e m o s t a s i s . The r o l e of u r i n a r y p l a sm inogen a c t i v a t o r o r u r o k i n a s e rema ins s p e c u l a t i v e , though i t can be r e a d i l y a p p r e c i a t e d t h a t an o rgan such as a k i dney w i t h i t s r i c h v a s c u l a t u r e and e x t e n s i v e network o f t u b u l e s , would be w e l l s e r v ed by a p r o t e i n a s e c apab l e of d eg r ad i ng c o a g u l a b l e p r o t e i n s . T h i s may a l s o be t he r o l e o f p l a sm inogen a c t i v a t o r s found i n the l u n g , i n c l o s e a s s o c i a t i o n w i t h e n d o t h e l i a l c e l l s and f i b r o b l a s t s . I t s r o l e i n i n f l a m m a t i o n and wound h e a l i n g can b e s t be a p p r e c i a t e d by v i r t u e of i t s a b i l i t y to a c t i v a t e the complement and k i n i n s y s t ems . P l a s m i n , gene ra t ed by the a c t i o n of p l a sm inogen a c t i v a t o r s on p l a sm i nogen , can gene ra t e a c t i v e complement C3 , wh i ch i n t u r n causes i n c r e a s e d v a s c u l a r p e r m e a b i l i t y and a c c u m u l a t i o n of PMN's . The a c t i v a t i o n of the k i n i n system r e s u l t s i n bo th i n c r e a s e d p e r m e a b i l i t y and i n c r e a s e d v a s o d i l a t i o n thus e n a b l i n g l e u k o c y t e s t o r e a ch an a r ea o f t h r o m b o s i s . 37 A r o l e f o r p l a sm inogen a c t i v a t o r s , i n ma l i g nan c y , s p e c i f i c a l l y the a b i l i t y o f n e o p l a s t i c c e l l s t o grow and i n vade s u r r o u n d i n g t i s s u e s , rema ins unp roven . I t i s t emp t i ng to s p e c u l a t e , t h a t tumour growth and i n v a s i v e n e s s i s dependent on f i b r i n o l y s i s and t hus i n d i r e c t l y on p l a sm inogen a c t i v a t o r s . T h i s would mean t h a t t h e r e s hou l d be a very c l o s e and d i r e c t r e l a t i o n s h i p between p l a sm inogen a c t i v a t o r p r o d u c t i o n and m a l i g n a n t t r a n s f o r m a t i o n . Wh i l e enhanced p l a sm inogen a c t i v a t o r a c t i v i t y i s seen i n a number of t r a n s f o rmed c e l l s ( 7 1 , 7 7 ) , a number o f m a l i g n a n t c e l l l i n e s wh ich have been p ropaga ted i n v i t r o f o r y e a r s , do not e xp r e s s p l a sm inogen a c t i v a t o r a c t i v i t y . The s u b c e l l u l a r d i s t r i b u t i o n o f PA i s not w e l l u n d e r s t o o d . Some worke r s assay the c y t o s o l ( 78 , 79 ) f o r PA w h i l e o t h e r s assay bo th the c y t o s o l and the l y s o s o m a l f r a c t i o n ( 8 0 ) . Recent work done by Laug e t a l on c u l t u r e d human f i b r o s a r c o m a c e l l s s ugge s t s t h a t PA i s a s s o c i a t e d w i t h the s l o w l y s e d imen t i n g f r a c t i o n o f G o l g i - d e r i v e d s e c r e t o r y o r c a r r i e r v e s i c l e s ( 5 6 ) . I n summary: t h e r e a re two d i v e r g e n t t h e o r i e s to e x p l a i n t he r o l e o f p l a sm inogen a c t i v a t o r a c t i v i t y PAA i n n e o p l a s i a . On the one hand PA i s c o n s i d e r e d not o n l y a marker f o r n e o p l a s i a but a l s o a measure of i t a g g r e s s i v e n e s s o r m e t a s t a t i c p o t e n t i a l . In s uppo r t o f t h i s t h e o r y t h e r e i s e v i d e n c e t h a t tumour p romo to r s ( 1 1 2 ) , DNA damage (113) and c a r c i n o g e n s i n du ce PA s y n t h e s i s . F u r t h e rmo r e the p l a s m i n gene r a t ed by the c l e a v age of p l a sm inogen by PA can a c t i v a t e c o l l a g e n a s e ( 82 , 83 ) though t h i s has been d i s p u t e d ( 8 4 ) . S u p p o r t e r s o f t h i s t h e o r y s i t e e v i d en ce o f 38 uncoordinated lys is and the abi l i ty of metastasizing tumour to breach the basement membranes surrounding blood capi l la r ies . On the other hand PA activity in tumours, especially human breast cancer is considered as a marker of the retention of differentiated function; by inference therefore, a tumour with less metastasizing potential than a tumour not capable of PA production. In support of this theory we have considerable evidence for hormonal control of PA (73,75). Work by Sutherland (78) and Thorson (79) indicates there is a positive correlation between PA activity and steroid receptors in human breast cancer. The latter being a measure of the degree to which a tumour retains i t s differentiated functions and structures. Recent work by Katzenellenbogen and colleagues (85) supports the contention that PAA may indeed serve as a marker for estrogen action in breast cancer c e l l s . Indeed this work and that by Yu (111) have shown a correlation between PA activity and c e l l growth. Interestingly, the same could not be said of PgR, often used as a measure of the extent to which the estrogen stimulation of the growth pathway remains intact . The enzymatic activity of plasmin was in the past determined using biological substrates such as casein or f i b r i n . These natural substrates are usually large protein molecules which are d i f f i c u l t to prepare in a pure native form. Further, the products formed by these reactions are usually d i f f i c u l t to detect since physiochemically they are very similar to the undigested substrate. Plasmin and plasminogen are a good example 39 of t h i s p r ob l em . In t he case of n a t u r a l s u b s t r a t e , the r e a c t i o n c o n d i t i o n s a re o f t e n q u i t e comp lex . Because o f t he se d i f f i c u l t i e s s y n t h e t i c s u b s t r a t e s , some m im i c k i n g the n a t u r a l s u b s t r a t e a re now more f r e q u e n t l y u s e d . Wh i l e a c l e a r r o l e f o r PA a c t i v i t y i n b r e a s t c ance r has not been e s t a b l i s h e d , i t s p o t e n t i a l as a p r o g n o s t i c i n d i c a t o r i n b r e a s t c a r c i noma i s b e i ng a c t i v e l y i n v e s t i g a t e d . v . METHODS FOR PLASMINOGEN ACTIVATOR ANALYSIS The f i r s t s y n t h e t i c s u b s t r a t e s c o n s i s t e d o f l y s i n e and /o r a r g i n i n e e s t e r s and am ides . These s u b s t r a t e s however l a c k e d bo th s e n s i t i v i t y and s e l e c t i v i t y . S e n s i t i v i t y was improved w i t h the i n t r o d u c t i o n o f c h r omopho r i c g roups such as p a r a n i t r o a n i l i n e (pNA) . S u b s t r a t e s e l e c t i v i t y and s e n s i t i v i t y were enhanced by r e p l a c i n g s i n g l e amino a c i d s w i t h a s h o r t p e p t i d e c h a i n . P l a s m i n s p l i t s the s u b s t r a t e H - D - V a l - L e u - L y s pNA (S2251) r a p i d l y . F r i b e r g e r e t a l (86) d e s c r i b e s the s e l e c t i o n and p o s i t i o n o f the v a r i o u s amino a c i d s and the e f f e c t on s e n s i t i v i t y and s e l e c t i v i t y f o r t h i s p a r t i c u l a r s u b s t r a t e . The f o l l o w i n g a re examples o f s y n t h e t i c s u b s t r a t e s . H-D-AA-Leu-Lys -pNA H-D-AA-P ro -Lys -pNA H-D-AA-Phe-Arg-pNA 40 AA- t h i s can be v a r i e d f r e e l y but an u n s u b s t i t u t e d D-amino a c i d i s p r e f e r r e d ; i t imp roves the s o l u b i l i t y of the s u b s t r a t e . Of the t h r e e f o r m u l a s above the f i r s t i s both the most s e n s i t i v e and demons t r a t e s the h i g h e s t s e l e c t i v i t y . S e l e c t i v i t y can be f u r t h e r enhanced by c hoo s i ng b u f f e r s o f c e r t a i n pH and i o n i c s t r e n g t h . 100% s e l e c t i v i t y i s u n f o r t u n a t e l y not a t t a i n a b l e , t hus o t h e r enzymes such as t r y p s i n w i l l c l e a v e most s y n t h e t i c s u b s t r a t e s . When p l a s m i n c a t a l y z e s the h y d r o l y s i s of the pep t i de -pNA bond, f r e e pNA i s r e l e a s e d . When pNA i s d e a c y l a t e d i n mode r a t e l y a k a l i n e o r n e u t r a l med ia , l e s s energy i s needed t o t r a n s f o r m the p i e l e c t r o n s to an e x c i t e d s t a t e . The de c r ea se i n energy b r i n g s about a red s h i f t i n the w a v e l e n g t h . The method i s t hu s based on the d i f f e r e n c e i n abso rbance between the pNA formed and the o r i g i n a l s u b s t r a t e . The amount o f pNA r e l e a s e d i s measured s p e c t r o p h o t o m e t r i c a l l y a t 405nm. At t h i s w a v e l e n g t h , the abso rbance of the s u b s t r a t e i s l e s s t han 1% of t h a t f o r f r e e pNA ( 8 7 ) . The f l u o r o m e t r i c m i c r o a s s a y of p l a sm inogen a c t i v a t o r s uses a mac r omo l e cu l e , p r o t am ine as the s u b s t r a t e . T h i s p o l y p e p t i d e can be used as a s u b s t r a t e f o r t r y p s i n , p l a s m i n , and t h r omb in ( 8 8 ) . As i s the case f o r q u a n t i t a t i o n o f PA w i t h S -2251 , the f l u o r o m e t r i c assay i s a two - s t e p a s s a y . P l a sm inogen a c t i v a t o r i n the. t e s t samp le , o r u r o k i n a s e used as a s t a n d a r d , c l e a v e s p l a sm inogen t o y i e l d the a c t i v e enzyme p l a s m i n . P l a s m i n i n t u r n d i g e s t s p e p t i d e bonds w i t h i n the N - t e r m i n a l b l o c k ed p r o t am ine s u b s t r a t e e xpo s i n g amino g r oup s . 41 F l u r a m , ( 4 - p h e n y l s p i r o - [ f u r a n - 2 ( 3 H ) , 1 1 - p h t h a l a n ] - 3 , 3 1 - d i o n e ) an amino group r e a g e n t , r e a c t s w i t h t he se newly exposed amino groups to y i e l d a f l u o r e s c e n t p r o d u c t . E x c i t a t i o n o c c u r s a t 390nm and e m i s s i o n i s r e c o r ded a t 480 nm. The amount of f l u o r e s c e n t p r oduc t e xp r e s s ed as r e l a t i v e f l u o r e s c e n t u n i t s (RFU) i s p r o p o r t i o n a l t o the q u a n t i t y o f p l a s m i n , wh ich i n t u r n i s dependent upon the q u a n t i t y of PA i n the samp le . Wh i l e u r o k i n a s e and presumab ly PA w i l l d i g e s t p r o t am ine ( s l o w l y ) and t hus be d i r e c t l y a s s a yed , u t i l i z i n g a two - s t e p method w i t h a c t i v a t i o n of p l a sm inogen to p l a s m i n has the advan tages o f s p e c i f i c i t y and a m p l i f i c a t i o n . Wi th a Km of 3 .6 x 42 _ 5 10 M f o r the a c t i o n o f u r o k i n a s e on p l a sm inogen (M.W. 90 ,000) e x c e s s i v e l y h i g h c o n c e n t r a t i o n s o f p l a sm inogen would be ne ce s s a r y t o r e a ch s a t u r a t i o n k i n e t i c s . P r o t am ine i s very s o l u b l e and i t s c o n c e n t r a t i o n can be r a i s e d h i g h enough t o s a t u r a t e p l a s m i n . Fo r t h i s assay a s u b s t r a t e c o n c e n t r a t i o n o f 20 mg/ml o r 5 mg/assay and a p l a sm inogen c o n c e n t r a t i o n of 0 .2 U/ml o r 0 .05 U/assay y i e l d e d a l i n e a r p l o t ove r a u r o k i n a s e c o n c e n t r a t i o n range of 10-150 uU/m l . P r o t a m i n e , u n l i k e s y n t h e t i c s u b s t r a t e s i s not s u b j e c t t o a t t a c k by amidases o r e s t e r a s e s , w h i l e o f f e r i n g the t e c h n i c a l c o n ven i e n c e s o f the low M.W. s y n t h e t i c s u b s t r a t e . U n l i k e the s i n g l e s i t e s y n t h e t i c s u b s t r a t e s , p r o t am ine o f f e r s numerous c l e a v a g e s i t e s , not a l l o f wh ich may r e s u l t i n the exposu re o f r e a c t i v e amino g r o u p s . Bo th f l u o r o m e t r i c and s p e c t r o p h o t o m e t r i c PA a s s a y s a r e c u r r e n t l y used by i n v e s t i g a t o r s . Recent advances i n s y n t h e t i c s u b s t r a t e p r o d u c t i o n have r e s u l t e d i n marked improvements i n bo th s e n s i t i v i t y and s p e c i f i c i t y , such t h a t the s p e c t r o p h o t o m e t r i c a s s a y s based on s y n t h e t i c s u b s t r a t e s a r e becoming more p o p u l a r . A3 v i . RESEARCH AIMS & INVESTIGATIVE SCOPE Wh i l e ER a n a l y s i s o f b r e a s t tumour samples i s g