Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Isolation of a cell surface antigen specific for human acute myelogenous leukemia cells Shipman, Robert Charles 1982

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A6_7 S49.pdf [ 2.84MB ]
Metadata
JSON: 831-1.0095769.json
JSON-LD: 831-1.0095769-ld.json
RDF/XML (Pretty): 831-1.0095769-rdf.xml
RDF/JSON: 831-1.0095769-rdf.json
Turtle: 831-1.0095769-turtle.txt
N-Triples: 831-1.0095769-rdf-ntriples.txt
Original Record: 831-1.0095769-source.json
Full Text
831-1.0095769-fulltext.txt
Citation
831-1.0095769.ris

Full Text

C-l. ISOLATION OP A CELL SURFACE ANTIGEN SPECIFIC FOR HUMAN ACUTE MYELOGENOUS LEUKEMIA CELLS by ROBERT CHARLES SHIPMAN B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f M i c r o b i o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Au g u s t 1982 0 R o b e r t C h a r l e s Shipman, 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f H\CROB\0LOG-V The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date SEPT. 3 . , 9 8 2 ABSTRACT Methods a r e d e s c r i b e d whereby t h r e e p r o t e i n bands (#1, #2 and #3), a p p a r e n t l y u n i q u e t o c e l l s o f p a t i e n t s w i t h a c u t e myelogenous l e u k e m i a (AML), were i s o l a t e d on n o n - r e d u c i n g p o l y -a c r y l a m i d e g e l s . A n t i s e r a r a i s e d a g a i n s t a l l t h r e e p r o t e i n s d e m o n s t r a t e d a b s o l u t e s p e c i f i c i t y , i n the ELISA, f o r a b s o r b e d c e l l membrane p r e p a r a t i o n s from c e l l s o f p a t i e n t s w i t h AML and d i d not r e a c t w i t h c o m p a r a b l e c e l l membrane p r e p a r a t i o n s from c e l l s o f normal i n d i v i d u a l s or t h o s e of p a t i e n t s w i t h lympho-p r o l i f e r a t i v e d i s o r d e r s . I n h i b i t i o n s t u d i e s showed t h a t AML band#1 (68,000 d a l t o n m o l e c u l a r w e i g h t ) m a t e r i a l was c a p a b l e o f t o t a l l y i n h i b i t i n g the r e a c t i o n , i n the ELISA, o f a l l t h r e e a n t i s e r a (anti-AML#1, #2 and #3) w i t h a b s o r b e d AML c e l l membrane p r e p a r a t i o n s . When AML c e l l membranes were examined i n SDS-PAGE, o n l y m a t e r i a l i d e n t i f i e d as AML band#1 was d e t e c t a b l e , i n d i c a t i n g t h a t AML bands#2 and #3 r e p r e s e n t e d a g g r e g a t e s o f AML band#1 or a r t i f a c t s of n o n - r e d u c i n g PAGE and the c e l l membrane p r e p a r a t i v e p r o c e d u r e s . AML band#1 m a t e r i a l was shown t o be homogenous by t w o - d i m e n s i o n a l g e l e l e c t r o -p h o r e s i s w i t h a m o l e c u l a r w e i g h t o f 68,000 d a l t o n s and a mean p i o f 7.16. AML band#1 m a t e r i a l was a l s o shown t o be c a p a b l e o f i n h i b i t i n g the r e a c t i o n o f anti-AML#1 a n t i s e r u m w i t h the p r o m y e l o c y t i c c e l l l i n e HL-60, as d e m o n s t r a t e d by FACS IV a n a l y s i s , whereas m a t e r i a l f r o m n o r m a l c e l l membranes c o u l d not d e m o n s t r a t e t h i s i n h i b i t i o n . i i i TABLE OF CONTENTS A b s t r a c t i i T a b l e of C o n t e n t s i i i L i s t o f T a b l e s . . i v L i s t o f F i g u r e s v L i s t o f A b b r e v i a t i o n s v i Acknowledgements v i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 9 R e s u l t s 19 D i s c u s s i o n 43 R e f e r e n c e s 46 i v L I S T OF TABLES T a b l e T i t l e Page I. R e s u l t s o f FACS IV a n a l y s i s o f HL-60 c e l l s w i t h a n t i s e r a p r e - i n c u b a t e d w i t h AML band#1, a b s o r b e d normal human c e l l membrane a n t i g e n or PBS p r i o r t o l a b e l l i n g and a n a l y s i s . 38 V L I S T OF FIGURES F i g u r e T i t l e Page 1A. Ontogeny of the c e l l l i n e a g e s i n v o l v e d i n human a c u t e myelogenous l e u k e m i a (AML) and o t h e r h e m a t o p o e t i c stem c e l l d i s o r d e r s . 6 1. ELISA o f i n d i v i d u a l e x t r a c t s of e i t h e r AML or n o rmal human p e r i p h e r a l b l o o d l e u c o c y t e s (PBL) w i t h a b s o r b e d a n t i - A M L a n t i s e r u m . 21 2. ELISA o f e x t r a c t s from AML and v a r i o u s bone marrow c e l l samples w i t h e i t h e r a n t i - A M L or a n t i - n o r m a l human a n t i s e r u m . 23 3. A n a l y t i c a l n o n - r e d u c i n g g e l p a t t e r n from membrane e x t r a c t s o f normal human PBL and b l a s t c e l l s from an AML p a t i e n t . 26 4. SDS-PAGE of a number o f a b s o r b e d membrane e x t r a c t s from a v a r i e t y o f c e l l s o u r c e s . 29 5. S p e c i f i c i t y o f v a r i o u s a n t i - A M L a n t i s e r a f o r a b s o r b e d AML e x t r a c t s i n the ELISA. 32 6. I n h i b i t i o n o f a n t i - A M L a n t i s e r a i n the ELISA w i t h v a r i o u s c o n c e n t r a t i o n s of AML band#1. 34 7. T w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l p r o f i l e o f AML band#1. 36 8 . I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i -n ormal human a n t i s e r a w i t h AML band#1 i n the FACS. 40 9. I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i -n ormal human a n t i s e r a w i t h a b s o r b e d normal human c e l l membrane a n t i g e n i n the FACS. 42 v i L I S T OF ABBREVIATIONS 1. ALL: A c u t e L y m p h o c y t i c Leukemia. 2. AML: A c u t e Myelogenous Leukemia. 3. AMML: A c u t e M o n o m y e l o c y t i c Leukemia. 4. APML: A c u t e P r o m y e l o c y t i c Leukemia. 5. CLL: C h r o n i c L y m p h o c y t i c Leukemia. 6. CML: C h r o n i c Myelogenous Leukemia. 7. DNA: D e o x y r i b o n u c l e i c A c i d . 8. ELISA: E n z y m e - l i n k e d Immunosorbent A s s a y . 9. F A C S ( I V ) : F l u o r e s c e n c e - a c t i v a t e d C e l l S o r t e r . 10. F I T C : F l u o r e s c e i n I s o t h i o c y a n a t e . 11. G-6-PD: G l u c o s e - 6 - p h o s h a t e D e h y d r o g e n a s e . 12. I E F : I s o e l e c t r i c F o c u s s i n g . 13. LAA: L e u k e m i a - a s s o c i a t e d A n t i g e n . 14. 2-ME: 2 - m e r c a p t o e t h a n o l . 15. PAGE: P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s . 16. 2D-PAGE: 2 - d i m e n s i o n a 1 P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s . 17. SDS-PAGE: S o d i u m - D o d e c y l - S u l p h a t e P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s . 18. PBS: P h o s p h a t e B u f f e r e d S a l i n e . 19. PBL: P e r i p h e r a l B l o o d L e u c o c y t e . 20. PCV: P o l y c y t h e m i a V e r a . 21. RNA: R i b o n u c l e i c A c i d . v i i ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my a p p r e c i a t i o n t o the many p e o p l e who " h e l p e d a l o n g the way " and e n a b l e d me t o c o m p l e t e t h i s t h e s i s w i t h a semblance o f s a n i t y s t i l l r e m a i n i n g . F i r s t and f o r e m o s t , Dr. J u l i a Levy f o r p r o v i d i n g o p p o r t u n i t y , s u p p o r t and g u i d a n c e from the b e g i n n i n g o f t h i s p r o j e c t . Thanks t o my f e l l o w g r a d u a t e s t u d e n t s f o r u n d e r s t a n d -i n g as o n l y g r a d u a t e s t u d e n t s c an and s p e c i a l t h a n k s t o Barb Angus and K e i t h S e i f e r t f o r p r o v i d i n g m o r a l (morel) s u p p o r t on the home f r o n t and a b a t i n g the m a e l s t r o m s o f c o n f u s i o n t h a t a r o s e . " . . . . F o r a f t e r w a r d s a man f i n d s p l e a s u r e i n h i s p a i n s , when he has s u f f e r e d l o n g and wandered l o n g . So I w i l l t e l l you what you ask and seek t o know." - Homer. 