e n e r a l l y a c c ep t ed as an i m p o r t a n t f a c t o r i n bo th t h e r apy s e l e c t i o n and p r o g n s i s , t h e r e i s a g row ing t r e n d towards the i n c l u s i o n o f PgR a s s a y s ( 3 8 , 5 3 ) . Wh i l e the methodo logy f o r both s t e r o i d a s s a y s i s w e l l c h a r a c t e r i z e d , a combined assay would overcome s e v e r a l p r o b l ems . F i r s t l y , the s i z e of tumour spec imens a v a i l a b l e f o r s t e r o i d a n a l y s i s r a r e l y exceeds 500 mg and t w i c e t h i s amount i s r e q u i r e d t o c a r r y out bo th a s say s a d e q u a t e l y . S e c ond l y , w i t h the w e l l documented h e t e r o g e n e i t y o f b r e a s t c a n c e r s , the a n a l y s i s of bo th r e c e p t o r s s h ou l d i d e a l l y be done on the same samp le . P l a sm inogen a c t i v a t o r a c t i v i t y as a measure o f hormona l dependence has i n the l a s t 3-4 y e a r s g ene r a t ed c o n s i d e r a b l e i n t e r e s t . The f i r s t e xpe r imen t s wh ich measured PAA used the 125 I - E 2 ~ f i b r i n p l a t e method, ( 77 , 89 ) a r e l a t i v e l y i n s e n s i t i v e cumbersome t e c h n i q u e , wh ich f u r t h e r m o r e i s not ve ry r e p r o d u c i b l e . A number o f t e c h n i q u e s have emerged s i n c e t h e n , wh i ch a re more s e n s i t i v e , l e s s cumbersome and more q u a n t i t a t i v e ( 7 9 , 8 8 - 9 1 ) . The purpose o f t h i s s tudy was to deve l op a method whereby a l l t h r e e p a r ame t e r s , ER,PgR and PA a c t i v i t y c o u l d be de t e rm ined from a s i n g l e 500 mg tumour samp le . Data thus ga t he r ed was examined t o de t e rm ine whether t h e r e i s a c o r r e l a t i o n between t h e s e t h r e e p a r ame t e r . A l s o the PA a c t i v i t y was examined f rom o l d e r tumours 44 f o r wh ich ER and p a t i e n t da t a r e g a r d i n g d i s e a s e f r e e i n t e r v a l was a v a i l a b l e , to de t e rm ine whether PA a c t i v i t y measurements a l one a r e o f p r o g n o s t i c v a l u e . The b a s i s f o r t h e ER and PgR a s say s was the DCC method e s t a b l i s h e d by J a cobson ( 2 6 ) . The f i r s t a spec t to be s t u d i e d was a b u f f e r a p p r o p r i a t e f o r bo th ER and PgR a s s a y s . The PgR assay was modeled on t h a t used f o r ER u s i n g 3H-R5020 as the l i g a n d and u n l a b e l e d R5020 as the c o m p e t i t i v e i n h i b i t o r . A l i t e r a t u r e s e a r c h y i e l d e d i n f o r m a t i o n f o r the recommended c o n s t i t u e n t s o f an a p p r o p r i a t e b u f f e r , 20mM TES-1.5mM EDTA-5mM mono th i og l y c e r o l - 5mM sodium molybdate-10% g l y c e r o l pH 7 .5 (TEMS) ( 7 , 2 4 , 2 5 ) . U s i n g t h i s b u f f e r , samples o f poo l ed human b r e a s t tumours were a n a l y z e d and compared w i t h t he same samples a n a l y z e d w i t h 10 mM T R I S - 1 . 5 mM EDTA-0.5 mM D L - D i t h i o t h r e i t o l pH 7 .5 (TED) b u f f e r , t h e b u f f e r employed r o u t i n e l y f o r ER a n a l y s i s . V e r i f i c a t i o n o f the e f f i c a c y o f TEMS as a b u f f e r f o r the PgR assay was a c comp l i s hed by u s i n g p oo l e d l y o p h i l i z e d t i s s u e , f o r wh ich the p r o g e s t e r o n e r e c e p t o r c o n t e n t was a l r e a d y known. The assay sys tem d e v i s e d by Jacobson (26) i n d i c a t e d t h a t the c y t o s o l i c p r o t e i n c o n c e n t r a t i o n a f f e c t e d the a s s a y . The p o s s i b l e e x i s t e n c e of a s i m i l a r e f f e c t f o r PgR was thus examined . P r o t e i n d e t e r m i n a t i o n s were based on the Lowry method (25) and a r a p i d U.V. method ( 9 2 ) . TEMS b u f f e r was found t o i n t e r f e r e 45 i n bo th t he se methods , though to a much g r e a t e r e x t e n t i n t he Lowry a s s a y . T h i s i n t e r f e r e n c e was s u c c e s s f u l l y c o r r e c t e d by ad j u s tmen t s t o the s t a n d a r d cu r ve and b l ank tubes f o r the Lowry a s say and ad j u s tmen t s t o the b l ank t ubes and changes i n the c a l c u l a t i o n s f o r the U.V. method. Optimum i n c u b a t i o n t ime f o r bo th ER and PgR a s s a y s were examined u s i n g TEMS b u f f e r and an i n c u b a t i o n t empe r a t u r e of 4°C. Fo r t h i s pu rpose poo l ed l y o p h i l i z e d t i s s u e was used and a l i q u o t s were removed a t c e r t a i n i n t e r v a l s f rom 0 .5 t o 18 .0 h o u r s . In o r d e r t o d i s t i n g u i s h between ER and PgR i n a combined a s s a y , one o f the r a d i o l a b e l s had t o changed . Repo r t s i n the 125 l i t e r a t u r e c i t e d I - E 2 as a s u i t a b l e a l t e r n a t e l i g a n d f o r ER d e t e r m i n a t i o n ( 1 6 , 9 3 - 9 5 ) . 125 The energy p r o f i l e f o r I c o n s i s t s o f two peaks , the l owe r energy peak , c o m p l e t e l y o v e r l a p p i n g t he s i n g l e 3 H peak . A s u i t a b l e s e t t i n g f o r the energy windows of the l i q u i d s c i n t i l l a t i o n c o u n t e r had to be found t o h e l p r e s o l v e t h i s p r o b l em . To v e r i f y t h a t t h i s had i n deed been a c c omp l i s h ed a number o f e x pe r imen t s were run on poo l e d b r e a s t t i s s u e i n wh i ch 125 ER was de t e rm ined s e p a r a t e l y u s i n g I - E 2 , P g R was de t e rm ined s e p a r a t e l y , u s i n g ' 5 H-R5020 and then bo th were de t e rm ined f rom the same assay m i x . Once a s u i t a b l e d u a l l a b e l assay had been e s t a b l i s h e d two methods f o r PA a c t i v i t y were s t u d i e d t o de t e rm ine wh ich would be most s u i t a b l e f o r u s e . The f i r s t method d e s c r i b e d by Ke s sne r and T r o l l (88) i s a f l u o r o m e t r i c method. T h i s assay i s based on the 46 d i g e s t i o n o f N - t e r m i n a l b l o c k ed p ro t am ine and the subsequent q u a n t i t a t i o n o f the exposed amino groups u s i n g the f l u o r o g e n i c amine r eagen t FLURAM ( 4 - p h e n y l s p i r o [ f u r a n - 2 ( 3 H ) , l - p h t h a l a n f - 3 , 3 d i o n e ) . The second method i s s p e c t r o p h o t o m e t r i c and makes use o f the s y n t h e t i c p e p t i d e s u b s t r a t e S2251 f rom K a b i . S2251 -( H - D - V a l - L e u - L y s - p N A ) - a c t s as a s u b s t r a t e f o r p l a s m i n , wh i ch s p l i t s o f f the p a r a n i t r o a n i l i n e (pNA) . The amount o f pNA r e l e a s e d i s p r o p o r t i o n a l t o the amount o f p l a s m i n wh ich i n t u r n i s p r o p o r t i o n a l t o the amount of p l a sm inogen a c t i v a t o r . Q u a n t i t a t i o n o f pNA i s d e r i v e d f rom abso rbance r e a d i n g s t a ken a t 405 nm. The s t a n d a r d cu r v e i s g ene r a t ed by i n c u b a t i n g known amounts o f s t r e p t o k i n a s e (SK) o r u r o k i n a s e (UK) and r e c o r d i n g the r e s u l t i n g abso rbance a t 405 nm. 47 I I .MATERIALS AND METHODS i . RECEPTOR ASSAY : MATERIALS C h e m i c a l s : TRIS ( t r i s [ h y d r o x y m e t h y l ] am inoe thane ) , TES ( N - T r i s - [ h y d r o x y m e t h y l ] - m e t h y l - 2 - a m i n o e t h a n e - s u l f o n i c a c i d ) , m o n o t h i g l y c e r o l , d i t h i o t h r e i t o l and DES were o b t a i n e d f rom S igma, S t . L o u i s , MO. EDTA ( e t h y l e n e d i a m i n e t e t r a c e t a t e ) and sodium mo lybda te were pu r chased f rom F i s h e r S c i e n t i f i c , F a i r Lawn N J . , w h i l e the g l y c e r o l was s u p p l i e d by Amer i can S c i e n t i f i c & C h e m i c a l , S e a t t l e WA. The r a d i o - l a b e l e d l i g a n d s , ' 5 H - E 2 , 125 3 I - E 2 and H-R5020 as w e l l as the u n l a b e l e d R5020 were o r d e r ed f rom New Eng l and N u c l e a r . The c h a r c o a l was s u p p l i e d by Schwarz -Mann, D i v i s i o n of Bec ton and D i c k i n s o n , M i s s i s s a u g a , O n t a r i o and the d e x t r a n was o b t a i n e d f rom Pha rmac i a , U p s a l a , Sweden. The l i q u i d s c i n t i l l a t i o n c o c k t a i l , Unoge l , was o r d e r e d f rom Bec ton and D i c k i n s o n . Equipment and Othe r S u p p l i e s : Eppendor f m i c r o t ube s were pu r chased f rom Br inkman I n s t r umen t s I n c . , Wes tbury , NY. The B e h r i n g M - P a r t i g e n A lbumin p l a t e s f o r a l bum in q u a n t i t a t i o n were o b t a i n e d f rom Hoechs t Canada I n c . , M o n t r e a l , Quebec. A Braun M i k r od i smembra t o r was used f o r t i s s u e p u l v e r i z a t i o n . T i s s u e s were s t o r e d i n a Mode l 8049 Hotpack U l t r a f r e e z e r , W a t e r l o o , O n t a r i o . L i q u i d s c i n t i l l a t i o n c o u n t i n g was c a r r i e d out i n a Beckman LS 9000 c o u n t e r Beckman I n s t r umen t s I n c . I r v i n e , CA . ) Fo r c e n t r i f u g a t i o n , c e l l u l o s e n i t r a t e tubes a v a i l a b l e f rom 48 Beckman, P a l o A l t o , CA. were used i n a S o r v a l AH 650 u l t r a c e n t r i f u g e r o t o r . i i . RECEPTOR ASSAY : METHOD The method used t h roughou t t h i s p r o j e c t i s based on the r e c e p t o r assay e s t a b l i s h e d by Jacobson ( 2 6 ) . T i s s u e to be a s sayed was s t o r e d a t - 8 0 °C . F o r t r i m m i n g , the samples a re kep t a t 4°C . Once the spec imen i s p a r t i a l l y thawed, e x t r a neou s f a t and c o n n e c t i v e t i s s u e , as w e l l as norma l t i s s u e i s removed. The r ema i n i n g t i s s u e i s cu t i n t o s m a l l cubes (about 2mm) and we i ghed . The weighed t i s s u e i s then t r a n s f e r r e d to a l a b e l e d p o l y p r o p y l e n e v i a l and r e f r o z e n i n l i q u i d n i t r o g e n . The r ema i n i n g p r o c edu r e s a re c a r r i e d out a t room t empe r a t u r e , w h i l e k eep i ng the t i s s u e samples a t 4°C . T e f l o n mo r t a r s and s t e e l b a l l s were p r e - c o o l e d i n l i q u i d n i t r o g e n . M o d i f i e d haemos ta t s s e r v ed as c lamps wh ich h e l d t he mo r t a r s and c o u l d be l a b e l e d . T i s s u e cubes were t r a n s f e r e d t o the a p p r o p r i a t e mo r t a r s and c o o l e d a g a i n i n l i q u i d n i t r o g e n . One mo r t a r a t a t ime was p l a c e d i n the Braun M i k r od i smembra t o r c l amp . P u l v e r i z a t i o n was c a r r i e d out a t the maximum s e t t i n g f o r 45 s e cond s . The mo r t a r was r e t u r n e d t o l i q u i d n i t r o g e n w h i l e the o t h e r samples were be i ng p r o c e s s ed i n the same manner. The p r o cedu r e was t hen r e pea t ed aga i n so t h a t each spec imen was s u b j e c t e d t o maximum throw f o r a t o t a l o f 90 seconds w i t h a p p r o x i m a t e l y t h r e e m inu te s o f c o o l i n g between p u l v e r i z a t i o n s . 49 A f t e r the second p u l v e r i z a t i o n was c omp l e t e , the mo r t a r s were l e f t a t room t empe r a t u r e and f i v e volumes of 20mM TES -1 . 5 mM EDTA-5mM mono th i og l y c e r o l - 5mM sodium mo lybda te - 10% g l y c e r o l pH 7 .5 b u f f e r (TEMS) was added, f o l l o w e d by m i x i n g on the v o r t e x to fo rm a homogenate. The homogenate i t s e l f was not a l l o w e d t o come t o room t e m p e r a t u r e . A poo l ed c o n t r o l , p u l v e r i z e d and l y o p h i l i z e d was s i m i l a r i l y r e c o n s t i t u t e d w i t h 25 volumes of TEMS b u f f e r . The t i s s u e homogenates and the c o n t r o l sample were t r a n s f e r r e d t o p r e - c o o l e d l a b e l e d c e l l u l o s e n i t r a t e c e n t r i f u g a t i o n t ube s ( 1 /2 X 2 i n c h e s ) and p l a c e d i n a p r e - c o o l e d sw i ng i n g bucke t o f a S o r v a l AH 650 u l t r a c e n t r i f u g e r o t o r . S i n c e the spec imens were i n v a r i a b l y of d i f f e r i n g s i z e s , l i q u i d p e t r o l e um wh i ch i s s i m i l a r i n d e n s i t y t o the tumour homogenate was used to top up a l l samples to the same f i n a l volume and t hus w e i g h t . The samples were c e n t r i f u g e d a t 39,000X g f o r 15 m i n u t e s . Upon c o m p l e t i o n o f c e n t r i f u g a t i o n , the samples were c a r e f u l l y removed and p l a c e d i n a p l e x i g l a s s h o l d e r , where the t ubes were pun c t u r ed w i t h a 20 gage need l e a t t a c h e d t o a 3 cc s y r i n g e and the s u p e r n a t a n t l a y e r was c a r e f u l l y w i thd rawn and then e x p e l l e d i n t o a c o o l e d l a b e l e d g l a s s t e s t tube kept on i c e . A p r e - i n c u b a t i o n p r o t e i n d e t e r m i n a t i o n was pe r fo rmed so t h a t the same p r o t e i n c o n c e n t r a t i o n , a p p r o x i m a t e l y 2mg/ml was used i n a l l a s s a y s . The method used was a U.V. method (80) based on the p r i n c i p l e t h a t most p r o t e i n s e x h i b i t a d i s t i n c t U.V. a b s o r p t i o n maximum at 280 nanometers due to the p r e sence o f t y r o s i n e and t r y p t o p h a n . Based on t h i s p r o t e i n e s t i m a t i o n the s u p e r n a t a n t 50 samples were d i l u t e d w i t h TEMS b u f f e r t o 2 mg/ml . A 250 u l sample o f t he d i l u t e d sample was then i n c u b a t e d w i t h t h r e e d i f f e r e n t l e v e l s o f 3 H - e s t r a d i o l , 1.5nM, 2.0nM and 5 .0 nM A l l doses were i n d u p l i c a t e . At t he 2 .0 nM l e v e l , 50 u l of DES was added p r i o r to a d d i t i o n o f t he l a b e l l e d l i g a n d . The assay se t up per tumour t h e r e f o r e was as f o l l o w s : Tube # TEMS D i l . S u p e r n . 4uM DES 3 H - E 2 E 2 ( u l ) ( u l ) ( u l ) ( u l ) (nM) 1,2 5,6 7 ,8 150 150 150 150 250 250 250 250 50 100 100 100 100 1.5 2.0 2.0 5.0 S c i n t i l l a t i o n v i a l s wh ich s e r ved as b l a n k s and backg round , were a l s o i n c l u d e d , as were v i a l s c o n t a i n i n g the t h r e e l e v e l s o f r a d i o a c t i v e l i g a n d , wh ich w i l l be r e f e r r e d to as the dose v i a l s . The b l a n k s i n c l u d e d a l l assay c o n s t i t u e n t s excep t the c y t o s o l and were a measure o f the background l e v e l o f 3 H - E 2 wh ich r ema ins subsequent t o the DCC t r e a t m e n t . The pe r c en t age of dose r ema i n i n g i s e s s e n t i a l l y l i n e a r , t hus n e c c e s s i t a t i n g on l y one dose d e t e r m i n a t i o n , f rom wh ich the o t h e r two can be c a l c u l a t e d The assay t ubes used were the 1.5 ml Eppendor f p o l y p r o p y l e n e m i c r o t e s t t u b e s , wh ich can be c o n v e n i e n t l y capped and h e l d i n 51 t r a n s f e r r a c k s d u r i n g the 16-18 hour i n c u b a t i o n a t 0 -4°C . A f t e r o v e r n i g h t i n c u b a t i o n , 500 u l c o l d d e x t r a n - c o a t e d c h a r c o a l [0.5% c h a r c o a l - 0.05% Dex t r a n 70] was added t o each assay t u b e . The recapped tubes were shaken b r i e f l y and r e t u r n e d t o 0-4°C f o r ano the r t h i r t y m i nu t e s , to i n s u r e the r emova l o f a l l unbound and l o o s e l y bound l a b e l by a d s o r p t i o n to the c h a r c o a l . The d e x t r a n reduces the a d s o r p t i o n o f p r o t e i n s w i t h o u t i n t e r f e r i n g w i t h a d s o r p t i o n o f f r e e s t e r o i d s . The t ubes were c e n t r i f u g e d a t 12,000Xg f o r 4 m inu tes and 500 u l o f the r e s u l t i n g c l e a r s upe r n a t a n t was p i p e t t e d i n t o g l a s s s c i n t i l l a t i o n v i a l s . Unoge l s c i n t i l l a t i o n c o c k t a i l was added t o each v i a l . A f t e r t ho rough m i x i n g the v i a l s were coun ted i n the l i q u i d s c i n t i l a t i o n c o u n t e r , w i t h a p p r o p r i a t e da t a r e d u c t i o n . The energy windows a re s e t as f o l l o w s : c h anne l one c h anne l two 0-397kEv 180-397kEv A quench cu r v e was e s t a b l i s h e d f o r the t r i t i a t e d l i g a n d s u s i n g a New Eng l and N u c l e a r k i t and the quench c o e f f i c i e n t s were used i n the c o u n t i n g p rog ram. Each sample was coun ted f o r t e n 3 m inu t e s and machine e f f i c i e n c y was mon i t o r ed u s i n g a H s t a n d a r d sample f rom New Eng l and N u c l e a r . I t i s a l s o p o s s i b l e to add the n o r m a l i z a t i o n f a c t o r t o the program such t h a t r e s u l t s a re a v a i l a b l e i n femtomo les e s t r a d i o l r a t h e r than coun t s pe r m inu te ( cpm) . The PgR assay was e s s e n t i a l l y the same as the ER a s s a y , 3 ex cep t f o r t h e l i g a n d s u s e d . H-R5020 and u n l a b e l e d R5020 52 r e p l a c e d the r a d i o l a b e l e d e s t r a d i o l and DES r e s p e c t i v e l y . I n the combined o r d u a l - l a b e l assay* i n c u b a t i o n t ime was f o u r h o u r s . Ten assay t ubes were r e q u i r e d per tumour samp le . Two f o r each of o f the t h r e e l i g a n d c o n c e n t r a t i o n s , as w e l l as two wh i ch c o n t a i n e d c o l d R5020 and two wh ich c o n t a i n e d DES, f o r the assessment of n o n - s p e c i f i c b i n d i n g f o r the PgR and ER d e t e r m i n a t i o n s r e s p e c t i v e l y . i i i . ISOTOPE SEPARATION 3 125 F i g u r e 9 i l l u s t r a t e s the energy p r o f i l e s of H and I . I t i s q u i t e e v i d e n t f rom t h i s i l l u s t r a t i o n t h a t the f i r s t peak o f the i o d i n e c o m p l e t e l y o v e r l a p s the s i n g l e t r i t i u m peak . F o r t u n a t e l y , the second r a d i o i o d i n e peak i s e s s e n t i a l l y d i s t i n c t f rom the t r i t i u m peak . Tak i ng advantage of t h i s f a c t and the d u a l c h a n n e l c a p a c i t y o f the Beckman LS 9000 the f o l l o w i n g s e t t i n g s were u s e d . F o r c h anne l one the l owes t s e t t i n g was 0 keV and the upper s e t t i n g was 700 keV. T h i s would t h e r e f o r e i n c l u d e the t o t a l t r i t i u m and r a d i o i o d i n e c o u n t s . Channe l two was a d j u s t e d t o i s o l a t e the second r a d i o i o d i n e peak . A l owe r s e t t i n g o f 400 keV and an upper s e t t i n g of 700 keV was f i n a l l y c ho s en . I t s h ou l d be men t i on a t t h i s p o i n t t h a t w i t h quench c o e f f i c i e n t s d e r i v e d f rom a quench cu r ve i t i s p o s s i b l e t o employ the Beckman da t a r e d u c t i o n package to gene r a t e da t a i n femtomoles r a t h e r than i n c oun t s pe r m i nu t e , (cpm) Numerous a t t emp t s were made to 125 e s t a b l i s h such a quench cu r ve f o r I , but the c o e f f i c i e n t s 53 o b t a i n e d f rom such a quench cu r ve when used i n the da t a r e d u c t i o n program r e s u l t e d i n l a r g e e r r o r s . S i n c e numerous e xpe r imen t s w i t h bo th r a d i o i o d i n e and t r i t i u m showed ve ry l i t t l e d i f f e r e n c e i n t he H number (60 -63 ) f rom tumour t o tumour , the d e c i s i o n was made t o abandon a t t emp t s to use the Beckman da t a r e d u c t i o n package and work s t r i c t l y w i t h cpm. To i l l u s t r a t e the a d v i s a b i l i t y o f t h i s d e c i s i o n , the f o l l o w i n g r e s u l t s f rom a poo l e d tumour sample a re g i v e n : ER= 191 .5 fm/mg TP ER= 318 .7 fm/mg TP ER= 200 .1 fm/mg TP Wi th the s e t t i n g s j u s t men t i oned , c h a n n e l two would 125 t h e o r e t i c a l l y y i e l d on l y I c oun t s though d e f i n i t e l y not a l l . T ak i ng advan tage of the 15 dose and b l ank v i a l s i n c l u d e d i n eve ry ER assay i t i s p o s s i b l e t o o b t a i n a r e a s o n a b l y a c c u r a t e SCR (samp le c h anne l s r a t i o ) wh i ch i s an i n d i c a t i o n o f the d i s t r i b u t i o n r a t i o o f the coun t s i n each c h a n n e l . On the b a s i s o f the SCR i t i s t hu s p o s s i b l e to c a l c u l a t e the number o f cpm i n 125 c h a n n e l one wh i ch a re due t o I . The d i f f e r e n c e t h e r e f o r e y i e l d s t he t r i t i u m c o u n t s . As an examp le : G i v en : c h a n n e l one c oun t s = 1751.40 : c h a n n e l two c oun t s = 589 .4 : average c h anne l two background f o r t h i s run = 12 : ave rage SCR f o r t h i s run = .463 125 I manual c a l c u l a t i o n "*" 2 5I d a t a r e d u c t i o n program 3 H w i t h da t a r e d u c t i o n program 54 Then (589 .4 - 12 .0 ) / . 4 6 3 = 1247 .1 T h i s r e p r e s e n t s the c oun t s i n c h a n n e l one wh ich o r i g i n a t e f rom the r a d i o i o d i n e . T h e r e f o r e 1751.4 - 1247 .1 = 504.3 c oun t s i n c hanne l one due t o t r i t u m . 125 Each new ba t ch o f I was coun ted t o de t e rm ine e f f i c i e n c y , a f i g u r e wh ich v a r i e d o n l y s l i g h t l y . (63.01% - 63.5%) D i v i s i o n o f cpm by e f f i c i e n c y y i e l d e d d i s i n t e g r a t i o n s per m inute (dpm). 125 W i th the r a t h e r s h o r t h a l f l i f e o f I i e . 60 days , the n o r m a l i z a t i o n f a c t o r was r e - c a l c u l a t e d f o r eve ry s i n g l e r u n . The n o r m a l i z a t i o n f a c t o r f o r 3 H w i t h a h a l f - l i f e o f 4 , 492 . 58 days was updated on l y once a month. The n o r m a l i z a t i o n f a c t o r i s c a l c u l a t e d f rom da ta p r o v i d e d by New Eng l and N u c l e a r w i t h each new l i g a n d , namely t he p r e p a r a t i o n da te and the s p e c i f i c a c t i v i t y a t t h a t t i m e . The f a c t o r i s g i v e n i n u n i t s o f dpm/fmole E^-Thus d i v i s i o n o f t he c a l c u l a t e d dpm by the n o r m a l i z a t i o n f a c t o r w i l l y i e l d the number of femtomoles o f e s t r a d i o l . i v . PROTEIN AND ALBUMIN DETERMINATION The p r e - i n c u b a t i o n p r o t e i n d e t e r m i n a t i o n was c a r r i e d out u s i n g an u l t r a v i o l e t method ( 8 0 ) . T h i s method i s based on the p r i n c i p l e t h a t most p r o t e i n s e x h i b i t a d i s t i n c t U.V. a b s o r p t i o n maximum a t 280 nanometers due to the p re sence of t y r o s i n e and t r y p t o p h a n . N u c l e i c a c i d s a l s o abso rb a t t h i s wave l eng th but t hey e x h i b i t a much s t r o n g e r peak a t 260 nanomete rs , w h i l e p r o t e i n does n o t . Based on t h i s d i f f e r e n c e , the i n t e r f e r e n c e o f 55 n u c l e i c a c i d s can be c o r r e c t e d f o r by c a l c u l a t i o n . The f o r m u l a f o r t h i s d e t e r m i n a t i o n i s : 1 . 5 5 X A b 2 8 0 - 0 . 