1 INTRODUCTION In the f i e l d s of tumour immunology and tumour v i r o l o g y t h e r e has been a g r e a t d e a l of s p e c u l a t i o n on the r e l a t e d n e s s of c a r c i n o g e n e s i s and v i r a l o n c o g e n e s i s . There has a l s o been a concommitant i n c r e a s e i n the amount of r e s e a r c h d i r e c t e d toward d e t e r m i n i n g the manner by which oncogenic v i r u s e s b r i n g about n e o p l a s t i c t r a n s f o r m a t i o n of normal c e l l s . Numerous papers i n the l i t e r a t u r e have d e a l t w i t h the i s o l a t i o n and c l o n i n g of DNA sequences from RNA tumour v i r u s e s which cause n e o p l a s t i c t r a n s f o r m a t i o n when used to t r a n s f e c t normal r e c i p i e n t c e l l s ( 1 - 5 ) . Many i n v e s t i g a t o r s have r e p o r t e d , u s i n g c l o n e d t r a n s f o r m i n g sequences as pr o b e s , the presence of normal c e l l u l a r genes homologous to the c l o n e d v i r a l oncogenes i n the DNA of normal, n o n - n e o p l a s t i c c e l l s ( 6 ) . Subsequent s t u d i e s have shown t h a t these normal c e l l u l a r genes are a c t i v e l y t r a n s c r i b e d i n n e o p l a s t i c c e l l s and t r a n s c r i p t i o n a l l y s i l e n t i n normal, n o n - n e o p l a s t i c c e l l s ( 7 ) . Other r e s e a r c h has suggested t h a t the t a r g e t s f o r t r a n s f o r m a t i o n , induced by v i r u s or c h e m i c a l c a r c i n o g e n s , comprise a s m a l l but d i s c r e t e s e t of c e l l u l a r oncogenes which are p r e s e n t , but i n a c t i v e , i n normal, n o n - n e o p l a s t i c c e l l s ( 8 ) . Experiments u s i n g sheared DNA from human neoplasms have demonstrated t h a t the t r a n s f o r m i n g p o t e n t i a l and u l t i m a t e n e o p l a s t i c phenotype are encoded i n the DNA of these n e o p l a s t i c c e l l s ( 9 ) . A d d i t i o n a l f i n d i n g s i n d i c a t e t h a t d i f f e r e n t neoplasms c a r r y d i f f e r e n t t r a n s f o r m i n g sequences 2 s u g g e s t i n g t h a t more t h a n one c e l l u l a r oncogene i s r e s p o n s i b l e f o r the v a r i e t y o f known human neoplasms ( 9 ) . Th e r e i s , a t p r e s e n t , some r e s e a r c h b e i n g d i r e c t e d toward the i s o l a t i o n of the p r o t e i n p r o d u c t s o f t h e s e c e l l u l a r o ncogenes and a l s o toward d e t e r m i n a t i o n o f t h e i r r o l e ( s ) i n the t r a n s f o r m a t i o n p r o c e s s ( 1 0 ) . T h e r e has been a c e r t a i n amount of s p e c u l a t i o n as t o whether or not t h e s e p r o t e i n s r e p r e s e n t gene p r o d u c t s o f the v i r u s e s t h e m s e l v e s or p r o d u c t s o f n o r m a l l y " s i l e n t " c e l l u l a r genes t r a n s c r i p t i o n a l l y a c t i v a t e d , d i r e c t l y or i n d i r e c t l y , by v i r a l i n t e g r a t i o n i n t o the h o s t c e l l DNA ( 1 1 ) . Other r e s e a r c h e r s have a t t e m p t e d t o i s o l a t e t r a n s f o r m a t i o n - s p e c i f i c or i n d u c e d p r o t e i n s from v i r a l l y t r a n s f o r m e d c e l l s . At l e a s t two l a b o r a t o r i e s have had some s u c c e s s but t h e q u e s t i o n o f f u n c t i o n s t i l l r e m a i n s unanswered ( 1 0 ) . The a c c e p t e d view a t the p r e s e n t t i m e i s t h a t t h e s e p r o t e i n s r e p r e s e n t n ormal growth r e g u l a t o r y p r o t e i n s . The i n a p p r o p r i a t e e x p r e s s i o n o f t h e s e p r o t e i n s , p r e c i p i t a t e d by v i r a l p r o m o t e r i n s e r t i o n or an a n a l o g o u s a c t i v a t i o n e v e n t , i n the c e l l c a u s e s an i m b a l a n c e i n the h o s t c e l l f e e d b a c k growth r e g u l a t o r y s y s t e m , the c o n s e q u e n c e s o f t h i s i m b a l a n c e r e p r e s e n t i n g the f i r s t s t e p ( s ) i n the t r a n s f o r m a t i o n p r o c e s s ( 6 ) . In r e g a r d t o human AML (Acute Myelogenous L e u k e m i a ) , t h e i n v o l v e m e n t o f o n c o g e n i c v i r u s e s or c h e m i c a l c a r c i n o g e n s as c a u s a t i v e a g e n t s i n l e u k e m i o g e n e s i s i s u n c l e a r . L i t t l e i s known a b o u t the d i s e a s e i t s e l f , a p a r t from i t s c l i n i c a l m a n i f e s t a t i o n s and p h y s i o l o g i c a l c o n s e q u e n c e s . The m a j o r i t y o f human l e u k e m i a r e s e a r c h has been c o n d u c t e d a t the c l i n i c a l 3 l e v e l and has d e a l t w i t h t h e c h r o n i c l e u k e m i a s r a t h e r t h a n the a c u t e l e u k e m i a s . Some of the r e s e a r c h w i t h human CML ( C h r o n i c Myelogenous Leukemia) has p r o v i d e d some u s e f u l s t a r t i n g p o i n t s f o r AML r e s e a r c h . CML has been shown t o be a c l o n a l l y - d e r i v e d d i s e a s e , i n t h a t c e l l s c h a r a c t e r i s t i c ofCCML a r e d e r i v e d from a s i n g l e stem c e l l i n t h e bone marrow o f the " p r e - l e u k e m i c " i n d i v i d u a l , w hich has become l e u k e m i c due t o t h e a c t i o n o f an e x t r i n s i c f a c t o r ( s ) . The c l o n a l o r i g i n of CML and o t h e r h e m a t o p o e t i c stem c e l l d i s o r d e r s c o r r e l a t e s v e r y w e l l w i t h t h e c u r r e n t t h e o r i e s on the o r i g i n s o f human c a n c e r s , s i n c e i t i s t h o u g h t t h a t c l o n a l d i s e a s e s would o r i g i n a t e from r a r e onco-g e n i c e v e n t s , such as s o m a t i c m u t a t i o n or v i r a l p r o m o t e r i n s e r t i o n n e x t t o a normal c e l l u l a r o ncogene, a c t i n g on a s i n g l e stem c e l l ( 1 2 ) . In CML t h i s a p p e a r s t o be t h e c a s e s i n c e o n l y s p e c i f i c c e l l l i n e a g e s a r e " l e u k e m i c " , shown by the p r e s e n c e o f a c h a r a c t e r i s t i c chromosomal anomaly o f chromosome 1 22 known as the P h i l a d e l p h i a chromosome (Ph ) as w e l l as the p r e s e n c e o f a s i n g l e t y p e of g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (Gd a or Gd b) i n t h e c e l l s o f G-6-PD h e t e r o z y g o t e s w i t h CML (13). 1 A l l c e l l s d e r i v e d from the l e u k e m i c stem c e l l p o s s e s s t h e Ph and the f a c t o r s c o n t r i b u t i n g t o t h e a p p e a r a n c e o f t h i s anomaly a r e most l i k e l y i n v o l v e d i n t h e c a u s e o f the d i s e a s e as w e l l . These f a c t o r s , t h e i r o r i g i n s and t h e i r t a r g e t s a r e s t i l l unknown ( 1 3 ) . The a l t e r a t i o n i n CML o c c u r s i n t h e m y e l o i d stem c e l l and i t i s s u p p o s e d t h a t the r e p l a c e m a n t o f t h e normal stem c e l l s by the l e u k e m i c stem c e l l s u l t i m a t e l y g i v e s r i s e t o a m y e l o i d stem c e l l p o p u l a t i o n exempt from normal growth c o n t r o l 4 mechanisms i n the bone marrow of leukemic i n d i v i d u a l s ( 1 4 , F i g u r e 1 A) . In AML the o v e r a l l p i c t u r e i s l e s s c l e a r . There have been s e v e r a l r e p o r t s i n the - l i t e r a t u r e c o n c e r n i n g l e u k e m i a -a s s o c i a t e d a n t i g e n s (LAAs) and AML i n which the AML-LAA has been d e s c r i b e d as a g l y c o p r o t e i n of 7 5,000-80,000 d a l t o n m o l e c u l a r weight ( 1 5 ) , a g l y c o p r o t e i n of 400,000 d a l t o n m o l e c u l a r weight (16) or c e l l stage dependent p r o t e i n s of 86,000 and 88,000 d a l t o n m o l e c u l a r weights ( 1 7 ) . The a n t i s e r a r a i s e d to these v a r i o u s p r o t e i n s show h i g h s p e c i f i c r e a c t i v i t y w i t h the a n t i g e n i t s e l f and c e l l l i n e s b e a r i n g the a n t i g e n on t h e i r c e l l s u r f a c e . S i g n i f i c a n t c r o s s - r e a c t i v i t y has a l s o been observed between these a n t i s e r a and c e l l s from r e l a t e d (AML, AMML and CML) and n o n - r e l a t e d (ALL, CLL, myelomas and lymphomas) stem c e l l d i s o r d e r s . T h i s spectrum of r e a c t i v i t y p r o b a b l y r e f l e c t s the presence of common stem c e l l a n t i g e n s , d i f f e r e n t i a t i o n a n t i g e n s of common LAAs p r e s e n t on the c e l l s u r f a c e of these m a l i g n a n t c e l l s . A l t h o u g h g e n e t i c s t u d i e s have not r e v e a l e d c h a r a c t e r i s t i c chromosomal anomalies, i n 1 AML analogous to the Ph i n CML, these s t u d i e s have i m p l i e d t h a t d e f i n e d g e n e t i c events are i n t i m a t e l y i n v o l v e d i n c r e a t i n g these anomalies and p r o b a b l y r e p r e s e n t c a u s a t i v e agents f o r the d i s e a s e as w e l l . In DNA t r a n s f e c t i o n e x p e r i m e n t s , u s i n g DNA from the p r o m y e l o c y t i c leukemia c e l l l i n e HL-60, i t has been demonstrated t h a t t h i s DNA i s ca p a b l e of t r a n s f o r m i n g r e c i p i e n t mouse NIH 3T3 c e l l s ( 1 8 ) . T h i s experiment s t r o n g l y s u p p o r t s the c o n t e n t i o n t h a t w i t h i n the 5 F i g u r e 1 A : Ontogeny of the c e l l l i n e a g e s i n v o l v e d i n human acute myelogenous leukemia (AML) and o t h e r hematopoetic stem c e l l d i s o r d e r s . AML: Acute Myelogenous Leukemia. CML: C h r o n i c Myelogenous Leukemia. ALL: Acute Lymphocytic Leukemia. CLL: C h r o n i c Lymphocytic Leukemia. PCV: P o l y c y t h e m i a Vera. AMML/APML: Acute Mono-/Promyelocytic Leukemia. 6 MYELOID STEM CELL GRANULOCYTES (Eosinophils, Basophils, Neutrophils) *AML ' ~ O MULTIPOTENT STEM CELL (Target for CML and AML causative agents) LYMPHOID STEM CELL o o MEGAKARYOCYTES (Platelets) * P l a t e l e t d e f i c i e n c y *CML *AMML/APML *Granuloma ° b ERYTHROCYTES (Red blood c e l l s ) *PCV (Polycythemia Vera) T-LYMPHOCYTES * T - c e l l lymphoma *T-ALL *T-CLL B-LYMPHOCYTES * B - c e l l lymphoma *B-ALL *B-CLL Disease states denoted by an a s t e r i s k (*). DNA of l e u k e m i c c e l l s r e s i d e s a g e n e ( s ) r e s p o n s i b l e f o r t h e o b s e r v e d t r a n s f o r m i n g a b i l i t y and f i n a l n e o p l a s t i c p h e n o t y p e of t h e s e c e l l s . A t the p r e s e n t t i m e , we have i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d what a p p e a r s t o be an A M L - s p e c i f i c c e l l s u r f a c e a n t i g e n (19,20) and have a l s o p r o d u c e d a h i g h t i t r e a n t i s e r u m t o t h i s marker i n r a b b i t s ( 2 0 ) . When t h i s a n t i s e r u m i s t e s t e d i n the ELISA o f FACS IV, i t shows a b s o l u t e s p e c i f i c i t y f o r AML c e l l s or c e l l membrane e x t r a c t s whereas no r e a c t i v i t y i s o b s e r v e d u s i n g c o m p a r a b l e c e l l s or c e l l membrane e x t r a c t s from normal i n d i v i d u a l s ( 2 0 , 2 1 ) . When t e s t e d i n the FACS IV, u s i n g a b r o a d e r p a n e l o f c e l l s a m p l e s , t h i s same a n t i s e r u m shows s p e c i f i c i t y f o r the bone marrow c e l l s and p e r i p h e r a l b l o o d l e u c o c y t e s o f b o t h AML and CML i n d i v i d u a l s . A g a i n , no r e a c t i v i t y i s seen w i t h u n r e l a t e d stem c e l l d i s o r d e r s or normal c e l l samples ( 2 2 ) . An i n t e r e s t i n g and s i g n i f i c a n t f i n d i n g i s t h a t t h i s a n t i s e r u m r e a c t s w i t h the p e r i p h e r a l b l o o d l e u c o c y t e s o f AML i n d i v i d u a l i n c l i n i c a l r e m i s s i o n ( 2 2 ) . T h i s r e s u l t i s s i g n i f i c a n t i n d e m o n s t r a t i n g the p r e s e n c e o f t h i s " m a l i g n a n c y m a r k e r " on the s u r f a c e o f p u t a t i v e l y normal c e l l s . The o b s e r v a t i o n t h a t t h e p r e s e n c e o f t h i s "marker" does not appear t o i n t e r f e r e w i t h the normal d i f f e r e n t i a t i o n o f t h e s e c e l l s i s a l s o i n t e r e s t i n g B o th o f t h e s e r e s u l t s i m p l y t h a t the e v e n t c a u s i n g the e x p r e s s i o n o f the AML-LAA on the c e l l s u r f a c e o f l e u k e m i c and r e m i s s i o n c e l l s does not r e p r e s e n t the o n c o g e n i c e v e n t r e s p o n s i b l e f o r the o n s e t o f l e u k e m i o g e n e s i s . A p a r t from showing t h a t the g e n e ( s ) r e s p o n s i b l e f o r p r o d u c t i o n o f the 8 AML-LAA i s exp r e s s e d i n AML, CML and r e m i s s i o n c e l l s which appear to be d i f f e r e n t i a t i n g n o r m a l l y , these s t u d i e s have shown AML to be a d i s e a s e of c l o n a l o r i g i n i n which the ma l i g n a n t c l o n e i s d e r i v e d from a p l u r i p o t e n t stem c e l l ( 2 2 ) . With our i s o l a t i o n of an AML-LAA and data from tumour v i r o l o g y s u g g e s t i n g the r e l a t e d n e s s between v i r a l t r a n s f o r m a t i o n and the i n d u c t i o n df t r a n s f o r m a t i o n - s p e c i f i c p r o t e i n s , i t i s tempting to r e g a r d the AML-LAA as a pr o d u c t of a v i r a l l y -a c t i v a t e d normal c e l l u l a r oncogene, a pr o d u c t of a recombinant oncogene (a gene composed of recombined normal c e l l u l a r and v i r a l oncogenes) or an a b e r r a n t l y expressed growth f a c t o r or r e c e p t o r . There i s , a t p r e s e n t , no evi d e n c e to support any of the above s u p p o s i t i o n s . S i n c e so l i t t l e i s known about the p r e c i s e c e l l u l a r mechanisms i n v o l v e d i n t r a n s f o r m a t i o n and u l t i m a t e l y what e f f e c t i s e x e r t e d by the oncogene p r o d u c t to b r i n g about t r a n s f o r m a t i o n , q u e s t i o n s r e g a r d i n g the natu r e and f u n c t i o n of the AML-LAA abound. Is the AML-LAA a normal growth r e g u l a t o r y gene(s) p r o d u c t ? Is t h i s gene(s) p r o d u c t overproduced or i n a p p r o p r i a t e l y e x pressed i n leukemic c e l l s ? Does e x p r e s s i o n of the AML-LAA u l t i m a t e l y r e s u l t i n leukemic t r a n s f o r m a t i o n ? Answers to the above q u e s t i o n s are not im m e d i a t e l y a v a i l a b l e and are the s u b j e c t s of ongoing r e s e a r c h . 9 MATERIALS & METHODS P r e p a r a t i o n o f i s o l a t e d c e l l membranes; 7 9 1x10 -10 AML c e l l s or normal PBLs were used t o p r e p a r e c e l l membranes by s o n i c a t i o n o f the c e l l sample f o r 2-3 min, i n 30 sec b u r s t s , on i c e a t a s e t t i n g o f 30% p r o b e i n t e n s i t y on a F i s h e r S c i e n t i f i c S o n i c D i s m e m b r a t o r ( M o d e l 300) f o l l o w i n g f r e e z i n g and t h a w i n g . C e l l d e b r i s was removed by c e n t r i f u g a t i o n of the c e l l sample a t 400xg f o r 10 min. C e l l membranes were p e l l e t e d from the s u p e r n a t e n t s by c e n t r i f u g a t i o n a t 50,000xg f o r 180 min. Membrane p e l l e t s were r e s u s p e n d e d i n m i n i m a l volumes o f PBS ( p h o s p h a t e b u f f e r e d s a l i n e , pH 7.2, 0.01M p h o s p h a t e ) and s o n i c a t e d as d e s c r i b e d above or e x t r a c t e d i n 3.0M KC1 o v e r n i g h t a t 4°C. The c e l l membrane sample was t h e n c e n t r i f u g e d a t 50,000xg f o r 180 min f o l l o w i n g s o n i c a t i o n or KC1 e x t r a c t i o n and d i a l y s i s a g a i n s t PBS. P e l l e t s were r e s u s p e n d e d t o a f i n a l volume o f 5.0 ml i n PBS and the t o t a l p r o t e i n c o n c e n t r a t i o n of b o t h the p e l l e t and s u p e r n a t e n t were d e t e r m i n e d by the method o f Lowry e t a l . ( 2 3 ) . Both the p e l l e t and s u p e r n a t e n t were a l s o t e s t e d f o r r e a c t i v e m a t e r i a l u s i n g s p e c i f i c a n t i s e r u m and the ELISA ( 2 4 ) . Feedback a n t i s e r u m ; A n t i s e r u m was p r e p a r e d a g a i membrane e x t r a c t s . R a b b i t s were as a n t i g e n , i n a 50% e m u l s i o n o n s t a p o o l o f normal PBL c e l l immunized w i t h . t h i s a n t i s e r u m , f F r e u n d ' s c o m p l e t e a d j u v a n t 1 0 i n f o u r d i s t a l i n t r a m u s c u l a r s i t e s i n 250 u l volumes. R a b b i t s were immunized m o n t h l y and> a f t e r a 6-8 week i m m u n i z a t i o n s c h e d u l e , b l e d w e ekly. A c t i v i t y o f t h i s a n t i s e r u m was e s t i m a t e d i n the ELISA ( 2 4 ) . T h i s a n t i s e r u m was used i n t t h e f f e e d b a c k p r o c e d u r e t o r a i s e an a n t i - A M L a n t i s e r u m . T h i s a n t i - A M L a n t i s e r u m was p r o d u c e d u t i l i z i n g t he f e e d b a c k method p r e v i o u s l y used t o r a i s e a n t i s e r u m s p e c i f i c f o r a human b r o n c h i o g e n i c c a r c i n o m a a n t i g e n (25) and a n t i s e r u m t o t u m o u r - a s s o c i a t e d a n t i g e n s i n the murine mastocytoma c e l l l i n e P815 ( 1 9 ) . The p r o c e d u r e was c a r r i e d o u t as f o l l o w s : A n t i - n o r m a l human a n t i s e r u m was r e a c t e d w i t h a 3.0M KC1 e x t r a c t o f AML c e l l membranes i n o r d e r t o d e t e r m i n e o p t i m a l p r o p o r t i o n s f o r immune p r e c i p i t a t i o n . T h i s r a t i o o f a n t i s e r u m and AML c e l l membrane e x t r a c t were mixed