7 4 X A b 2 6 0 X d i l u t i o n f a c t o r A Lowry p r o t e i n d e t e r m i n a t i o n (25) was c a r r i e d out t o e s t a b l i s h t he c o n c e n t r a t i o n of bo th the d i l u t e d and the c o n c e n t r a t e d c y t o s o l . Two r e a c t i o n s a re i n v o l v e d i n t he d e t e r m i n a t i o n : (1) an i n i t i a l i n t e r a c t i o n o f p r o t e i n and C u 2 + i n a l k a l i ( r e l a t e d t o t he b i u r e t r e a c t i o n ) ; (2) a r e d u c t i o n o f the p h o s p h o t u n g s t i c and phosphomo lybd i c a c i d s to molybdenum b l u e and t u n g s t e n b l u e , bo th by t he C u - p r o t e i n complex and by the t y r o s i n e and t r y p t o p h a n of the p r o t e i n . The two amino a c i d s g i v e a c o l o u r i n t he absence of Cu , but the r e s t of the p r o t e i n 2 "i* g i v e s no c o l o u r w i t h o u t Cu Immunod i f f u s i on p l a t e s were used to assay f o r a l b u m i n . The p r i n c i p l e of s i n g l e r a d i a l i m m u n o d i f f u s i o n i s the r e a c t i o n of the a n t i g e n ( a l bum in ) w i t h i t s s p e c i f i c p r e c i p i t a t i n g a n t i s e r u m . S m a l l w e l l s a re made i n an a n t i b o d y - c o n t a i n i n g g e l and the w e l l s a re f i l l e d w i t h a n t i g e n (unknowns and s t a n d a r d s ) . The a n t i g e n d i f f u s e s out i n t o the g e l t o form c i r c u l a r p r e c i p i t i n l i n e s . The d i ame t e r o f t he p r e c i p i t i n r i n g i s p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the a n t i g e n . A s t a n d a r d cu r ve was g ene r a t ed u s i n g known amounts o f a n t i g e n , i n t h i s case a l b u m i n . A p l o t of the squa re s of the r i n g d i ame t e r s vs the a l bum in c o n c e n t r a t i o n y i e l d e d a s t r a i g h t l i n e . The c o n c e n t r a t i o n o f a l bum in i n the 56 unknown sample can t hus be de te rm ined f rom the i n t e r c e p t on the s t a n d a r d c u r v e . The B e h r i n g M - P a r t i g e n A lbumin p l a t e s used had a c o n c e n t r a t i o n range of 40-580 m g / d l . v . PLASMINOGEN ACTIVATOR ACTIVITY ASSAYS : MATERIALS C h e m i c a l s : U r o k i n a s e , s u c c i n i c a nh yd r i d e and p r o t am ine s u l p h a t e ( f rom sa lmon) were o b t a i n e d f rom S igma, S t . L o u i s MO. Human p l a sm inogen was o b t a i n e d f rom Sigma and f rom K a b i , Pha rmac i a Canada I n c . , D o r v a l , Quebec. S2251 was a l s o o b t a i n e d f rom K a b i . S t r e p t o k i n a s e was s u p p l i e d by B e h r i n g I n s t i t u t e , Hoechs t Canada, M o n t r e a l , Quebec. The f l u o r e s c a m i n e r eagen t F l u r am was o b t a i n e d f rom Hof fman-LaRoche , V a u d r e u i l , Quebec. B a r b i t a l was pu r cha sed f rom F i s h e r S c i e n t i f i c , F a i r Lawn, N J . Equ ipment : S o n i c a t i o n was c a r r i e d out u s i n g a B i o s o n i k I I I U l t r a s o n i c System s o n i c a t o r . An Aminco Bowman f l u o r o m e t e r (Amer i can I n s t r umen t Co. I n c . S i l v e r S p r i n g , MD.) was used i n t he f l u o r o m e t r i c method. v i . PLASMINOGEN ACTIVATOR ACTIVITY ASSAYS : METHOD Two methods f o r q u a n t i f y i n g p l a sm inogen a c t i v a t o r a c t i v i t y were examined . One method uses a s y n t h e t i c chromogen ic s u b s t r a t e , w h i l e the o t h e r i s a f l u o r o m e t r i c method. Bo th methods a re two s t ep methods i n wh ich the p l a sm inogen a c t i v a t o r a c t s on p l a sm i n ogen , g e n e r a t i n g p l a s m i n , wh ich i n t u r n r e a c t s 57 w i t h the g i v e n s u b s t r a t e . P r e p a r a t i o n o f c y t o s o l i c f r a c t i o n s was as d e s c r i b e d f o r the s t e r o i d a s s a y , such t h a t the same c y t o s o l used f o r ER and PgR a n a l y s i s was a l s o used f o r the p l a sm inogen a c t i v a t o r a c t i v i t y (PAA) a n a l y s i s . L y sosoma l f r a c t i o n s were p r epa r ed e s s e n t i a l l y as d e s c r i b e d by Renn i e ( 9 6 ) . The p e l l e t wh ich r ema ins a f t e r the 105 ,000xg c e n t r i f u g a t i o n was r e - su spended i n TEMS b u f f e r . The sample was s o n i c a t e d w i t h f o u r 15 - second p u l s e s o f a B i o s o n i k I I I U l t r a s o n i c System s o n i c a t o r a t a s c a l e s e t t i n g o f 60 . The sample was kep t on i c e d u r i n g t h i s t r e a tmen t and was s ub s equen t l y t r a n s f e r r e d to an Eppendor f m i c r o t ube and c e n t r i f u g e d a t 15 ,000xg f o r 5 m i n u t e s . The r e s u l t i n g s u p e r n a t a n t i s the l y s o s o m a l f r a c t i o n . The p r o t o c o l f o r the f l u o r o m e t r i c assay i s as d e s c r i b e d by Ke s sne r (88) but w i t h some m o d i f i c a t i o n s . The human p l a sm inogen and u r o k i n a s e were d i l u t e d i n 50 mM b a r b i t a l , 100 mM NAC1 pH 8 .0 b u f f e r (VS b u f f e r ) , t o y i e l d s t o c k s o l u t i o n s of 0 .1 Sigma U n i t s pe r ml f o r u r o k i n a s e and 5.0 Sigma u n i t s pe r ml f o r p l a s m i n o g e n . S t o c k s o l u t i o n s were then s t o r e d a t 4°C u n t i l u s e . 50mM ba r b i t a l - l OOmM N a C l , 50mM C a C l 2 pH 8 .0 (VC b u f f e r ) was used whenever p r o t e a s e a c t i v i t y was a s s e s s e d . The s u b s t r a t e , s u c c i n y l a t e d p r o t am ine ( S - P ) , was p r epa r ed as f o l l o w s . S u c c i n i c a n h y d r i d e , 300 mg i n 2 ml o f ace tone and p ro t am ine s u l f a t e , 1600 mg i n 30 ml VS b u f f e r were p r e p a r e d . The s u c c i n i c a nhyd r i d e i s added drop by drop t o t he p ro t am ine s u l f a t e , a t the same t ime m a i n t a i n i n g the pH by the a d d i t i o n of 10 N NaOH. The volume i s 58 a d j u s t e d t o 40 mis and a l i q u o t s o f the r eagen t a re s t o r e d i n the -80°C f r e e z e r u n t i l r e q u i r e d . The f l u o r e s c a m i n e r eagen t F l u r am was p r epa r ed f o r use by d i s s o l v i n g i t i n a ce tone to y i e l d a c o n c e n t r a t i o n o f 2 mg/ml . B o r a t e b u f f e r , used to quench the r e a c t i o n s , was 0 .2 M b o r i c a c i d , pH 8 . 0 . Fo r the p ro t am ine s u l p h a t e s t a nda r d c u r v e , s t o c k p ro t am ine (40 ug/ml ) was s e r i a l l y d i l u t e d to y i e l d c o n c e n t r a t i o n s f rom 5.0 ug/ml t o 40 u g / m l . A z e r o s t a nda r d c o n t a i n e d on l y VS b u f f e r . 50 u l o f each c o n c e n t r a t i o n was p i p e t t e d i n t o Eppendor f m i c r o t u b e s . 200 u l o f VS b u f f e r was added f o r a f i n a l volume of 250 u l . 50 u l was w i thd rawn f rom each s t a nda r d tube and d i l u t e d w i t h 2 ml o f b o r a t e b u f f e r t o wh ich 50 u l o f F l u r am was added w i t h r a p i d m i x i n g . Samples were then read on an Aminco Bowman s p e c t r o p h o t o m e t e r , w i t h an e x c i t a t i o n wave l eng th of 390 nm and an e m i s s i o n wave l eng th o f 480 nm. F l u o r e s c e n c e was p l o t t e d a g a i n s t p r o t am ine c o n c e n t r a t i o n to gene ra t e a s t a nda r d c u r v e . S i n c e t h e r e were no i n t e r f e r i n g peaks i n the immed ia te v i c i n i t y , h i g h s e n s i t i v i t y was sought a t the expense o f h i g h r e s o l u t i o n . S l i t #1 on the e x c i t a t i o n s i d e was s e t a t 2 .5 nm, w h i l e a l l o t h e r s were s e t a t 4 . 0 nm. The h i g h v o l t a g e s e t t i n g was kept a t 800 v o l t s and the s e n s i t i v i t y was s e t a t 80%. A c u v e t t e w i t h a one c e n t i m e t r e p a t h l e n g t h was u s ed . The u r o k i n a s e s t a nda r d cu r ve s e t up was somewhat d i f f e r e n t . 0-75 u l o f u r o k i n a s e wo r k i ng s t o c k ( 0 . 5 mU/ml) was p i p e t t e d i n t o m i c r oeppendo r f t e s t t u b e s . 50 u l o f p l a sm inogen ( e q u i v a l e n t t o 0 . 05U /a s s ay ) was added t o each t u b e . VS b u f f e r was added to 59 y i e l d a t o t a l volume of 125 u l . The tubes were then p l a c e d i n a 37° C wate r ba th and i n c u b a t e d at t h i s t empe ra tu r e f o r one hou r . A f t e r one hour 125 u l o f s u c c i n y l a t e d p ro t am ine a l s o at a t empe r a t u r e o f 37°C was added to each t u be , a l l o w i n g 20 seconds between t u b e s . At e x a c t l y f o u r m inu te s a f t e r the a d d i t i o n o f s u c c i n y l a t e d p r o t a m i n e , 50 u l was w i thd rawn and p i p e t t e d i n t o two ml o f b o r a t e b u f f e r . T h i s was f o l l o w e d immed i a t e l y by the a d d i t i o n of 50 u l of F l u ram w i t h v o r t e x i n g . At t h i r t y - f o u r m inu te s a f t e r a d d i t i o n o f s u b s t r a t e , a second 50 u l sample was w i thd rawn f rom each tube i n t u r n and added t o bo r a t e b u f f e r , f o l l o w e d by the r a p i d a d d i t i o n o f F l u r a m . Read ings were t a ken on the s p e c t r o f l u o r o m e t e r as d e s c r i b e d above . The d i f f e r e n c e between the r e a d i n g s a t f o u r m inu te s and t ho se at t h i r t y - f o u r m inu te s i n r e l a t i v e f l u o r e s c e n c e u n i t s (RFU) was p l o t t e d a g a i n s t the u r o k i n a s e c o n c e n t r a t i o n . The s e t - u p f o r the assay was as f o l l o w s : u l U r o k i n a s e u l VS b u f f e r u l p l a sm inogen u l S-P 0 75 50 125 5 70 50 125 12 . 5 62 .5 50 125 25 50 50 125 35 40 50 125 50 25 50 125 75 00 50 125 60 The s p e c t r o p h o t o m e t r i c method was based on the work o f Tho r sen ( 7 9 ) , Overw ien (91) and G i l b o a ( 9 0 ) . The enzyme used f o r the s t a n d a r d was s t r e p t o k i n a s e , the s u b s t r a t e was S2251. The b u f f e r s used were 50 mM TES pH 8 .0 ( b u f f e r A) and 50 mM TES-150 mM NaC l pH 7 .4 ( b u f f e r B ) . The s t r e p t o k i n a s e work ing s o l u t i o n was p r epa r ed f rom the l y o p h i l i z e d powder when needed . A l i q u o t s o f r e c o n s t i t u t e d enzyme, even when f r o z e n , were not found t o be s t a b l e . P l a sm inogen was d i s s o l v e d i n water and the pH a d j u s t e d to 3.0 w i t h h y d r o c h l o r i c a c i d so t h a t the f i n a l c o n c e n t r a t i o n was 1.0 C U / m l . ( C a s e i n U n i t ) A l i q u o t s of the p l a sm inogen were kept f r o z e n u n t i l r e q u i r e d . S2251 was d i s s o l v e d i n b u f f e r B a t a c o n c e n t r a t i o n of 1.67 mg/ml o r 3 .02 mM. The K m f o r p l a s m i n and S2251 i s 3 . 5 x l 0 ~ 4 M . The S2251 was kept a t 4°C u n t i l needed . The assay was s e t up as f o l l o w s : 0 - 60 u l o f s t r e p t o k i n a s e wo r k i ng s t o c k (500 IU /m l ) was p i p e t t e d i n t o eppendor f m i c r o t u b e s . B u f f e r was added to make up a t o t a l volume of 150 u l . 100 u l o f p l a sm inogen was then added t o each t u be , and the t ubes were then p l a c e d i n a 37°C wa t e r ba t h f o r one hou r . A f t e r one hour 50 u l was w i thd rawn and t r a n s f e r r e d to a second Eppendor f tube c o n t a i n i n g 50 u l o f S2251 and 150 u l of b u f f e r B a t 37°C . I n c u b a t i o n of t h i s m i x t u r e c o n t i n u e d f o r 30 m i n u t e s , a t wh i ch t ime 1.00 ml o f 10% a c e t i c a c i d was added to quench the r e a c t i o n . As was the case f o r the f l o u r o m e t r i c u r o k i n a s e method, 20 seconds between a d d i t i o n s and /o r w i t h d r a w a l s f rom r e s p e c t i v e t u b e s , was judged adequate t i m e . In t h i s way i t was p o s s i b l e t o keep the r e a c t i o n t ime t o p r e c i s e l y 30 m inu tes f o r each assay 61 t u b e . The samples were read a f t e r 15 - 30 m inu tes on a s p e c t r o p h o t o m e t e r . The assay s e t up was as f o l l o w s : u l SK s t o c k u l B u f f e r A u l P l a sm inogen s t o c k 0 150 100 7 .5 142 .5 100 15 135 100 35 115 100 50 100 100 60 90 100 A f t e r one hour i n c u b a t i o n at 37 C , 50 u l i s w i thd rawn f rom each tube and added t o 50 u l o f S2251 and 150 u l o f b u f f e r B i n Eppendor f t ubes i n c u b a t e d a t 37°C. When a c t u a l samples of b r e a s t c y t o s o l were a s s a y e d , 50 u l was i n c u b a t e d w i t h and w i t h o u t 100 u l p l a sm inogen , and enough b u f f e r A t o g i v e a f i n a l volume of 250 u l . The assay t ubes w i t h ou t p l a sm inogen a l l o w e d f o r q u a n t i t a t i o n o f p r o t e a s e a c t i v i t y . 62 I I I .RESULTS AND DISCUSSION VERIFICATION OF PROFICIENCY WITH THE ESTROGEN RECEPTOR ASSAY S i x p r e v i o u s l y a n a l y z e d b r e a s t tumours o f s u f f i c i e n t q u a n t i t y were removed f rom the -80°C f r e e z e r and the ER assay as d e s c r i b e d above was p e r f o r m e d . L i n e a r r e g r e s s i o n a n a l y s i s gave a c o r r e l a t i o n c o e f f i c i e n t r = 0.998 where y = 0.899x + 1 8 . 6 . These r e s u l t s i n d i c a t e d t h a t s u f f i c i e n t p r o f i c i e n c y w i t h the assay had been a c h i e v e d . TEMS BUFFER VS TED BUFFER IN THE ESTROGEN RECEPTOR ASSAY A s tudy o f t he l i t e r a t u r e i n d i c a t e d t h a t the a d d i t i o n of 10% g l y c e r o l i s e s s e n t i a l f o r the PgR assay and t h a t the f u r t h e r i n c l u s i o n o f sod ium mo ldybdate was a d v i s a b l e ( 7 , 2 4 , 2 5 ) . The r e s u l t i n g b u f f e r w i l l be r e f e r r e d t o as TEMS and i t c o n s i s t s o f t he f o l l o w i n g : 20 mM TES - 1 . 5 mM EDTA-5mM mono th i og l y c e r o l - 5mM sodium molybdate-10% g l y c e r o l pH 7 . 5 . To v e r i f y i t s s u i t a b i l i t y f o r use i n the ER a s s a y , poo l ed tumour t i s s u e was as sayed i n bo th TEMS and TED u s i n g the s t anda r d assay p r o t o c o l . TED (20mM TRIS-0.5mM EDTA-0.5mM d i t h i o t h r e i t o l pH 7 .5 ) was the b u f f e r o r i g i n a l l y used i n the ER assay d e s i gned by Jacobson ( 2 6 ) . 63 Tab l e I : B u f f e r Compar i son Time B u f f e r ER( fmo les /mg CP) P r o t e i n (mg/assay) Kd (mo l e s / L ) ( h r ) 2 .0 TED 90 .0 .425 -18 TED 86 . 5 .425 3 . 4 x I O " 1 0 2.0 TEMS 98 .6 .675 -18 TEMS 102 .675 4 . 9 x I O " 1 0 Compar i son of ER and Kd v a l u e s u s i n g the same sample i n d i f f e r e n t b u f f e r . R e p r e s e n t a t i v e da t a shown i n t a b l e one i l l u s t r a t e s t h a t TEMS b u f f e r i s an a p p r o p r i a t e a l t e r n a t e f o r TED i n the ER a s s a y . Sodium mo lybda te (5mM) was i n c l u d e d i n the d u a l - l a b e l assay b u f f e r f o r i t s r e pu t e d s t a b i l i z i n g e f f e c t on bo th ER and PgR. A number of r e p o r t s have appeared i n t he l i t e r a t u r e i n d i c a t i n g the v a l u e o f sod ium mo lybda te i n r e d u c i n g r e c e p t o r d e g r a d a t i o n ( 2 4 , 9 7 - 1 0 1 ) . I n i t i a l l y , t he s u g g e s t i o n was made t h a t mo lybda te e x e r t e d t h i s s t a b i l i z i n g e f f e c t by i n h i b i t i n g a l k a l i n e phospha tase ( 9 8 ) , but subsequent work has shown t h a t enzyme i n h i b i t i o n i s not the mechanism by wh i ch mo lybda te p r e v e n t s r a p i d r e c e p t o r d e g r a d a t i o n . The e x a c t mechanism by wh ich mo lybda te s t a b i l i z e s the r e c e p t o r has ye t t o be c l a r i f i e d , but the s u g g e s t i o n i s t h a t i t p r e v e n t s the d i s s o c i a t i o n of the 7-8S mo ie t y i n t o 4-5S s u b u n i t s and i t may a c c o m p l i s h t h i s by b i n d i n g to 64 t he n a t i v e t h i o l g roups of the r e c e p t o r t hus f o rm ing a b r i d g e between s u b u n i t s ( 2 4 , 9 7 ) . The i n c l u s i o n o f g l y c e r o l and m o n o t h i o g l y c e r o l i n the b u f f e r was found t o cause i n t e r f e r e n c e w i t h the Lowry and U.V. p r o t e i n d e t e r m i n a t i o n s . U.V. p r o t e i n d e t e r m i n a t i o n s were c a r r i e d out on a doub l e beam Cary 118 S p e c t r o p h o t o m e t e r . U s i ng the b u f f e r i n the r e f e r e n c e c e l l l a r g e l y e l i m i n a t e d the i n t e r f e r e n c e . The i n t e r f e r e n c e by g l y c e r o l and m o n o t h i o g l y c e r o l i n the Lowry assay was not unexpec t ed , s i n c e o t h e r worke r s had r e p o r t e d t h i s p rob lem as w e l l ( 1 0 2 - 1 0 4 ) . T h i s p rob lem was overcome by 1) r e a d i n g a l l samples a g a i n s t a b l ank c o n s i s t i n g of TEMS b u f f e r , 2) c o n s t r u c t i n g the p r o t e i n s t a n d a r d c u r v e w i t h the a d d i t i o n o f 10 u l o r 20 u l of b u f f e r to each s t a n d a r d p r o t e i n c o n c e n t r a t i o n . I t was found t h a t a d i f f e r e n c e o f 10 u l o f b u f f e r made a d i f f e r e n c e i n the s t a n d a r d c u r v e . T h e r e f o r e , the p r o t e i n c o n t e n t i n c y t o s o l samples o f 10 u l vo lumes were a lways de t e rm ined f rom the 10 u l - s t a n d a r d c u r v e , and 20 u l samples f rom the 20 u l - s t a n d a r d c u r v e . D i l u t e d c y t o s o l s u s u a l l y r e q u i r e d 20 u l f o r Lowry d e t e r m i n a t i o n and c o n c e n t r a t e d c y t o s o l r e q u i r e d 10 o r on o c c a s i o n 5 u l to b r i n g i t i n t o the range of the Lowry s t a n d a r d c u r v e . PROGESTERONE RECEPTOR ASSAY The p r o g e s t e r o n e assay p r o t o c o l was based on the ER a s s a y , w i t h a p p r o p r i a t e m o d i f i c a t i o n s . The o b v i o u s major d i f f e r e n c e i s the l i g a n d u s e d . Wh i l e the f i r s t c h o i c e might l o g i c a l l y be p r o g e s t e r o n e , i t was not chosen f o r a number of r e a s o n s . Most 65 i m p o r t a n t among t he se i s the f a c t t h a t p r o g e s t e r o n e i s not on l y s p e c i f i c f o r i t s c y t o s o l i c r e c e p t o r , but i t a l s o b i nd s t o c o r t i c o s t e r o i d - b i n d i n g g l o b u l i n (CBG) , g l u c o c o r t i c o i d r e c e p t o r , and t o a l e s s e r e x t e n t sex hormone b i n d i n g g l o b u l i n (SHBG). Wh i l e p r o g e s t e r o n e r e c e p t o r a f f i n i t y f o r PgR i s h i g h , i t i s not h i g h enough, such t h a t c o n s i d e r a b l e d i s s o c i a t i o n i s r e p o r t e d t o o c cu r d u r i n g p r o c edu r e s s u i t a b l e f o r ER ( 1 0 2 , 1 1 4 ) . The p rob lems of a f f i n i t y and s p e c i f i c i t y have been l a r g e l y s o l v e d by t he i n t r o d u c t i o n of the s y n t h e t i c p r o g e s t i n R5020. T h i s s t e r o i d has a g r e a t e r a f f i n i t y f o r PgR than p r o g e s t e r o n e and p o s s e s s e s l i t t l e a t t r a c t i o n f o r o t h e r b i n d i n g p r o t e i n s ( 1 1 5 ) . 66 N o n - s p e c i f i c b i n d i n g was a s s e s s ed by i n c l u d i n g a 500 f o l d ex ce s s o f u n l a b e l e d R5020. U s i n g R5020 and the ER p r o t o c o l , r a t mammary tumour was a s s a y e d . I n c u b a t i o n t ime was 1.5 hours at 0 -4° C. Fou r doses were used - 194 pM, 424 pM, 632 pM and 944 pM. The assay p r o t e i n was 0 .325 mg or 0 .65 mg / mL. Wool f p l o t a n a l y s i s y i e l d e d a PgR of 88 .3 femtomo les pe r mg c y t o s o l p r o t e i n w i t h a Kd o f -12 4 . 6 - 1 0 M, w h i l e a S c a t c h a r d a n a l y s i s gave a PgR of 92 .3 -12 femtomoles/mg c y t o s o l p r o t e i n and a Kd o f 5 . 2 -10 M. The r e s u l t o b t a i n e d p r e v i o u s l y u s i n g a d i f f e r e n t assay system (110) and Woolf p l o t a n a l y s i s was 77 fm/mg CP w i t h a Kd of 3 .0 x 10 " " 1 2 M. T h i s v a l u e r e p r e s e n t s the ave rage o f 12 a s s a y s The K d ' s compared very -12 -12 w e l l , i e . 5x10 mo l e s / L u s i n g my assay vs 3x10 m o l e s / L . Re cep t o r c on t en t ( B m a x ) d e t e r m i n a t i o n s a l s o agreed very w e l l between t he se two assay p r o c e d u r e s ; 88 vs 77 femtomoles / mg t o t a l p r o t e i n r e s p e c t i v e l y . TIME COURSE OF BINDING FOR PGR A s tudy o f optimum i n c u b a t i o n t ime was then u n d e r t a k e n . F o r t h i s pu r po se , p oo l e d human b r e a s t tumour was c ho s en . The tumour c y t o s o l was i n c u b a t e d w i t h t h r e e d i f f e r e n t c o n c e n t r a t i o n s o f l a b e l e d l i g a n d and the i n c u b a t i o n was t e r m i n a t e d at v a r i o u s i n t e r v a l s w i t h t he a d d i t i o n o f DCC. The r e s u l t s a re g i v e n i n t a b l e two, and a re g raphed i n f i g u r e f i v e , i l l u s t r a t i n g t h a t maximum b i n d i n g o c c u r s at 1.5 h o u r s . A f t e r t h a t t h e r e i s a s l i g h t , though not s i g n i f i c a n t d e c r ea se up t o 18 h o u r s . The ER assay pe r fo rmed under the same 67 INCUBATION TIKE (hours) F i g . 5 The e f f e c t of incubation time on progesterone receptor a n a l y s i s . S p e c i f i c Binding (#) Non-specific Binding (^ ) Total Binding 68 c o n d i t i o n s ( see f i g u r e s i x ) shows maximum b i n d i n g at 4 hours w i t h subsequent l o s s on l o n g e r i n c u b a t i o n , though a g a i n t h i s i s not a s i g n i f i c a n t r e d u c t i o n . The 1.5 hour maximum f o r PgR and a 4 hour maximum f o r ER are i n agreement w i t h r e s u l t s o b t a i n e d by o t h e r s (102,105). On the b a s i s of these r e s u l t s an i n c u b a t i o n time of 3 hours was chosen f o r the d u a l - l a b e l assay. Table I I : I n c u b a t i o n Time f o r PgR Time PgR T o t a l NSB S p e c i f i c BI Kd ( h r ) fmol/mg TP fmol/mg TP fmol/mg TP fmol/mg TP % moles/L 1.0 54.2 34 5.5 28.5 84 6.5-10- 1 0 1.5 60.9 37 5.2 31.8 86 6.4-10" 1 0 2.0 51.6 35.2 4.7 30.5 87 4.1-10- 1 0 4.0 42.2 34.7 5.7 29.1 84 1.2-10- 1 0 A n a l y s i s of PgR b i n d i n g over a f o u r hour p e r i o d . BI r e f e r s t o s p e c i f i c bound / t o t a l bound x 100. The above data i s g r a p h i c a l l y r e p r e s e n t e d i n f i g u r e f i v e . I n c u b a t i o n t i m e s f o r the ER assay u s i n g TEMS b u f f e r are r e p r e s e n t e d i n f i g u r e s i x . 69 2'.0 7A> tsTo BTO lo' .O 15.'o INCUBATION TIME ( hours ) F i g . 6 Time course of binding f o r the estrogen receptor assay. The e f f e c t of time on t o t a l binding i n the estrogen receptor assay. 70 OPTIMUM PROTEIN CONCENTRATION FOR PgR Jacobsen (26) n o t i c e d t h a t b i n d i n g of ' 3 H - E 2 d i d not i n c r e a s e l i n e a r i l y w i t h i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n . T h i s i s dubbed the " p r o t e i n e f f e c t " . Because of t h i s , i t i s d e s i r a b l e to d i l u t e each c y t o s o l sample t o the same, opt imum, p r o t e i n c o n c e n t r a t i o n . T h i s optimum p r o t e i n c o n c e n t r a t i o n was found to be 0 .6 - 1.2 mg / ml f o r the ER a s s a y . Fo r t h i s r e a s o n , a p r e - i n c u b a t i o n p r o t e i n d e t e r m i n a t i o n i s made on each samp le , a l l o w i n g d i l u t i o n to the a p p r o p r i a t e p r o t e i n c o n c e n t r a t i o n f o r the a s s a y . The PgR assay was t h e r e f o r e a l s o examined to see i f such an e f f e c t e x i s t e d . Poo l ed human b r e a s t t i s s u e was used w i t h p r o t e i n c o n c e n t r a t i o n s o f 0 .42 - 1.55 mg/ml. Three doses were u sed , 180 pM,450 pM and 975 pM. Tab l e I I I : E f f e c t o f P r o t e i n on PgR P r o t e i n SB NSB PgR BI mg/assay fmol/mg TP fmol/mg TP fmol/mg CP % .210 45 12 .4 100 78 .370 53 11 .9 85 .8 82 .510 45 9 .9 63 .2 82 .700 48 9 .4 63 .8 84 .775 51 9 .6 67 84 Changes i n the v a r i o u s b i n d i n g pa rame te r s o f the p r o g e s t e r o n e r e c e p t o r w i t h a l t e r a t i o n s i n the assay p r o t e i n c o n c e n t r a t i o n .  71 140 Fig. 7 "Protein Effect" on the Progesterone Assay. The effect of protein concentration on progesterone receptor analysis. 72 60" z 40 o 2 20 0.2 0.4 0.6 O.t PROTEIN CONCENTRATION (mg / assay) F i g . 8 " P r o t e i n E f f e c t " and t h e P r o g e s t e r o n e A s s a y . E f f e c t of the a s s a y p r o t e i n c o n c e n t r a t i o n on S p e c i f i c B i n d i n g and (•) N o n - s p e c i f i c B i n d i n g . 73 F i g u r e s seven and e i g h t i l l u s t r a t e the above d a t a . A p o s s i b l e e x p l a n a t i o n f o r t h i s " p r o t e i n e f f e c t " i s t h a t w i t h i n c r e a s i n g amounts o f p r o t e i n , some of t h e se p r o t e i n s , e s p e c i a l l y the n o n - s p e c i f i c p r o t e i n s , b i nd to the w a l l s o f the t e s t tube and t hu s a re u n a v a i l a b l e f o r i n t e r a c t i o n w i t h the l a b e l e d l i g a n d . T h i s same e f f e c t might appear to be p r e s e n t i n the PgR assay when one f i r s t examines f i g u r e s e ven . Here the Woolf p l o t e x t r a p o l a t e d PgR c o n c e n t r a t i o n s a re p l o t t e d a g a i n s t the c o n c e n t r a t i o n of p r o t e i n i n the a s s a y . There appears t o be a dec r ea se i n b i n d i n g up t o 0 .5 mg pe r a s s a y , a f t e r wh ich the b i n d i n g rema ins e s s e n t i a l l y c o n s t a n t . Note however, the r e l a t i v e l y l a r g e r e r r o r a t the l ower p r o t e i n c o n c e n t r a t i o n s . I f the da t a i s p l o t t e d as s p e c i f i c - b i n d i n g and n o n - s p e c i f i c b i n d i n g v s . c o n c e n t r a t i o n of p r o t e i n t h i s appa ren t " e f f e c t " i s d i m i n i s h e d . The da t a i n t a b l e t h r e e r e v e a l s a s l i g h t de c r ea se i n NSB w i t h i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n ove r t he range 0 . 