t o g e t h e r and a l l o w e d t o s t a n d a t 4°C f o r 24 h, f o l l o w i n g which the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n , and more a n t i - n o r m a l human a n t i s e r u m was added t o the s o l u b l e m i x t u r e t o b r i n g i t t o a f i n a l c o n c e n t r a t i o n o f 1 0 - f o l d e x c e s s of a n t i - n o r m a l human a n t i s e r u m . The m i x t u r e was a l l o w e d t o s t a n d f o r a f u r t h e r 24 h and t h e n c e n t r i f u g e d . The s u p e r n a t e n t , c o n t a i n i n g a n t i - n o r m a l human a n t i b o d i e s , s o l u b l e c o m p l e x e s o f a n t i - n o r m a l a n t i b o d y and no r m a l a n t i g e n i c components of the AML c e l l membrane e x t r a c t and p o s s i b l y the t u m o u r - a s s o c i a t e d a n t i g e n ( s ) o f AML c e l l s , was used t o immunize r a b b i t s . The s o l u b l e m a t e r i a l was p r e c i p i t a t e d from the s u p e r n a t e n t w i t h alum and a d m i n i s t e r e d i n 1.0 ml volumes (0.5 ml i n t r a m u s c u l a r l y and 0.5 ml i n t r a p e r i t o n e a 1 1 y ) t o young a d u l t f e m a l e a l b i n o 11 r a b b i t s . R a b b i t s so immunized were b l e d a f t e r a 4-6 week i n t e r v a l , f o l l o w i n g which t h e y were immunized and b l e d w e e kly. The a n t i s e r u m r e c o v e r e d from t h e s e r a b b i t s was a b s o r b e d t w i c e on immunoadsorbent c o l u m n s , p r e p a r e d from normal PBL membrane e x t r a c t s , p r i o r t o t e s t i n g i n the ELISA. Anti-AML a n t i s e r u m (anti-AML#1 ,#2,#3) : F r a c t i o n s c o n t a i n i n g e l u t e d p r o t e i n , from p r e p a r a t i v e p o l y a c r y l a m i d e g e l s , shown t o have a n t i g e n i c a c t i v i t y i n t h e ELISA were used t o immunize young a d u l t f e m a l e a l b i n o r a b b i t s . The e l u t e d m a t e r i a l was mixed w i t h F r e u n d ' s c o m p l e t e a d j u v a n t i n a 50% e m u l s i o n t o a t o t a l volume o f 1.0 ml and i n j e c t e d i n t r a m u s c u l a r l y i n t o f o u r d i s t a l s i t e s . R a b b i t s were g i v e n a n o t h e r i m m u n i z a t i o n 4 weeks l a t e r and b l e d 1 week a f t e r the s e c o n d i m m u n i z a t i o n . T h i s a n t i s e r u m , f o l l o w i n g t e s t i n g i n t h e ELISA, was f o u n d t o have i n s i g n i f i c a n t l e v e l s o f a n t i - n o r m a l a c t i v i t y and t h e r e f o r e was not a d s o r b e d on immunoadsorbent columns p r e p a r e d w i t h normal c e l l membrane e x t r a c t s . An a n t i -n o r mal a n t i s e r u m was a l s o p r e p a r e d by h y p e r i m m u n i z i n g r a b b i t s w i t h a normal human c e l l membrane p r e p a r a t i o n and b l e e d i n g t h e s e r a b b i t s w e e k l y , f o l l o w i n g s e v e r a l m o n t h l y i m m u n i z a t i o n s . Immunoadsorbents; Immunoadsorbents c o n t a i n i n g a n t i - n o r m a l human a n t i s e r u m or n o rmal c e l l membrane e x t r a c t s were p r e p a r e d as f o l l o w s : A n t i s e r u m r a i s e d i n r a b b i t s t o a p o o l o f normal PBL c e l l membranes or a n t i g e n e x t r a c t e d from t h e s e same normal PBL 1 2 membranes was c o u p l e d t o cy a n o g e n bromide a c t i v a t e d S e p h a r o s e 4B ( P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , Sweden) (26,27). P r i o r to u s e, the immunoadsorbent columns were e q u i l i b r a t e d w i t h b o r a t e b u f f e r e d s a l i n e , pH 8.5. Samples were l a y e r e d o n t o t h e columns and c y c l e d o v e r t w i c e , d u r i n g a 90 min p e r i o d , u s i n g b o r a t e b u f f e r e d s a l i n e . A l l c e l l membrane samples were t r e a t e d i n t h i s manner p r i o r t o t e s t i n g . T h i s t r e a t m e n t was fo u n d t o remove a p p r o x i m a t e l y 90% o f the p r o t e i n p r e s e n t i n the c e l l membrane p r e p a r a t i o n s . The "column p a s s " f r a c t i o n s , p r e s u m a b l y e n r i c h e d f o r t u m o u r - a s s o c i a t e d c e l l membrane components, were used f o r s u b s e q u e n t s t u d i e s and i m m u n i z a t i o n s t o p r o d u c e a d d i t i o n a l a n t i s e r u m . Some a n t i - A M L a n t i s e r u m p r e p a r a t i o n s were c y c l e d o v e r immunoadsorbent columns c o n t a i n i n g i m m o b i l i z e d normal c e l l membrane components. T h i s t r e a t m e n t was f o u n d t o remove the m a j o r i t y o f t h e a n t i - n o r m a l human a c t i v i t y p r e s e n t i n t h e s e a n t i - A M L a n t i s e r a , as d e t e c t e d by the ELISA. The a b s o r b e d a n t i - A M L a n t i s e r a were used f o r f u r t h e r s t u d i e s i n the ELISA and FACS IV. Immunoadsorbent columns were p u r g e d a f t e r each a d s o r p t i o n by washing w i t h 0.1N HC1 f o l l o w e d by e q u i l i b r a t i o n w i t h PBS, pH 7.2. E n z y m e - l i n k e d immunosorbent a s s a y ( E L I S A ) ; A n t i s e r a were a s s a y e d u s i n g the e n z y m e - l i n k e d immuno-s o r b e n t a s s a y (ELISA) f o l l o w i n g a p r e v i o u s l y d e s c r i b e d p r o t o c o l (2 ). B r i e f l y , e l u t e d r e a c t i v e m a t e r i a l from n a t i v e p o y a c r y l a m i d e g e l s or a n t i g e n i n t h e form of a b s o r b e d AML or normal c e l l membrane e x t r a c t s was c o a t e d o n t o ELISA m i c r o t i t r e 13 p l a t e s (Cooke E n g i n e e r i n g , A l e x a n d r i a , Va.) a t 300 ng/ml i n 0.05M c a r b o n a t e b u f f e r , pH 9.6, i n 100 u l volumes f o r 18 h a t 4°C. P l a t e s were washed t h r e e t i m e s w i t h PBS-Tween 20 b u f f e r and r e a c t e d w i t h t h e t e s t a n t i s e r u m f o r 60 min a t room t e m p e a t u r e and washed an a d d i t i o n a l t h r e e t i m e s w i t h PBS-Tween 20 b u f f e r . Sheep a n t i - r a b b i t I g , which had been g l u t a r a l d e h y d e c o u p l e d t o a l k a l i n e p h o s p h a t a s e (Sigma Type V I I ) ( 2 8 ) , was t h e n added t o the ELISA p l a t e s i n 100 u l volumes a t a 1/1500 d i l u t i o n . R e a c t i o n was a l l o w e d t o p r o c e e d a f u r t h e r 90 min a t room t e m p e r a t u r e f o l l o w i n g which the p l a t e s were washed a g a i n w i t h PBS-Tween 20 b u f f e r and d e v e l o p e d f o r a l k a l i n e p h o s p h a t a s e a c t i v i t y w i t h p - n i t r o p h e n y l p h o s p h a t e - N a (Sigma 104) f o r 45 min a t room t e m p e r a t u r e . ELISA p l a t e s were r e a d f o r a b s o r b a n c e a t 405 nm i n a T i t e r . t e k M u l t i s k a n (Flow L a b o r a t o r i e s , I n g l e w o o d , Ca..). i P l a t e s were b l a n k e d a g a i n s t a row o f w e l l s c o n t a i n i n g a n t i g e n a l o n e and a l l t e s t s were ru n a t l e a s t t w i c e and i n t r i p l i c a t e on each o c c a s s i o n . The ELISA was a l s o used t o c a a r y o u t i n h i b i t i o n s t u d i e s w i t h AML band#1 m a t e r i a l . A l l a n t i s e r a (anti-AML#1 , #2 , #3) were n o r m a l i z e d f o r r e a c t i v i t y w i t h t h e a b s o r b e d AML c e l l membrane p r e p a r a t i o n and d i l u t i o n s o f each a n t i s e r a were s e l e c t e d so t h a t a l l y i e l d e d e q u i v a l e n t a b s o r b a n c e s a t 405 nm i n the ELISA ori-.this a n t i g e n a f t e r 45 min i n c u b a t i o n a t room t e m p e r a t u r e . Each a n t i s e r u m was p r e - i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of AML band#1 m a t e r i a l (from 0.0 t o 3.0 ug/ml) f o r 18 h a t 4°C, a f t e r which a l l p r e p a r a t i o n s were t e s t e d f o r r e a c t i v i t y on ELISA p l a t e s which had been p r e - c o a t e d w i t h a b s o r b e d AML c e l l 1 4 membrane e x t r a c t a t 300 ng/ml. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE); E l e c t r o p h o r e s i s was c a r r i e d o u t a c c o r d i n g t o the p r o c e d u r e o f Laemmli ( 2 9 ) , e x c e p t t h a t i n some i n s t a n c e s t h e sodium d o d e c y l s u l p h a t e (SDS) and 2 - m e r c a p t o e t h a n o l (2-ME) were o m i t t e d . Samples o f a b s o r b e d c e l l membrane e x t r a c t s were a p p l i e d to 7.5% p o l y a c r y l a m i d e gel :s, f o r b o t h a n a l y t i c a l and p r e p a r a t i v e p r o c e d u r e s , and e l e c t r o p h o r e s e d a t 70V f o r 5-6 h. P r e p a r a t i v e g e l s were run u s i n g a o n e - t o o t h comb (1.5 mm x 10.0 cm) and l o a d i n g 250-500 ug o f p r o t e i n o n t o the g e l . M o l e c u l a r w e i g h t d e t e r m i n a t i o n s were a c c o m p l i s h e d by c o - e l e c t r o p h o r e s i n g samples w i t h p r o t e i n s t a n d a r d s , c o n t a i n i n g b o v i n e serum a l b u m i n (67,000 d a l t o n ) , o v a l b u m i n (43,000 d a l t o n ) , c a r b o n i c a n h y d r a s e (30,000 d a l t o n ) , t r y p s i n i n h i b i t o r (20,100 d a l t o n ) and l y s o z y m e (14,400 d a l t o n ) . G e l s were s t a i n e d w i t h e i t h e r C o omassie B l u e , a c c o r d i n g t o the p r o c e d u r e o f F a i r b a n k s e t a l . ( 3 0 ) , or s i l v e r s t a i n e d a c c o r d i n g t o Wray e t a 1. (31) . For p r e p a r a t i v e g e l s a s e c t i o n o f the g e l was removed and s t a i n e d , the r e m a i n d e r b e i n g s t o r e d a t -70°C u n t i l i t was r e q u i r e d f o r s l i c i n g and e l u t i o n o f p r o t e i n . E l u t i o n o f p r o t e i n ; S t a i n e d and u n s t a i n e d p o r t i o n s o f p r e p a r a t i v e g e l s were l i n e d up and i l l u m i n a t e d from below u s i n g a l i g h t box. Bands were c u t from r e g i o n s o f the u n s t a i n e d g e l c o r r e s p o n d i n g t o l o c a t i o n s on the s t a i n e d s e c t i o n o f the g e l shown t o c o n t a i n 1 5 d e t e c t a b l e p r o t e i n . G e l s l i c e s were p l a c e d i n s m a l l , c apped g l a s s t u b e s and c r u s h e d w i t h a s p a t u l a . 2.0 ml o f PBS were added t o each tube and e l u t i o n was e f f e c t e d by e n d - o v e r - e n d m i x i n g i n a Labquake r o t a t o r ( L a b i n d u s t r i e s , B e r k e l e y , Ca.) o a t 4 C f o r 48 h. E l u t e d p r o t e i n was c o l l e c t e d by vacuum f i l t r a t i o n and p r o t e i n c o n c e n t r a t i o n s o f a l l f r a c t i o n s were d e t e r m i n e d u s i n g the method o f Lowry e t a l . ( 2 3 ) . T e s t i n g of the e l u t e d m a t e r i a l f o r a n t i g e n i c a c t i v i t y was a c c o m p l i s h e d by u s i n g a r a b b i t a n t i - A M L a n t i s e r u m (19,20) and t h e ELISA. T w o - d i m e n s i o n a l p o l y a c r y l a m i d e r.gel ^ e l ec t r ophor e s i s (2D-PAGE) ; G e l p u r i f i e d AML band#1 m a t e r i a l was a n a l y s e d on 2D-PAGE a c c o r d i n g t o t h e p r o c e d u r e o f O ' F a r r e l l e t a l . ( 3 5 ) . In b r i e f , a 100 u l sample c o n t a i n i n g 36 u l o f AML band#1 (40.0 u g / m l ) , 2.25 u l o f a 1.0M T r i s - H C l s o l u t i o n , pH 6.8 (0.05M f i n a l c o n e ) , 2.25 u l 2-ME (5% f i n a l c o n e ) , 4.5 u l o f a 20% SDS s o l u t i o n (2% f i n a l c o n e ) , 5 u l o f 40% LKB A m p h o l i n e s (2% f i n a l c o n e ) , 8 u l o f a 10% T r i t o n X-100 s o l u t i o n (8% f i n a l c o n e ) and 55 mg s o l i d u r e a (8M f i n a l cone.) was added t o a s i n g l e i s o e l e c t r i c f o c u s s i n g tube g e l (4% p o l y a c r y l a m i d e , 2 mm x 11.5 cm) and f o c u s s e d a t 400V u n t i l the " v o l t - h o u r s " (V-h) t o t a l l e d between 7,500 and 10,000V-h. A f t e r i s o e l e c t r i c f o c u s s i n g ( I E F ) , t h e tube g e l was e x t r u d e d from the c o a t e d tube by a t t a c h i n g a s h o r t segment of Tygon t u b i n g t o the top o f the tube and u s i n g a 1.0 ml s y r i n g e f i l l e d w i t h d i s t i l l e d water a t the o t h e r end o f t h e t u b i n g t o push the g e l o n t o a c l e a n g l a s s p l a t e . The tube g e l was t h e n p r e p a r e d f o r r u n n i n g i n the s e c o n d d i m e n s i o n 1 6 by e q u i l i b r a t i n g i t w i t h SDS-PAGE r u n n i n g b u f f e r (384mM g l y c i n e , 50mM T r i s - H C l and 0.1% SDS), pH 6.8, and 5% 2-ME f o r a t l e a s t 2 h w i t h g e n t l e a g i t a t i o n on a J u n i o r O r b i t Shaker a t room t e m p e r a t u r e . A f t e r e q u i l i b r a t i o n , the tube g e l was s e t on top of t h e s t a c k i n g g e l bed (3% p o l y a c r y l a m i d e , pH 6.8) and s e c u r e d i n t o p o s i t i o n by a l l o w i n g a 1% a g a r o s e - . s o l u t i o n i n r u n n i n g b u f f e r t o s o l i d i f y o v e r t h e tube g e l - s t a c k i n g g e l c o n t a c t a r e a . The s e c o n d d i m e n s i o n was t h e n r u n under p r e v i o u s l y d e s c r i b e d c o n d i t i o n s ( c f . a b o v e ) , a n d when the r u n was c o m p l e t e d , s t a i n e d a c c o r d i n g t o the p r o c e d u r e o f Wray e t a l . ( 3 1 ) . M o l e c u l a r w e i g h t s t a n d a r d s were i n c l u d e d i n the s e c o n d d i m e n s i o n by f o r m i n g a w e l l i n the 1% a g a r o s e o v e r l a y u s i n g a p l a s t i c w e l l -f o r m e r (5 mm x 1.5 mm ;x 3.0 cm). The w e l l - f o r m e r was removed when the a g a r o s e o v e r l a y had s o l i d i f i e d and m o l e c u l a r w e i g h t p r o t e i n s t a n d a r d s were added b e f o r e the r u n n i n g o f the s e c o n d d i m a n s i o n commenced. C e l l l i n e ; The human p r o m y e l o c y t i c c e l l l i n e HL-60 was o b t a i n e d from Dr. R.C. G a l l o , N.C.I., B e t h e s d a , Md. I t was m a i n t a i n e d i n RPMI 1640 s u p p l e m e n t e d w i t h 10% f e t a l c a l f serum i n a h u m i d i f i e d 37°C i n c u b a t o r i n 10% CO.,. C e l l s were h a r v e s t e d when the c e l l d e n s i t y r e a c h e d 1 x 10^/ml, washed i n s e r u m - f r e e medium p r i o r t o c e l l membrane p r e p a r a t i o n or washed i n serum-c o n t a i n i n g medium and l a b e l l e d f o r FACS IV a n a l y s i s , - a s d e s c r i b e d below. 1 7 F l u o r e s c e n t a n t i b o d i e s ; F l u o r e s c e i n - l a b e l l e d D E A E - p u r i f i e d g o a t a n t i - r a b b i t IgG was p r e p a r e d a c c o r d i n g t o a s t a n d a r d p r o c e d u r e ( 3 2 ) . B r i e f l y , the g o a t a n t i b o d y (10 mg/ml) was d i a l y z e d a g a i n s t 0.15M N a C l , a f t e r which i t was d i a l y z e d f o r 5 h a g a i n s t 0.5M b i c a r b o n a t e b u f f e r e d s a l i n e , pH 8.5, and f i n a l l y a g a i n s t 0.05M b i c a r b o n a t e b u f f e r e d s a l i n e , pH 9.2. The r e a c t i o n was t e r m i n a t e d by d i a l y s i s a g a i n s t 0.02M PBS, pH 7.2, a t 4°C f o r 4 h. Unbound f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) was removed from F I T C - I g G c o n j u g a t e s by p a s s a g e o f t h e m a t e r i a l o v e r Sephadex G-25 ( P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a ^ Sweden). A r a t i o o f f l u o r o c h r o m e t o p r o t e i n (F:P) of^r4 was d e t e r m i n e d by s p e c t r o p h o t o m e t r i c a n a l y s i s ( 3 3 ) . C e l l l a b e l l i n g ; HL-60 c e l l s were h a r v e s t e d and washed t w i c e i n PBS c o n t a i n i n g 5% f e t a l c a l f serum, f o l l o w i n g which 1 x 10 c e l l s were i n c u b a t e d f o r 1.5 h on i c e w i t h 0.2 ml o f a n t i s e r u m (normal r a b b i t serum, a n t i - A M L or a n t i - n o r m a l human a n t i s e r u m ) d i l u t e d t o 1/10 i n PBS, pH 7.2. A l l a n t i s e r a were c e n t r i f u g e d b e f o r e use t o e l i m i n a t e a g g r e g a t e s . C e l l s were washed t h r e e t i m e s i n PBS and 0.2 ml o f f l u o r e s c e i n a t e d g o a t a n t i - r a b b i t IgG a t 1/20 was added t o each c e l l p e l l e t . C e l l s were i n c u b a t e d a f u r t h e r 1.5 h on i c e , washed once i n PBS, and t h e n c e n t r i f u g e d t h r o u g h 100% f e t a l c a l f serum. The c e l l s were f i n a l l y s u s p ended i n 1.0 ml PBS c o n t a i n i n g 5% f e t a l c a l f serum f o r FACS IV a n a l y s i s . 