2 - 0 . 5 mg / a s s a y . C o n c e n t r a t i o n s g r e a t e r than 0 .5 mg/assay g i v e the same NSB. The s p e c i f i c b i n d i n g on the o t h e r hand rema ins e s s e n t i a l l y unchanged. These r e s u l t s i n d i c a t e t h a t PgR i s l i n e a r i l y r e l a t e d to p r o t e i n c o n c e n t r a t i o n , wh ich i s i n agreement w i t h r e s u l t s o b t a i n e d by o t h e r wo rke r s ( 1 0 2 ) . . Fo r the combined ER-PgR assay the t a r g e t p r o t e i n c o n c e n t r a t i o n was t hu s chosen as 0 .5 mg/assay t o meet the r e q u i r e m e n t s o f t he ER d e t e r m i n a t i o n . 74 USE OF 125I-ESTRADI0L AS THE RADIOLABELED LIGAND As the f i n a l goal is to determine ER and PgR simultaneously, a radioisotope other than tritium had to be used. The 125 radioisotope chosen was I and the radioligand was thus 125 I - E 2 . This radioligand was f i r s t synthesized by Hochberg in 1979 (16). Other workers (93,95) have used i t sat isfactori ly 3 125 in place of H - E 2 < While I is a gamma emitter, the secondary electrons react with the s c i n t i l l a t i o n cocktail and i t can thus also be counted as a beta emitter. The efficiency is thus considerably reduced, but i t is s t i l l greater than that for 125 tritium, 63.5% vs 40.5% respectively. I _ E"2 o f s P e c i f i c activity 200 mCi/mmole was obtained from New England Nuclear. 3 125 Figure nine i l lustrates the energy profiles of H and I when counted as beta emitters in the Beckman LS 9000. The f i r s t experiments were conducted by counting tritium and 125 I seperately. The energy window for tritium was set at 0-397 keV while that for 1 2 5 I was set at 0-700 keV to accomodate both peaks. A number of experiments were performed using both single tumour samples and lyophylized pooled tumours. The results are given in table four. Examination of this data 125 and figure ten, verif ies that I - E-2 ^ s a n a P P r ° P r i a t e ligand for ER determinations. 75 600 700 800 900 1000 CHANNEL Fig. 9 Energy Profiles of Tritium and -Iodine. Illustrates the complete overlap of the f i r s t iodine peak by tritium. 77 3 125 Tab l e IV : ER D e t e r m i n a t i o n s U s i ng H - E 2 and I - E 2 Sample L a b e l ER ( fmol /mgTP) Kd (mo l a r ) BI (%) l y o p h i l i z e d 3H 191 .5 3.2 x 1 0 " 1 0 99 " 125-1 200 .1 A .5 x 1 0 " 1 0 95 f r o z e n p o o l 3H 80 .9 A .5 x l O - 1 0 95 " 125-1 86 .9 6.2 x 1 0 - 1 0 91 Poo l ed human b r e a s t t i s s u e assayed f o r ER u s i n g i o d i n a t e d and t r i t i a t e d e s t r a d i o l . I n c u b a t i o n t ime was t h r e e h o u r s .  The da t a f o r the l y o p h i l i z e d p o o l i s i l l u s t r a t e d i n f i g u r e t e n . DUAL-LABEL ASSAY FOR STEROID RECEPTOR DETERMINATION 125 Hav ing e s t a b l i s h e d t h a t I - E - 2 ^s a S L J i t a b l e r ep l a cemen t f o r 3 H - E 2 i n d e t e r m i n i n g e s t r o g e n r e c e p t o r c on t en t of human b r e a s t tumours , the f i n a l s t ep was to e s t a b l i s h a method whereby t r i t i u m 125 c oun t s o r i g i n a t i n g form R5020 c o u l d be d i s t i n g u i s h e d f rom I coun t s o r i g i n a t i n g f rom the e s t r a d i o l , when the two r a d i o l i g a n d s a re p r e s en t i n the same samp le . The p rob lem i s w e l l i l l u s t r a t e d i n f i g u r e n i ne where i t i s e v i d e n t t h a t the s i n g l e t r i t i u m peak o v e r l a p s the e n t i r e f i r s t peak o f r a d i o i o d i n e . F o r t u n a t e l y the second peak of r a d i o i o d i n e 78 i s d i s t i n c t f rom the t r i t i u m peak. The f i r s t e xpe r imen t s were s e t up to e s t a b l i s h t h a t the t r i t i u m and r a d i o i o d i n e c o u l d be a c c u r a t e l y de t e rm ined u s i n g the s e t t i n g s d e s c r i b e d . To t h i s end each expe r imen t was conduc ted u s i n g poo l e d l y o p h i l i z e d human b r e a s t tumour t i s s u e wh ich was d i v i d e d i n t o t h r e e equa l p a r t s . One t h i r d of the t i s s u e 3 125 sample was l a b e l e d w i t h on l y H-R5020, one t h i r d w i t h I ~ E -2 125 3 o n l y and the f i n a l t h i r d was l a b e l e d w i t h bo th I and H. I n c u b a t i o n t ime was t h r e e hours at 0-4°C f o r a l l t h r e e . A l l doses were i n t r i p l i c a t e , w i t h the NSB de t e rm ined i n d u p l i c a t e . S i n c e NSB i s l i n e a r w i t h dose on l y one d e t e r m i n a t i o n i s n e c c e s s a r y , f rom which the o t h e r s can then be d e r i v e d . In the d u a l l a b e l assay the NSB f o r e s t r a d i o l and the NSB f o r PgR was de t e rm ined s e p a r a t e l y so t h a t the assay tubes f o r NSB d e t e r m i n a t i o n s f o r e s t r a d i o l c o n t a i n e d on l y 125 3 I - E 2 and DES, w h i l e those f o r PgR c o n t a i n e d on l y H-R5020 and u n l a b e l e d R5020. R e s u l t s f rom t h i s s e r i e s o f e x pe r imen t s i s g i v e n i n t a b l e f i v e . Tab l e V : D u a l - L a b e l and S i n g l e - L a b e l Compar i son C o n d i t i o n PgR ER ( fmo les /mg TP ( fmole/mg TP) 1. s i n g l e - l a b e l assay 51 .7 59 .9 d u a l - l a b e l a ssay 51 .1 59 .3 2. s i n g l e - l a b e l assay 66 .5 87 .4 d u a l - l a b e l a ssay 65 .3 92 .1 Two e xpe r imen t s where ER and PgR were de t e rm ined s e p a r a t e l y ( s i n g l e - l a b e l ) and s i m u l t a n e o u s l y ( d u a l - l a b e l ) f rom the same poo l e d human b r e a s t t i s s u e .  79 DOSE (pM) F i g 11a Estrogen Receptpr Assay using d u a l - l a b e l (•) and s i n g l e - l a b e l (*•) a n a l y s i s . 80 200 400 600 DOSE ( pM) Fig. l i b Progesterone Receptor Assay using dual-label (•) and single-label (-&) analysis. 81 Tab l e f i v e g i v e s examples of r e c e p t o r d e t e r m i n a t i o n s based on AQC and H# m o n i t o r i n g . Manua l c a l c u l a t i o n s based on sample c h a n n e l r a t i o are d e s c r i b e d i n M a t e r i a l s and Methods . F i g u r e s 11a and l i b i l l u s t r a t e the r e s u l t s o b t a i n e d and demons t ra te e x c e l l e n t 125 s e p a r a t i o n o f the t r i t i u m and I c oun t s u s i n g the d e s c r i b e d p r o t o c o l . T h i s was r epea t ed t o c o n f i r m the a c cu r a c y of the method. Fo r t he PgR d u a l - l a b e l vs s i n g l e l a b e l a s say , ( n=12 ) the l i n e a r r e g r e s s i o n c o r r e l a t i o n c o e f f i c i e n t r = 0 . 9 8 . Fo r ER (n=8) r = 0 . 9 9 . T h i s compares f a v o u r a b l e w i t h r e s u l t s o b t a i n e d by o t h e r worke r s u s i n g s i m i l a r methods ( 1 0 6 - 1 0 8 ) . STEROID RECEPTORS IN BREAST CARCINOMA Us i ng the p r o c e d u r e s f o r the d u a l l a b e l assay as i n d i c a t e d a number of o l d e r tumours which had been as sayed p r e v i o u s l y , and f o r wh i ch s u f f i c i e n t r e s i d u a l tumour r ema i ned , were r e - a s s a y e d f o r bo th ER and PgR. The r e s u l t s f rom the d u a l l a b e l assay and the o r i g i n a l ER assay a re g i v e n i n t a b l e s i x . TABLE V I : ER and PgR i n I n d i v i d u a l Tumour Samples Tumour Number PgR ER O r i g i n a l ER Time ( fmo l /mg TP) ( fmol/mg TP) ( fmol/mg TP) ( e l a p s e d mos.) 1443 1.7 0 .8 0 .6 52 1683 1.7 1.7 3 .8 48 1701 120 .1 100 .1 290 48 82 1712 1 5 . 1 . 2 0 . 1 23 .2 48 1822 146 74 .8 70 .2 34 1833 63 .6 52 70 34 2340 12 .8 2 .1 1.7 39 2450 318 .2 ND 230 37 2456 ND ND 5 37 2510 0 .6 ND 31 36 1928 ND 0.6 1.2 45 1934 ND 129 .5 89 . 1 45 1959 619 .3 457 .1 478 .1 44 1961 47 .3 301 .7 219 .6 44 1962 1000 90 163 44 1990 ND 312 .6 279 44 2135 NSB NSB 400 42 2273 ND 432 .5 513 40 2134 121 208 .2 297 42 2136 10 . 1 13 .9 17 .6 42 2140 NSB 0 .7 1.0 42 2172 NSB 1.3 2.0 42 2228 22 . 8 1.5 2 .01 41 2335 5.7 41 . 1 55 .6 39 2380 121 .6 118 132 .6 38 2436 188 ND 253 38 2468 NSB ND 1.4 37 2489 1000 197 168 .5 37 2504 457 .3 45 78 37 83 3398 199 .3 289 233 12 3446 2 .5 10 .5 77 35 3455 1.6 1.0 2.0 23 3466 1.4 1.0 3 .2 23 3503 409 61 .5 63 23 3557 2.5 NSB NSB 22 3582 11 .7 NSB NSB 22 3599 21 .2 65 .3 58 .2 22 3882 NSB NSB 5 18 S imu l t a neou s ( d u a l - l a b e l ) d e t e r m i n a t i o n of ER and PgR f rom s e l e c t e d tumours . The o r i g i n a l assay va l ue f o r ER i s a l s o shown as w e l l as the t ime wh ich has e l a p s e d between the f i r s t d e t e r m i n a t i o n and the d u a l - l a b e l a ssay d e t e r m i n a t i o n .  L i n e a r r e g r e s s i o n a n a l y s i s of the o r i g i n a l ER da t a v e r s u s the r e - a s s a y e d ER da t a y i e l d s a c o r r e l a t i o n c o e f f i c i e n t r of 0 .984 by o r t h o g o n a l r e g r e s s i o n a n a l y s i s . In the p l o t of o r i g i n a l ER vs r e - a s s a y e d ER, the v a r i a b l e s are bo th dependent r a t h e r than i ndependen t vs dependent and f o r t h i s r ea son o r t h o g o n a l r e g r e s s i o n a n a l y s i s was emp loyed . U n l i k e l i n e a r r e g r e s s i o n a n a l y s i s wh i ch d e r i v e s a c o r r e l a t i o n c o e f f i c i e n t based on v e r t i c a l m i n i m a l i z a t i o n , o r t h o g o n a l r e g r e s s i o n i s based on p e r p e n d i c u l a r m i n i m a l i z a t i o n . See a p p e n d i x . F i g u r e 12A i s a s c a t t e r g r a m of t h i s d a t a , wh ich i l l u s t r a t e s t h a t w i t h p r ope r s t o r a g e c o n d i t i o n s ( i e . -70°C) v a l i d ER d e t e r m i n a t i o n s a re p o s s i b l e up to f i v e y e a r s l a t e r . F i g u r e 12B i l l u s t r a t e s ER vs PgR f o r the tumours i n t a b l e s i x . The c o r r e l a t i o n c o e f f i c i e n t was found to be 0 .480 w i t h P=0.01 and y = 1.18x + 6 2 . 7 . O the r worke r s have r e p o r t e d a c o r r e l a t i o n c o e f f i c i e n t r = 0 .394 ( 4 5 ) . 84 1.0 10.0 100.0 ORIGINAL EU (fmoles / mg total protein) Fig. 12a A comparison of re-assay vs original estrogen receptor content of various tumours. (•) 0 - 2 2 months elapsed between assays (#•) 23 - 27 months elapsed between assays (3f») 28 - 37 months elapsed between assays (•) 38 - 47 months elapsed between assays (O) 48 - 54 months elapsed between assays 85 1.0 10.0 100.0 ER (fmoles / rag t o t a l p r o t e i n ) Fig. 12b Progesterone vs Estrogen Receptor Content in older breast tumours assayed for receptor content using the dual-label assay. 86 STEROID RECEPTOR ANALYSIS AND QUALITY CONTROL Fo r the s e r i e s o f e xpe r imen t s j u s t d e s c r i b e d , a sample o f l y o p h i l i z e d poo l ed human b r e a s t tumour t i s s u e was used as a c o n t r o l f o r eve ry r u n . Fo r t he seven s e t s o f e xpe r imen t s c ondu c t e d , the mean was 44 fmoles/mg TP +/ - 8 .6 fmoles/mg TP. PLASMINOGEN ACTIVATOR ACTIVITY DETERMINED FLUOROMETRICALLY Us i ng the method of Ke s sne r and T r o l l (88) as d e s c r i b e d i n M a t e r i a l s and Methods , e xpe r imen t s were de s i gned to i n v e s t i g a t e a s u i t a b l e s t a nda r d c u r v e . As the s u b s t r a t e f o r t h i s method i s s u c c i n y l a t e d p r o t a m i n e , i t was d e c i d ed to use p ro t am ine s u l p h a t e of v a r y i n g c o n c e n t r a t i o n s to gene ra t e a s t a n d a r d c u r v e . C o n c e n t r a t i o n s f rom 0 - 4 0 ug/ml were found to be l i n e a r w i t h abso rbance when p l o t t e d a g a i n s t r e l a t i v e f l u o r e s c e n c e u n i t s . L i n e a r r e g r e s s i o n a n a l y s i s y i e l d e d a c o r r e l a t i o n c o e f f i c i e n t r = 0.996 w i t h y = 2 .1x + 1 .6 . Day to day v a r i a b i l i t y was a c c e p t a b l e , as shown i n t a b l e s e v e n . The g raph of the p r o t am ine s u l p h a t e s t a n d a r d cu r ve i s i l l u s t r a t e d i n f i g u r e 13 . 