1 8 F l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r (FACS) IV a n a l y s i s ; T w e n t y - f i v e t h o u s a n d c e l l s from each c e l l sample were a n a l y z e d on a B e c t o n D i c k i n s o n FACS IV u s i n g the 488 nm wave-l e n g t h o f the S p e c t r a P h y s i c s Model 164-05 a r g o n l a s e r a t a power s e t t i n g o f 400 mW. The s t a n d a r d f i l t e r f o r FITC a n a l y s i s was used ( 5 2 0 - l o n g p a s s f i l t e r ) . The FACS IV was s t a n d a r d i z e d u s i n g g l u t a r a l d e h y d e - f i x e d c h i c k e n r e d b l o o d c e l l s (34) and f l u o r e s c e n t m o n o d i s p e r s e d c a r b o x y m e t h y l a t e d m i c r o s p h e r e s (d = 1.75 um 0.02 SD; P o l y s c i e n c e s I n c . , W a r r i n g t o n , P a . ) . A l l a n t i - A M L a n t i s e r a were t i t r a t e d a g a i n s t the HL-60 c e l l l i n e i n the FACS IV and compared w i t h the r e a c t i v i t y o f the p o s i t i v e c o n t r o l ( a n t i - n o r m a l human) a n t i s e r u m . A l l a n t i - A M L a n t i s e r a showed a p p r o x i m a t e l y the same r e a c t i v i t y as the p o s i t i v e c o n t r o l a n t i s e r u m on the HL-60 c e l l s and as a r e s u l t , i n i n h i b i t i o n s t u d i e s , o n l y anti-AML#1 a n t i s e r u m was u s e d . Both t e s t a n t i s e r u m (anti-AML#1) and c o n t r o l a n t i s e r a ( normal r a b b i t serum and a n t i - n o r m a l human) were a b s o r b e d w i t h AML band#1 m a t e r i a l f o r 18 h a t 4°C w i t h the a n t i s e r a d i l u t e d 1/10 and t h e i n h i b i t i n g a n t i g e n a t 40.0 ug/ml. A n t i s e r a were s u b s e q u e n t l y t e s t e d on HL-60 i n "..'the FACS IV. These i n h i b i t i o n a s s a y s , u s i n g the same a n t i s e r a , were r e p e a t e d u s i n g a b s o r b e d normal human c e l l membrane e x t r a c t s as w e l l . 1 9 RESULTS A n t i s e r u m was p r e p a r e d a g a i n s t AML, u s i n g the a n t i b o d y f e e d b a c k p r o c e d u r e , and t e s t e d i n the ELISA a g a i n s t b o t h AML and normal human c e l l m e m b r a n e - e x t r a c t s . R e p r e s e n t a t i v e r e s u l t s a r e shown i n F i g u r e 1, i n which KC1 e x t r a c t s o f 16 AML and 12 no r m a l c e l l samples were a s s a y e d i n the ELISA u s i n g a b s o r b e d a n t i - A M L a n t i s e r u m . These r e s u l t s show t h a t the a n t i - A M L a n t i -serum r e a c t e d w i t h a l l 16 AML c e l l e x t r a c t s whereas no e q u i v a l e n t r e a c t i v i t y was o b s e r v e d w i t h any o f the 12 normal c e l l e x t r a c t s . These r e s u l t s a r e a l s o i n d i c a t i v e o f the f a c t t h a t t h i s a n t i s e r u m i s r e c o g n i z i n g an a n t i g e n common t o AML c e l l s w h ich i s not d e t e c t a b l e i n normal c e l l e x t r a c t s , a t l e a s t i n t h i s a s s a y p r o c e d u r e . The p o s s i b i l i t y t h a t t h i s a n t i -serum was d e t e c t i n g a b l a s t c e l l a n t i g e n or a d i f f e r e n t i a t i o n a n t i g e n not f o u n d on l y m p h o i d c e l l s was i n v e s t i g a t e d by e x a m i n i n g e x t r a c t s o f bone marrow c e l l s , on the a s s u m p t i o n t h a t t h i s c e l l p o p u l a t i o n would c o n t a i n b l a s t c e l l s o f the m y e l o i d c e l l l i n e a g e . The r e s u l t s shown i n F i g u r e 2 i n d i c a t e t h a t t h e a n t i g e n i s not p r e s e n t i n d e t e c t a b l e l e v e l s i n e i t h e r c e l l p o p u l a t i o n , r e g a r d l e s s o f whether t h e bone marrow sample came from c l i n i n c a l l y normal i n d i v i d u a l s or t h o s e w i t h v a r i o u s h e m a t o p o e t i c stem c e l l d i s o r d e r s . In s u b s e q u e n t s t u d i e s , s o n i c a t e d c e l l membrane p r e p a r a t i o n s o f a p o o l o f normal human w h i t e b l o o d c e l l membranes and an e q u i v a l e n t p r e p a r a t i o n o f AML b l a s t c e l l s were u s e d . A f t e r 20 F i g u r e 1: ELISA o f i n d i v i d u a l e x t r a c t s o f e i t h e r AML or n ormal p e r i p h e r a l b l o o d l e u c o c y t e s (PBL) w i t h a b s o r b e d a n t i -AML a n t i s e r u m . AML c e l l e x t r a c t s : (O). Normal c e l l e x t r a c t s : ( A ) . Anti-AML a n t i s e r u m a t 1:100 d i l u t i o n . 2 1 0.3 1.0 3.0 ANTIGEN CONCENTRATION u g ^ m ! 22 F i g u r e 2: ELISA o f e x t r a c t s from AML and v a r i o u s bone marrow c e l l samples w i t h e i t h e r a n t i - A M L or a n t i - n o r m a l human a n t i -serum. a) A n t i g e n p r e p a r a t i o n s from c e l l e x t r a c t s o f two p a t i e n t s w i t h AML. b) I n d i v i d u a l bone marrow c e l l e x t r a c t s from p a t i e n t s w i t h a v a r i e t y o f h e m a t o p o e t i c stem c e l l d i s o r d e r s as f o l l o w s : 1. Normal bone marrow. 2. Lymphoma p a t i e n t w i t h m a l i g n a n t c e l l i n f i l t r a t i o n i n the bone marrow. 3. M u l t i p l e myeloma p a t i e n t w i t h m a l i g n a n t c e l l i n f i l t r a t i o n i n the bone marrow. 4. Lymphoma p a t i e n t w i t h m i n i m a l m a l i g n a n t c e l l i n f i l t r a t i o n i n the bone marrow. 5. A c u t e l y m p h o c y t i c l e u k e m i a p a t i e n t w i t h m a l i g n a n t c e l l i n f i l t r a t i o n i n t h e bone marrow. 6. Normal bone marrow. A l l c e l l e x t r a c t s were used a t 3.0 ug/ml. Anti-AML and a n t i - n o r m a l human a n t i s e r a were used a t 1:100 d i 1 u t i o n ; A nti-AML a n t i s e r u m : ) • A n t i - n o r m a l human a n t i s e r u m : 2 . 0 , i.ok O 0 ^ o o o G O O O D D O O O a a a a o a a a a a a a o o o o o o o o a a o a a • o o a o • • o • a • o o o a • a o o o o o o a a a o a a o a • a a a • a a a a a o a o o o o o o o O D D o a a o o o a a a o o o 0 o • a o • • o • o o o o o o O • D O O O - E l l . A M I 0 2 3 4 5 BONE MARROW'* 24 a b s o r p t i o n o f t h e s e c e l l m e m b r a n e e x t r a c t s o n a n i m m u n o a d s o r b e n t c o l u m n , c o n t a i n i n g a n t i - n o r m a l h u m a n a n t i s e r u m , a n d r u n n i n g o n a 7 . 5 % p o l y a c r y l a m i d e g e l , u n d e r n o n - r e d u c i n g c o n d i t i o n s , a b a n d p a t t e r n e m e r g e d i n w h i c h t h e n o r m a l c e l l m e m b r a n e p r e p a r a t i o n d i s p l a y e d a s i n g l e p r o t e i n b a n d a n d t h e AML c e l l m e m b r a n e p r e p a r a t i o n s h o w e d t h e p r e s e n c e o f 4 u n i q u e p r o t e i n b a n d s ( F i g . 3 ) . P r e p a r a t i v e g e l s w e r e r u n o n t h e AML c e l l m e m b r a n e s a m p l e a n d e a c h o f t h e f o u r p r o t e i n b a n d s w e r e e l u t e d a n d e x a m i n e d f o r r e a c t i v i t y w i t h t h e f e e d b a c k a n t i - A M L a n t i s e r u m i n t h e . '„ -. E L I S A , ( 1 9 ) . T h i s e x a m i n a t i o n s h o w e d AML b a n d s # 1 , # 2 a n d #3 t o be r e a c t i v e w h e r e a s AML b a n d # 4 s h o w e d no s i g n i f i c a n t r e a c t i v i t y a b o v e b a c k g r o u n d . C o n s e q u e n t l y , -AML b a n d # 3 m a t e r i a l was u s e d f o r p r e l i m i n a r y s t u d i e s a n d m a t e r i a l e l u t e d f r o m p r e p a r a t i v e g e l s was u s e d t o r a i s e s p e c i f i c a n t i s e r u m i n r a b b i t s . T h i s a n t i s e r u m ( a n t i - A M L # 3 ) s h o w e d e s s e n t i a l l y a b s o l u t e s p e c i f i c i t y i n r t h e E L I S A f o r c e l l e x t r a c t s f r o m p a t i e n t s w i t h m y e l o -p r o l i f e r a t i v e d i s e a s e a n d d i d n o t r e a c t w i t h e q u i v a l e n t c e l l e x t r a c t s f r o m i n d i v i d u a l s w i t h n o a p p a r e n t d i s e a s e o r w i t h l y m p h o p r o l i f e r a t i v e d i s o r d e r s . F u r t h e r w o r k , u s i n g t h i s a n t i -AML a n t i s e r u m w i t h b o n e m a r r o w c e l l s f r o m a v a r i e t y o f p a t i e n t s i n t h e F A C S I V s h o w e d , s i m i l a r l y , t h a t o n l y c e l l s o f p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e d i s e a s e f l u o r e s c e d i n t h e p r e s e n c e o f t h i s a n t i s e r u m ( 2 1 ) . T h e a n t i - A M L # 3 a n t i s e r u m was a l s o f o u n d t o r e a c t s t r o n g l y w i t h AML b a n d s # 1 a n d # 2 , e l u t e d f r o m t h e same p r e p a r a t i v e g e l ( 2 0 ) . T h i s i n d i c a t e d t h a t AML b a n d s # 1 , # 2 a n d #3, a t l e a s t , s h a r e d common a n t i g e n i c s i t e s a n d t h a t t h i s a n t i g e n i c i d e n t i t y F i g u r e 3 e x t r a c t s pa t i e n t . A n a l y t i c a l n o n - r e d u c i n g g e l of n o rmal human PBL and b l a s t p a t t e r n from membrane c e l l s from an AML l a n e a: M o l e c u l a r w e i g h t s t a n d a r d s . l a n e b: Normal human PBL membrane e x t r a c t . l a n e c : AML b l a s t c e l l membrane e x t r a c t . B oth c e l l membrane p r e p a r a t i o n s had been a b s o r b e d t w i c e on an immunoadsorbent column c o n t a i n i n g a n t i - n o r m a l human a n t i b o d y , p r i o r t o PAGE. G e l s t a i n e d i n Coomassie B l u e a c c o r d i n g t o F a i r b a n k s e t a l . (1971). 