87 10 20 T 30 "T~ AO PROTAMINE SULPHATE ( u g / ml) Fig. 13 Protamine Sulphate Standard Curve. Standard curve used in the fluorometric micro-assay for plasminogen activator activity determination. 88 TABLE V I I : Day to Day V a r i a t i o n i n F l u o r o m e t r i c Assay p ro tam ine s u l p h a t e RFU RFU S .D . mg/ml 1 2 0 0 .87 1.07 0 .14 5 11 .5 11 .5 0 10 22 24 .5 1.76 15 33 29 . 5 2 .47 20 46 . 5 46 .7 0 .14 30 61 .0 62 .9 1.34 40 67 . 5 _ _ Data i l l u s t r a t e s the i n t e r - a s s a y v a r i a t i o n w i t h the f l u o r o m e t r i c method f o r p l a sm inogen a c t i v a t o r a c t i v i t y d e t e r m i n a t i o n .  The s t a b i l i t y o f the f l u o r e s c e n t p r oduc t was s t u d i e d o ve r a p e r i o d of two and a h a l f h o u r s , to v e r i f y t h a t the p r oduc t i s s t a b l e f o r a t l e a s t two h o u r s . The r e s u l t s a re i l l u s t r a t e d i n f i g u r e 14 . The e f f e c t o f v a r y i n g the c o n c e n t r a t i o n of p r o t e i n on the f l u o r o m e t r i c assay was s t u d i e d . Three d i f f f e r e n t c o n c e n t r a t i o n s of bov i ne serum a l bum in (BSA) were added t o the the a s s a y . The r e s u l t s a re g i v e n i n t a b l e e i g h t . 89 TABLE V I I I : E f f e c t of P r o t e i n on F l u o r o m e t r i c Assay ug/ml p ro tam ine 10 ug p r o t e i n 50 ug p r o t e i n 100 ug p r o t e i n pe r ml per ml per ml RFU RFU RFU 0 11 .5 50 77 10 49 75 20 98 .5 Data i l l u s t r a t e s the i n c r e a s e i n f l u o r e s c e n c e w i t h the a d d i t o n o f bov i ne serum a l b u m i n . The da ta i n t a b l e e i g h t shows t h a t d i f f e r e n c e s i n p r o t e i n c o n c e n t r a t i o n do i ndeed have an e f f e c t on the assay r e s u l t s . The a d d i t i o n of 10 ug of bov i ne serum a l bum in (BSA) to the assay at the 0 l e v e l ( i e . no p ro t am ine s u l p h a t e p r e s e n t ) caused an i n c r e a s e i n f l u o r e s c e n c e f rom 0 .87 to 11 .5 r e l a t i v e f l u o r e s c e n c e u n i t s (RFU) . T h i s i s not s u r p r i s i n g s i n c e the mechanism of the assay i s the r e a c t i o n of f l u r a m w i t h f r e e amino groups to produce a f l u o r e s c e n t p r o d u c t . There seems, however , to be ano the r mechanism a t work h e r e , s i n c e the same amount o f BSA added t o 10 ug / ml p ro t am ine s u l p h a t e does not r e s u l t i n an i n c r e a s e i n RFU of 10 .6 to 3 3 . 6 , but r a t h e r i n a much g r e a t e r i n c r e a s e t o an RFU of 49 . S i m i l a r i l y , a t the 20 ug / ml l e v e l of p ro tam ine s u l p h a t e , the a d d i t i o n of 10 ug of BSA r e s u l t e d i n 90 50 ' 1 0 L lJ <_> cn l i ! 30 CP 1 20 3 D ~ 20 mg/ml p r o t a m i n e s u l p h a t e 10 mg/ml p r o t a m i n e s u l p h a t e 5 mg/ml p r o t a m i n e s u l p h a t e 0 mg/ml p r o t a m i n e s u l p h a t e 1.0 2.0 2.5 TIME (HOURS) F i g - 14 S t a b i l i t y o f the F l u o r e s c e n t E n d - P r o d u c t . E x a m i n a t i o n o f t h e s t a b i l i t y o f t he f l u o r e s c e n t e n d - p r o d u c t o v e r a p e r i o d o f 2.5 h o u r s , a t f o u r d i f f e r e n t l e v e l s o f p r o t a m i n e s u l p h a t e s t a n d a r d . 91 an i n c r e a s e d f l u o r e s c e n c e f rom 46 .6 RFU to 98 .5 RFU. r a t h e r the 57 .2 as would be e x p e c t e d . On the b a s i s o f t he se r e s u l t s i t was d e c i d ed t h a t each c y t o s o l i c sample would have to be d i l u t e d t o the same p r o t e i n c o n c e n t r a t i o n , namely 1 mg/ml . U r o k i n a s e was a l s o used as the b a s i s f o r a s t a nda r d c u r v e . S tock u r o k i n a s e was d i l u t e d i n TEMS b u f f e r t o y i e l d c o n c e n t r a t i o n s per ml of 0 - 150 u U n i t s and was c a r r i e d t h r ough the same s t e p s as the samples t o be a n a l y z e d . Assay t ubes w i t h ou t p l a sm inogen were i n c l u d e d to a l l o w q u a n t i t a t i o n of i n t r i n s i c p r o t e o l y t i c a c t i v i t y . S u c c i n y l a t e d p ro t am ine was o m i t t e d i n ano the r se t of assay t ubes to a l l o w q u a n t i t a t i o n o f d i g e s t i o n of endogenous p r o t e i n s . And f i n a l l y u r o k i n a s e was o m i t t e d t o p e rm i t d e t e c t i o n o f any c o n t a m i n a t i n g p l a s m i n . The g raph f o r the u r o k i n a s e s t a n d a r d cu r ve i s shown i n f i g u r e f i f t e e n . The e q u a t i o n f o r the l i n e i s y= 0.45x + 12 and the l i n e a r r e g r e s s i o n c o r r e l a t i o n c o e f f i c i e n t r = 0 .989 ove r the range 0 -150 uU /m l . One u n i t w i l l a c t i v a t e t h a t amount o f p l a sm inogen wh ich w i l l p roduce a A 2 7 5 o f 1 , 0 p e r m l P e r m i n u t e a t P H 7 - 5 a n d 37°C when measu r i ng p e r c h l o r i c a c i d s o l u b l e p r o d u c t s f rom a - c a s e i n . PLASMINOGEN ACTIVATOR ACTIVITY DETERMINED SPECTROPHOTOMETRICALLY The f i n a l p l a sm inogen a c t i v a t o r assay to be examined was the s p e c t r o p h o t o m e t r i c assay u s i n g the s y n t h e t i c s u b s t r a t e S - 2251 . S t r e p t o k i n a s e was used to gene ra t e a s t a n d a r d c u r v e . The assay p r o cedu r e i s d i s c u s s e d i n d e t a i l i n M a t e r i a l s and Methods . The s t r e p t o k i n a s e s t a n d a r d cu r v e i s graphed i n f i g u r e s i x t e e n . A p l o t of 92 8 0 -60 -AO 20 -I 1 1 1 1 1 1 1 -10 30 50 70 90 110 130 150 UROKINASE (Sigma U n i t s / ml) Fig. 15 Urokinase Standard Curve. An alternate standard curve for the determination of plasminogen activator activity using the fluorescent micro-assay. 93 .400. —I— 120 140 — I-20 40 1 & 100 160 SK IU / ml F i g . 16 Streptokinase Standard Curve for the determination of plasminogen activator act ivi ty using a spectrophotometry assay. o/. abso rbance a t 405 nm v e r s u s s t r e p t o k i n a s e (SK) c o n c e n t r a t i o n was found to be l i n e a r ove r the range 0 - 160 I U / m l . The e q u a t i o n f o r the l i n e i s y = 1.58x + 111 , w i t h r = 0 . 999 . The s t a b i l i t y o f the c o l o u r e d end p r oduc t was checked by r e a d i n g the samples at 0 .5 hours and aga i n at 24 h o u r s . S i x d i f f e r e n t c o n c e n t r a t i o n s o f s t r e p t o k i n a s e were p r e p a r e d . The expe r imen t was pe r f o rmed as u s u a l and the absorbance de t e rm ined t h i r t y m inu te s a f t e r quench ing w i t h a c e t i c a c i d . The tubes were then capped and l e f t o v e r n i g h t . A f t e r t w e n t y - f o u r hours the sample abso rbance was a ga i n c h e c k e d . L i n e a r r e g r e s s i o n a n a l y s i s y i e l d e d a c o r r e l a t i o n c o e f f i c e n t r o f 0 .999 w i t h the e q u a t i o n o f the l i n e be i ng y = 0.98x + 7 . 6 . T - t e s t a n a l y s i s gave a p v a l u e o f 0 . 0 0 1 , f o r n=6. R e p r o d u c i b i l i t y of the s t a n d a r d cu r v e was found to be e x c e l l e n t . Even so , a s t a nda r d cu r v e was run w i t h eve r y ba t ch o f PA a c t i v i t y d e t e r m i n a t i o n s . A poo l ed l y o p h i l i z e d sample of b r e a s t tumour was u t i l i z e d as a c o n t r o l f o r t h i s p l a sm inogen a c t i v a t o r a s s a y . P l a sm inogen a c t i v a t o r a c t i v i t y (PAA) as w e l l as n o n - s p e c i f i c p r o t e a s e a c t i v i t y ( P rA ) was a s s e s s ed p r e - and p o s t - l y o p h i l i z a t i o n . The r e s u l t s a re g i v e n i n t a b l e e l e v e n . 95 TABLE IX : Effect of Lyophilization Pre-Lyophilization Post-Lyophilization Total Abs. (405nm) 264 191 PrA Abs. (405nm) 153 90 Protein mg/ml 3.51 3.2 IU SK/mg protein 19.5 15.6 Plasminogen activator activity and protease activity in a pooled sample of human breast tissue before and after lyophilization u t i l i z i n g the spectrophotometric method.  Lyophilization of the sample did not appear to significantly affect PAA, though i t apparently decreased the non-specific protease activity as i l lustra ted above. Control samples were stored at -70°C for over 6 months, and withdrawn intermittently, whenever the plasminogen activator assay was performed. The PAA of the four pooled samples run during this time period were 25.3 IU/mg protein, 24.5 IU/mg protein, 29.3 IU/mg protein and 26.2 IU/mg protein. The mean value was 26.3 IU/mg protein with a range of 24.5 - 29.3 A control sample lef t at 4°C for one month, showed considerable decline in PAA to 15.3 IU/mg protein. The most dramatic decrease in PAA was seen when a control sample was lef t at room temperature for 48 hours. The remaining activity was only 3.06 IU/mg protein. 96 A s i n g l e r e c o v e r y expe r imen t was a l s o p e r f o rmed , i n wh i ch a sample of known a c t i v i t y was s p i k e d w i t h i n c r e a s i n g amounts o f s t r e p t o k i n a s e . The c a l c u l a t e d r e c o v e r y was 92%. A d i l u t i o n s tudy was a l s o pe r fo rmed u s i n g a sample o f c o n t r o l . R e c o n s t i t u t e d p o o l i n TEMS b u f f e r was made up to a f i n a l c o n c e n t r a t i o n of 2 mg/ml p r o t e i n . The r e s u l t s of t h i s e xpe r imen t a re g i v e n i n t a b l e t e n . The p r e d i c t e d a c t i v i t y v a l u e s a re based on the v a l u e o b t a i n e d f rom the u n d i l u t e d sample o f c o n t r o l . TABLE X : D i l u t i o n Study B u f f e r : P o o l 1 1 1 1 4 3 2 1 P r e d i c t e d A c t i v i t y IU SK/ml 46 .4 43 .5 38 .6 29 A c t u a l A c t i v i t y IU SK/ml 45 . 6 40 .0 35 . 5 30 Based on the r e s u l t s o b t a i n e d the assay would t hus appear to be l i n e a r w i t h d i l u t i o n . Hav ing t h o r o u g h l y s t u d i e d a l l t h r e e methods, i t was d e c i d ed t h a t the chromogen i c assay was t o be used f o r the b r e a s t c an ce r s t u d y . Wh i l e a l l t h r e e methods pe r fo rmed w e l l , the ch romogen i c assay was chosen because o f the g r e a t e r s t a b i l i t y o f the measured end p r oduc t (24 hours vs 2 .5 hou r s f o r the f l u o r o m e t r i c a s s a y ) . A f u r t h e r advan tage was the f a c t t h a t the t e s t samples d i d not have t o be 97 d i l u t e d to the same p r o t e i n c o n c e n t r a t i o n (1 mg/ml f o r the f l u o r o m e t r i c a s s ay ) b e f o r e a s s a y i n g f o r PAA. The g r e a t e r s e n s i t i v i t y o f the f l u o r o m e t r i c method was not found t o be ne ce s sa r y when a s s a y i n g PA a c t i v i t y i n b r e a s t c y t o s o l s . F u r t h e r m o r e , i t s hou l d be noted t h a t w i t h the f l u o r o m e t r i c assay the s u b s t r a t e s u c c i n y l a t e d p ro t am ine has numerous c l e a v age s i t e s . I t can not be assumed t h a t a l l o f t he se c l e a v a g e s i t e s a re c a p a b l e o f f o rm i ng a f l u o r e s c e n t p r o d u c t . Wi th the K a b i s u b s t r a t e S2251 , each c l e a v age r e s u l t s i n the r e l e a s e o f p a r a - n i t r o a n a l i n e and a c o r r e s p o n d i n g i n c r e a s e i n abso rbance a t 405 nm. PLASMINOGEN ACTIVATOR ACTIVITY IN HUMAN BREAST CARCINOMA Us i ng the s p e c t r o p h o m e t r i c method a number of tumours wh ich were a n a l y z e d f o r PgR and ER c o n t e n t , were a l s o a n a l y z e d f o r PAA. The u n d i l u t e d c y t o s o l wh i ch rema ined a f t e r s t e r o i d r e c e p t o r d e t e r m i n a t i o n s , was used f o r PAA a n a l y s i s . The p e l l e t wh ich remained a f t e r r emova l of the s upe r n a t a n t ( c y t o s o l ) was d i s r u p t e d by s o n i c a t i o n and the r e s u l t i n g homogenate was c e n t r i f u g e d . The r e s u l t i n g s upe r n a t a n t ( l y s o s o m a l ) was a l s o a n a l y z e d f o r PAA. The r e s u l t s , by ER number a re g i v e n i n t a b l e e l e v e n . P r o t e i n d e t e r m i n a t i o n s were aga i n done by the method of Lowry ( 2 5 ) . A l l a c t i v i t i e s a re e xp r e s s ed as IU SK/mg p r o t e i n . P r o t e a s e a c t i v i t y was mon i t o r ed f o r each samp le , but as i t was ve ry low i n a lmos t a l l s a m p l e s , v a l u e s a re not g i v e n . I f p r o t e a s e a c t i v i t y was h i g h , i t i s denoted i n the t a b l e as +. 98 TABLE XI : PAA i n B r e a s t Tumour Samples Sample # C y t o s o l i c PAA Lysosoma l PAA IU SK/mg TP IU SK/mg ' 1443 5.4 0 1683 3 .5 93 .2 1701 0 ND 1712 0 ND 1744 155 .6 ND 1833 12 .5 0 2340 93 . 1 > 150 2450 117 ND 2456 94 .7 103 3446 0 2.0 2335 ND 77 .0 3599 44 .3 0 2765 130 .7 > 150 2172 ND 0 2411 95 7.4 3882 0 0 1962 81 .8 0 2380 78 .5 >150 2808 0 0 3398 127 .3 34 .4 99 3767 0 0 2729 29.2 0 2723 0 0 2489 ND 42.4 2468 ND 53 2733 12.8 87 1961 40.6 0 3557 44.8 0 2135 ND 57 2140 ND 31.4 2480 78.5 0 2504 ND 46 2741 49 0 2732 72.5 >150 2273 ND 0 2510 0 ND 1928 0 ND 1934 106.5 ND 1959 74.4 ND 1990 16.8 ND 2134 315 ND 2737 71.4 ND 2738 108.9 ND 2780 0 ND 2811 ND 167 3455 ND ND 3466 68.7 ND 3503 97 ND 100 The r e s u l t s here show c o n s i d e r a b l e v a r i a b i l i t y . In some i n s t a n c e s t h e r e i s c o n s i d e r a b l e a c t i v i t y i n the c y t o s o l and none o r ve ry l i t t l e i n the l y s o s o m a l f r a c t i o n . The r e v e r s e i s a l s o s e en . In the fo rmer c a s e , i t may be t h a t the l y s o s o m a l PA p r ema t u r e l y l e a k e d i n t o the c y t o s o l i c compar tment . A p r e l i m i n a r y s tudy i n d i c a t e d t h a t t h i s i s an u n l i k e l y s e n a r i o , s i n c e i n t e s t s conduc ted on a few tumours b e f o r e s t a r t i n g the major s t u d y , i n d i c a t e d t h a t a c i d phospha tase was not d e t e c t a b l e i n the c y t o s o l a f t e r p r e p a r a t i o n , t hus s u g g e s t i n g t h a t the l y sosomes were s t i l l i n t a c t . However, t h i s s hou l d i d e a l l y have been checked f o r each tumour , t hus p r e c l u d i n g the random oc cu r ance o f a damaged l y s o s o m a l membrane. Ano the r p o s s i b i l i t y i s t h a t d u r i n g the s o n i c a t i o n s t e p , not o n l y the l y s o s o m a l membrane became r u p t u r e d , but the l y s o s o m a l PA may have been damaged as w e l l . In v iew of t he se p rob lems and i n c o n s i d e r a t i o n of the f a c t t h a t t h e r e were a g r e a t e r number of da t a p o i n t s a v a i l a b l e f o r the c y t o s o l i c PA , o n l y the c y t o s o l i c PA was u t i l i z e d f o r the c o r r e l a t i o n s t u d i e s . TABLE X I I : P o l y t r o n vs D ismembrator - T h e i r E f f e c t on ER & PAA ER PAA PrA fmol/mg TP IU SK/mg TP IU SK/mg TP P o l y t r o n 83 25 .3 4 .4 D i smembrator 91 24 .7 10.6 101 T h i s da t a v e r i f i e s t h a t ER and PAA d e t e r m i n a t i o n s do not va ry s i g n i f i c a n t l y based on the d i s r u p t i o n p r o cedu r e c ho s en . W i th the d i smembra to r the p r o t e a s e a c t i v i t y was a p p a r e n t l y g r e a t e r . PLASMINOGEN ACTIVATOR DETERMINATIONS AND QUALITY CONTROL Fo r each and eve ry s e r i e s of PAA d e t e r m i n a t i o n s a s t a n d a r d cu r ve was run at the same t i m e . Compar i son between the v a l u e s f o r the s t r e p t o k i n a s e s t a n d a r d cu r ve o b t a i n e d a f t e r a l a p s e o f e i g h t months shows r ema rkab l e s t a b i l i t y w i t h i n the same l o t number. L i n e a r r e g r e s s i o n a n a l y s i s y i e l d e d a c o r r e l a t i o n c o e f f i c e n t r of 0 . 9 98 . The e q u a t i o n o f the l i n e was y = 0.95x + 4 . 8 . The t - t e s t f o r e q u a l i t y o f the means gave p <0 . 001 f o r n=6. PLASMINOGEN ACTIVATOR ACTIVITY AND STEROID HORMONE RECEPTORS To de te rm ine i f t h e r e was a c o r r e l a t i o n between s t e r o i d r e c e p t o r s and PAA, a l o g - l o g p l o t was c o n s t r u c t e d o f PAA i n IU/mg p r o t e i n v e r su s PgR i n femtomoles/mg t o t a l p r o t e i n and ER i n femtomoles/mg t o t a l p r o t e i n . L i n e a r r e g r e s s i o n a n a l y s i s gave a c o r r e l a t i o n c o e f f i c i e n t r = 0 .51 (p 0 .05) f o r p r o g e s t e r o n e r e c e p t o r , w i t h the e q u a t i o n o f the l i n e be i ng y = 0.7x + 2 9 . 8 . - F o r the e s t r o g e n r e c e p t o r , r=0 .39 ( p = . l ) w i t h y = 0.22x + 3 3 . 2 . ( see f i g u r e s 17 and 18) 102 1.0 10.0 100.0 PgR (fmol.es / mg t o t a l p r o t e i n ) F i g . 17 P l a s m i n o g e n A c t i v a t o r A c t i v i t y compared t o P r o g e s t e r o n e R e c e p t o r C o n t e n t of tumours a n a l y z e d f o r r e c e p t o r c o n t e n t by t h e d u a l - l a b e l method and a c t i v a t o r a c t i v i t y u s i n g the s y n t h e t i c s u b s t r a t e S 2251. 103 F i g . 18 Plasminogen Ac t i v a t o r A c t i v i t y compared to Estrogen Receptor Content of tumours analyzed f o r receptor content by the d u a l - l a b e l method and a c t i v a t o r a c t i v i t y using the synthetic substrate S 2251. 104 PA ACTIVITY AND RECEPTOR CONTENT VS SURVIVAL F o r e i g h t e e n of the p a t i e n t s f o r whom ER, PgR and PAA was d e t e r m i n e d , s u r v i v a l d a t a was o b t a i n a b l e . Three s u r v i v a l c u r v e s were c o n s t r u c t e d f rom t he se t h r e e p a r ame t e r s . F i g u r e 19 compares p a t i e n t s w i t h tumours d e v o i d o f PAA (n=7) w i t h t ho se whose tumours d i d c o n t a i n PAA ( n = l l ) . F i g u r e 20 compares p a t i e n t s whose tumours were p o s i t i v e f o r PgR ( g r e a t e r t han 10 fmoles/mg TP) w i t h t hose whose tumours were n e g a t i v e f o r PgR. And f i n a l l y , f i g u r e 21 compares the s u r v i v a l of p a t i e n t s whose tumours were p o s i t i v e f o r ER ( g r e a t e r than 10 fmoles/mg TP) w i t h t ho se whose tumours were n e g a t i v e f o r ER. The ER v a l u e s i n t h i s case were the r e - a s s a y e d v a l u e s . The s u r v i v a l c u r v e s were gene r a t ed a c c o r d i n g to the p r o d u c t - l i m i t method ( 109 ) , and were compared u s i n g the l o g - r a n k or Man t e l t e s t . Fo r the compa r i s on of the ER+ s u r v i v a l c u r v e w i t h the ER- s u r v i v a l cu r ve ( f i g . 2 1 ) P = 0 . 1 0 . Fo r PgR+ vs PgR- s u r v i v a l ( f i g . 2 0 ) P = 0 . 7 0 , w h i l e t h a t f o r PAA s u r v i v a l a n a l y s i s gave a P = 0 . 6 8 . Wh i l e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s a re not found i n any c a s e , t h e r e i s some i n d i c a t i o n o f a t r e n d i n ER+ vs ER- p a t i e n t s . 105 PA A + "S "a 5 « 50 60 KWIHS AFTER SFGEFV F i g . 19 Survival curves of patients with breast cancer. Analysis of s u r v i v a l on the basis of plasminogen activator a c t i v i t y of the tumour cytosol. 106 100 I I I P g R + 50 P g R -10 20 30 10 50 60 ramfi AFTER SURGERY Fig. 20 Survival curves of patients with breast cancer. Analysis of survival on the basis of progesterone receptor content of the tumour cytosol. 107 50 E R — M 20 5) fa & W '••am AFTER SLW£RY F i g . 21 Survival curves of patients with breast cancer. Analysis of s u r v i v a l on the basis of estrogen receptor content of the tumour cy t o s o l . 108 IV . CONCLUSION U s i n g ^ I - l a b e l e d e s t r a d i o l and ^H - l a be l e d R5020 a d u a l l a b e l method was e s t a b l i s h e d f o r the s imu l t a n eou s d e t e r m i n a t i o n of ER and PgR i n human b r e a s t c a r c i n o m a s . Compar i son of the d u a l - l a b e l assay w i t h the c o n v e n t i o n a l s i n g l e - l a b e l assay demons t r a t e s an e x c e l l e n t c o r r e l a t i o n w i t h r = 0 .98 f o r the PgR assay and r = 0 .99 f o r the ER a s s a y . E x am i na t i o n o f the o r i g i n a l d a t a on ER d e t e r m i n a t i o n s f o r 38 b r e a s t tumours and the r e s u l t s of the d u a l - l a b e l a s se s smen t , i l l u s t r a t e s t h a t the e s t r o g e n r e c e p t o r i s s t a b l e ove r a p e r i o d of a t l e a s t f i v e y e a r s , p r o v i d e d the spec imen i s a p p r o p r i a t e l y s t o r e d . O r t h ogona l r e g r e s s i o n a n a l y s i s o f i n i t a l ER d e t e r m i n a t i o n vs d u a l - l a b e l d e t e r m i n a t i o n a t a l a t e r da te g i v e s r = 0 . 984 . S t e r o i d r e c e p t o r s and PAA were examined to de t e rm ine whether or not t h e r e i s a r e l a t i o n s h i p between t he se p a r ame t e r s . I n 1980 S u t h e r l a n d (78) conduc ted a s i m i l a r e x pe r imen t , but the s t e r o i d r e c e p t o r s were de t e rm ined i n d i v i d u a l l y and PAA was assayed u s i n g 125 an I - f i b r i n p l a t e method. H i s c o r r e l a t i o n c o e f f i c i e n t f o r PAA vs PgR was 0 .42 w h i l e t h a t f o r PAA vs ER was 0 . 3 0 . Two y e a r s l a t e r , Thorsen (79) r e pea t ed t h i s work u s i n g a s i n g l e p o i n t s a t u r a t i o n DCC assay f o r s t e r o i d d e t e r m i n a t i o n s and a ch romogen i c assay u t i l i z i n g S2251 f o r PAA d e t e r m i n a t i o n s . He r e p o r t e d a ve ry s t r o n g c o r r e l a t i o n between these p a r ame t e r s . The Spearman ' s c o e f f i c i e n t o f rank c o r r e l a t i o n r was 0 .71 f o r PgR and 0 .64 f o r 109 ER. Thorsen also reported that PAA discriminates between PgR positive (greater than 5 femtomoles/mg protein) and PgR negative tumours, irrespective of ER content. The results from this study corroborate his findings. Also, a single tumour negative for carcinoma was found to contain no detectable PAA, nor did a tumour histopathologically identified as cystosarcoma phyllodes show any PAA. Unlike the work of Thorsen (79) and Sutherland (78) who determined receptor content and PAA on recently collected breast tumour tissue, this work was carried out on much older tissue samples. While comparison between original assay ER and later ER was possible, there was no data available on the PAA of the samples at the time of original assay. Nevertheless, PAA shows a weak positive correlation with steroid receptor content. This may indicate that when properly stored PAA remains stable just as estrogen receptor appears to. Survival curve analysis of patients on the basis of ER. PgR, and PA did not reveal any s ta t i s t i ca l ly significant differences. However, due to the small sample size i t would not be prudent to dismiss the prognostic potential of these three parameters, simply because of a lack of s t a t i s t i c a l significance. Furthermore, one should bear in mind that this data is based on "re-assayed" values. While the s tabi l i ty of the ER receptor seems assured, there are no data available on the s tabi l i ty of PAA over time periods as great as five years. For PgR, receptor 110 s t a b i l i t y ove r l o n g p e r i o d s has a l s o not been e s t a b l i s h e d . 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Annals of Surg. 197:123 (1983) 122? v,i A P P E N D I X ORTHOGONAL REGRESSION: The s l o p e i s g i v e n as : b = 2M /M y + < / ( m _ m 2 + y r j p - 2 ' " xx yy) xy And the i n t e r c e p t as : a = y -bx Where M = E ( x - x ) ( y - y ) And M = E ( x - x ) 2 , M = E ( y - y ) 2 xx yy The c o r r e l a t i o n c o e f f i c i e n t i s : r = nExy - ( E x ) ( E y ) / nEx 2 - ( E x ) 2 n£y 2 - ( E y ) 2 T - T E S T : t = Y - X / a Y - X where a Y - X = / (0 -D )7n (n-1) ]2'3 

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