67.000 • 43.000 • 30.000 • 20,100 • 27 m i ght be r e f l e c t e d i n s t r u c t u r a l s i m i 1 i a r i t i e s as w e l l . Because of t h e c o n s i d e r a b l e c r o s s - r e a c t i v i t y between AML bands#1,#2 and #3, t h e p o s s i b i l i t y t h a t AML bands#2 and#3 m i g h t r e p r e s e n t a g g r e g a t e d forms o f t h e l o w e s t m o l e c u l a r w e i g h t c o n s t i t u e n t (AML band#1) was c o n s i d e r e d . In o r d e r t o d e t e r m i n e whether s i m i l a r g e l band p a t t e r n s would d e v e l o p when e x t r a c t s from o t h e r AML c e l l membrane p r e p a r a t i o n s were r u n , or i f a n a l o g o u s p r o t e i n bands would be s e e n i n c e l l membrane p r e p a r a t i o n s from p a t i e n t s w i t h l y m p h o p r o l i f e r a t i v e d i s o r d e r s , a p a n e l o f c e l l membrane samples were p r e p a r e d , a b s o r b e d and r u n on 7.5% SDS-PAGE ( F i g . 4 ) . These g e l s a p p e a r t o c o n f i r m t h a t AML bands#2 and #3, o b s e r v e d o r i g i n a l l y , were p r o b a b l y a r t i f a c t s o f the p r e p a r a t i v e p r o c e d u r e s and r e p r e s e n t e d a g g r e g a t e s o f AML band#1 m a t e r i a l , s i n c e the common f e a t u r e between e x t r a c t s o f c e l l s from p a t i e n t s w i t h AML i s t h e p r e s e n c e o f AML band# 1 . Membrane p r e p a r a t i o n s from normal c e l l s or t h o s e from i n d i v i d u a l s w i t h h e m a t o p o e t i c stem c e l l d i s o r d e r s , o t h e r t h a n AML, do not p o s s e s s t h i s p r o t e i n band. I t i s i n t e r e s t i n g t o note t h a t the APML/AMML ( a c u t e p r o - / m o n o m y e l o c y t i c l e u k e m i a ) c e l l membrane sample c o n t a i n s a band t h a t i s s l i g h t l y l o w e r i n m o l e c u l a r w e i g h t t h a n AML band# 1 . T h i s may r e p r e s e n t an u n m o d i f i e d form of AML band#1 i n t h a t APML/AMML c e l l membrane p r e p a r a t i o n s r e a c t , i n the ELISA, w i t h the anti-AML# 1 a n t i s e r u m , w h i c h may be i n d i c a t i v e o f s t r u c t u r a l s i m i l a r i t i e s . In o r d e r t o t e s t f u r t h e r t h e c o n t e n t i o n t h a t AML bands#2 and #3 r e p r e s e n t a g g r e g a t e s o f AML band # 1 , a n t i s e r a t o a l l t h r e e 28 F i g u r e 4: SDS-PAGE o f a number o f a b s o r b e d membrane e x t r a c t s from a v a r i e t y o f c e l l s o u r c e s . l a n e s a-d: A b s o r b e d membrane e x t r a c t s o f f o u r i n d i v i d u a l AML c e l l s a m p l e s . l a n e e : A b s o r b e d membrane e x t r a c t o f an AMML c e l l sample, l a n e f : M o l e c u l a r w e i g h t s t a n d a r d s . l a n e s g - i : A b s o r b e d membrane e x t r a c t s o f a p r e - C L L , an ALL bone marrow and a normal human b u f f y c o a t c e l l s a m p l e s , r e s p e c t i v e l y . G e l s t a i n e d u s i n g the s i l v e r s t a i n i n g method o f Wray e t a l . (1981). 29 30 p r o t e i n bands were r a i s e d i n r a b b i t s . The s p e c i f i c i t y o f each a n t i s e r u m was t e s t e d i n the ELISA u s i n g a b s o r b e d AML c e l l membrane samples and an e q u i v a l e n t p r e p a r a t i o n o f normal human w h i t e b l o o d c e l l membranes. R e s u l t s ( F i g . 5 ) show t h a t a l l the a n t i s e r a r e a c t s p e c i f i c a l l y w i t h t h e AML c e l l membrane sample and do not r e a c t w i t h the normal human p r e p a r a t i o n . In o r d e r t o i n v e s t i g a t e the p o s s i b i l i t y t h a t each a n t i s e r u m was d i r e c t e d to s i m i l a r , i f not i d e n t i c a l , a n t i g e n s , a l l p r e s e n t i n AML band #1, i n h i b i t i o n a s s a y s were p e r f o r m e d u s i n g AML band#1 m a t e r i a l as t h e i n h i b i t o r . As shown i n F i g u r e 6, a l l the a n t i s e r a were 100% i n h i b i t e d by the same c o n c e n t r a t i o n o f AML band#1 (about 1.0 u g / m l ) . T h i s r e s u l t s u p p o r t s the c o n t e n t i o n t h a t a l l t h r e e a n t i s e r a a r e d i r e c t e d t o the same a n t i g e n i c d e t e r m i n a n t s and t h a t the t h r e e p r o t e i n bands i n the AML samples s h a r e a n t i g e n i c and p o s s i b l y s t r u c t u r a l i d e n t i t y . S i n c e AML band#1 i s f o u n d t o be u n i q u e l y p r e s e n t i n a l l p r e p a r a t i o n s t e s t e d from i n d i v i d u a l s w i t h m y e l o p r o l i f e r a t i v e d i s e a s e , t h e c h a r a c t e r and s t r u c t u r e of AML band#1 and i t s o r i g i n become more i m p o r t a n t . In o r d e r t o e s t a b l i s h t h a t AML band#1 c o n s t i t u t e d a s i n g l e component, 2D-PAGE was p e r f o r m e d on a number o f p u r i f i e d AML band#1 p r e p a r a t i o n s from a v a r i e t y o f AML c e l l membrane s a m p l e s . A r e p r e s e n t a t i v e g e l p a t t e r n i s shown i n F i g u r e 7 i n which i t can be s e e n t h a t AML band#1 i s composed o f a s i n g l e p r o t e i n component o f a p p r o x i m a t e l y 68,000 d a l t o n m o l e c u l a r w e i g h t w i t h a mean p i o f 7.16. The " t a i l i n g " or s t r e a k i n g seen i n F i g u r e 7 i s p r o b a b l y due t o d i f f e r e n t i a l g l y c o s y l a t i o n o f t h e AML band #1 m a t e r i a l . 3 1 F i g u r e 5: S p e c i f i c i t y o f v a r i o u s a n t i - A M L a n t i s e r a f o r a b s o r b e d AML e x t r a c t s i n t h e ELISA. The a n t i s e r a were t i t r a t e d on ELISA w e l l s c o n t a i n i n g 300 ng/ml of e i t h e r a b s o r b e d AML a n t i g e n or a b s o r b e d normal human a n t i g e n ; a n t i -AML #1 a n t i s e r u m (on AML a n t i g e n ) ( • ) • II (on n ormal a n t i g e n ) ( A ) a n t i -AML# 2 a n t i s e r u m (on AML a n t i g e n ) ( • ) -II (on normal a n t i g e n ) ( O ) a n t i -AML#3 a n t i s e r u m (on AML a n t i g e n ) ( • ) • II (on normal a n t i g e n ) ( • ) 3 2 - L O G OF D ILUTION 33 F i g u r e 6 : I n h i b i t i o n o f a n t i - A M L a n t i s e r a i n t h e E L I S A w i t h v a r i o u s c o n c e n t r a t i o n s o f AML b a n d#1. A n t i - A M L a n t i s e r a w h i c h h a d b e e n p r e - i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f AML band#1 w e r e t e s t e d f o r r e a c t i v i t y o n a b s o r b e d AML a n t i g e n i n t h e E L I S A . E L I S A w e l l s c o n t a i n e d 3 0 0 n g / m l o f t h e a b s o r b e d AML a n t i g e n . T h e a n t i s e r a a r e a s f o l l o w s ; a n t i - A M L # 1 a n t i s e r u m ( A )• a n t i - A M L # 2 a n t i s e r u m ( • ) . a n t i - A M L # 3 a n t i s e r u m ( • ) . D a t a a r e p r e s e n t e d a s p e r c e n t i n h i b i t i o n . -2.5 -1.5 -0.5 0.0 0.5 (0.003 0.03 0.3 1.0 3.0 u g / m i ) LOG CONC. INHIBITOR 35 F i g u r e 7: T w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l p r o f i l e o f AML band* 1 . l a n e a: M o l e c u l a r w e i g h t s t a n d a r d s . l a n e b: 5 ug o f AML band#1 m a t e r i a l was l o a d e d o n t o an IEF tube g e l c o n t a i n i n g 2% LKB A m p h o l i n e s . The sample was f o c u s s e d a t 400V f o r 24h, a f t e r w hich i t was e x t r u d e d and e q u i l i b r a t e d w i t h SDS-PAGE r u n n i n g b u f f e r . The IEF tube g e l was p l a c e d o n t o a 7.5% PA g e l and e l e c t r o p h o r e s e d a t 70V f o r 5h. The f i n i s h e d g e l was t h e n s i l v e r s t a i n e d a c c o r d i n g t o the method o f Wray e t a l . (1981). The l i n e s seen on the g e l between 67,000 and 55,000 d a l t o n m o l e c u l a r w e i g h t a r e a r t i f a c t s o f the SDS-PAGE p r o c e d u r e . These same l i n e s a p p e a r on 'blank g e l s ' ( g e l s r u n w i t h o u t any p r o t e i n p r e s e n t ) and t h e r e f o r e r e p r e s e n t a r t i f a c t s o f , e i t h e r the SDS-PAGE r u n n i n g c o n d i t i o n s or the s i 1 v e r - s t a i n i n g p r o c e d u r e . 36 37 E a r l i e r s t u d i e s u s i n g the anti-AML#3 a n t i s e r u m on p a t i e n t s ' c e l l s i n the FACS IV have shown t h a t t h i s a n t i s e r u m d e t e c t e d a c e l l s u r f a c e component p r e s e n t o n l y on c e l l s of p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e d i s o r d e r s ( 2 0 ) . T h i s a n t i s e r u m a l s o r e a c t e d w i t h the p r o m y e l o c y t i c l e u k e m i a c e l l l i n e HL-60 i n the FACS IV. S t u d i e s w i t h anti-AML#1 a n t i s e r u m showed t h a t i t a l s o r e a c t e d w i t h HL-60 and a p p e a r e d t o r e a c t w i t h p a t i e n t s ' c e l l s i n a manner i d e n t i c a l t o t h e anti-AML#3 a n t i s e r u m . To e s t a b l i s h t h a t the m a t e r i a l w i t h which anti-AML#1 was r e a c t i n g on HL-60 was i n d e e d AML band#1, an i n h i b i t i o n a s s a y i n t h e FACS IV was c a r r i e d o u t . I t can be seen t h a t AML band#1 m a t e r i a l s u c c e s s f u l l y b l o c k s the r e a c t i o n of anti-AML#1 a n t i s e r u m w i t h HL-60 c e l l s ( F i g . 8 a and T a b l e I ) . C o n v e r s e l y , normal human c e l l membrane p r e p a r a t i o n s s u c c e s s f u l l y b l o c k the r e a c t i v i t y o f the a n t i -n o r mal human a n t i s e r u m w i t h HL-60 ( F i g . 9 b and T a b l e I ) . The p a r t i a l i n h i b i t i o n o f b o t h the a n t i - n o r m a l human a n t i s e r u m by AML band#1 ( F i g . 8 b and T a b l e I) and the anti-AML#1 a n t i s e r u m by the normal human c e l l membrane p r e p a r a t i o n ( F i g . 9 a and T a b l e I) can be a c c o u n t e d f o r by the p o s s i b i l i t y t h a t AML band #1 i s c o m p r i s e d o f b o t h l e u k e m i a - a s s o c i a t e d and normal c e l l u l a r components. The p r e s e n c e o f b o t h ' l e u k e m i c ' and 'no r m a l ' c e l l u l a r m o i e t i e s t o g e t h e r on t h e same m o l e c u l e most p r o b a b l y a r i s e s as a r e s u l t o f ' l e u k e m i c - t r a n s f o r m a t i o n ' o f normal h e m a t o p o e t i c stem c e l l ( s ) . 38 T a b l e 1. R e s u l t s o f FACS IV a n a l y s i s o f HL-60 c e l l s w i t h a n t i s e r a p r e - i n c u b a t e d w i t h AML band#1, a b s o r b e d normal human c e l l membrane a n t i g e n or PBS p r i o r t o l a b e l l i n g and a n a l y s i s . Number o f C e l l s A n t i s e r u m I n h i b i t o r F l u o r e s c i n g Normal r a b b i t serum - 3787 " normal a n t i g e n 4127 AML band#1 34 8 5 A n t i - n o r m a l human - 22,392 " n o r m a l a n t i g e n 3560 AML band#1 20,927 Anti-AML #1 - 13,175 " normal a n t i g e n 13,370 AML band#1 4547 39 F i g u r e 8: I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i - n o r m a l human a n t i s e r a w i t h AML band#1 i n the FACS. p a n e l a: I n h i b i t i o n o f anti-AML#1 by AML band#1 m a t e r i a l i n the FACS IV u s i n g HL-60 c e l l s . The upper d i s t r i b u t i o n p a t t e r n shows the f l u o r e s c e n c e o f HL-60 c e l l s w i t h anti-AML#1 a n t i s e r u m . The lo w e r d i s t r i b u t i o n p a t t e r n shows HL-60 c e l l s w i t h anti-AML#1 t h a t has been p r e -i n c u b a t e d w i t h AML band#1. p a n e l b: F a i l u r e o f AML band#1 t o i n h i b i t t h e f l u o r e s c e n c e o f HL-60 c e l l s r e a c t e d w i t h a n t i - n o r m a l human a n t i s e r u m . The upper d i s t r i b u t i o n p a t t e r n shows the f l u o r e s e n c e of HL-60 c e l l s w i t h the a n t i - n o r m a l human a n t i s e r u m . The l o w e r d i s t r i b u t i o n p a t t e r n shows HL-60 c e l l s w i t h a n t i - n o r m a l human a n t i s e r u m t h a t has been p r e -i n c u b a t e d w i t h AML band#1. 4 1 F i g u r e 9: I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i - n o r m a l human a n t i s e r a w i t h a b s o r b e d normal human c e l l membrane a n t i g e n i n the FACS. p a n e l a: F a i l u r e o f a b s o r b e d normal human c e l l membrane a n t i g e n t o i n h i b i t the f l u o r e s c e n c e of HL-60 c e l l s r e a c t e d w i t h anti-AML#1 a n t i s e r u m . The upper f l u o r e s c e n c e p a t t e r n i n d i c a t e s r e a c t i v i t y o f t h e anti-AML#1 a n t i s e r u m i n the a b s e n c e o f normal human a n t i g e n . The lo w e r f l u o r e s c e n c e p a t t e r n shows t h e r e a c t i v i t y o f anti-AML#1 a n t i s e r u m w i t h HL-60 i n the p r e s e n c e o f normal human a n t i g e n . p a n e l b:, I n h i b i t i o n o f a n t i - n o r m a l human a n t i s e r u m by normal human c e l l membrane a n t i g e n i n the FACS IV u s i n g HL-60 c e l l s . The upper l i n e i n d i c a t e s f l u o r e s e n c e i n the a b s e n c e o f normal human a n t i g e n , whereas t h e low e r l i n e d e m o n s t r a t e s the i n h i b i t i o n s e e n i n the p r e s e n c e o f the normal human c e l l membrane a n t i g e n . 43 DISCUSSION The d a t a p r e s e n t e d h e r e , and t h a t p u b l i s h e d p r e v i o u s l y (19, 20) , show t h a t the a n t i s e r a p r e p a r e d i n r a b b i t s by i n j e c t i o n o f g e l p u r i f i e d p r o t e i n from a b s o r b e d membrane e x t r a c t s o f AML c e l l s r e a c t e x c l u s i v e l y , i n the ELISA, w i t h AML c e l l membrane p r e p a r a t i o n s and not w i t h e q u i v a l e n t p r e p a r a t i o n s o f c e l l membranes from e i t h e r c l i n i c a l l y n ormal i n d i v i d u a l s or t h o s e w i t h l y m p h o p r o l i f e r a t i v e d i s o r d e r s . The s p e c i f i c i t y o f t h i s a n t i - A M L a n t i s e r a was c o n f i r m e d i n f u r t h e r s t u d i e s , i n which bone marrow c e l l s and PBL from p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e d i s o r d e r s r e a c t e d w i t h the a n t i s e r a i n the FACS IV whereas c o m p a r a b l e c e l l s from normal i n d i v i d u a l s or t h o s e w i t h a v a r i e t y of l y m p h o p r o l i f e r a t i v e d i s o r d e r s d i d not ( 2 1 ) , ..... i n d i c a t i n g t h a t the a n t i g e n i n q u e s t i o n was p r e s e n t on the c e l l s u r f a c e . In a p r e v i o u s p u b l i c a t i o n ( 2 0 ) , we showed t h a t a b s o r b e d membrane p r e p a r a t i o n s o f AML c e l l s , when run on n o n - r e d u c i n g PAGE, d e m o n s t r a t e d the p r e s e n c e o f 3 a p p a r e n t l y u n i q u e , immuno-l o g i c a l l y c r o s s - r e a c t i v e p r o t e i n bands. At t h i s t i m e , we have c l a r i f i e d , t o some e x t e n t , the n a t u r e o f t h e s e p r o t e i n s i n v a r i o u s ways. When a b s o r b e d AML c e l l membranes a r e r u n on SDS-PAGE, o n l y the l o w e s t m o l e c u l a r w e i g h t band (AML band!1) i s c o n s i s t e n t l y p r e s e n t . T h i s band has been p r e s e n t i n a l l c e l l membrane p r e p a r a t i o n s examined from c e l l s o f p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e d i s o r d e r s and has not y e t been d e t e c t e d i n i d e n t i c a l c e l l membrane p r e p a r a t i o n s o f c e l l s f r o m normal 44 i n d i v i d u a l s or t h o s e from p a t i e n t s w i t h l y m p h o p r o l i f e r a t i v e d i s o r d e r s . A n t i s e r a r a i s e d t o each of the 3 p r o t e i n bands p r e s e n t i n the a b s o r b e d AML c e l l membrane sample a r e i n h i b i t a b l e , i n the ELISA, w i t h g e l p u r i f i e d AML band#1 m a t e r i a l . These d a t a c o n f i r m t h e c o n t e n t i o n t h a t t h e h i g h e r m o l e c u l a r w e i g h t p r o t e i n bands seen i n some AML c e l l membrane p r e p a r a t i o n s r e p r e s e n t a g g r e g a t e s o f AML band#1 i n a s s o c i a t i o n w i t h some o t h e r c e l l u l a r m o i e t i e s . The m o l e c u l a r w e i g h t s o f the 4 p r o t e i n bands seen i n F i g u r e 3 a r e 56,000, 80,000, 110,000 and 240,000 d a l t o n s m o l e c u l a r w e i g h t r e s p e c t i v e l y . The m o l e c u l a r w e i g h t o f AML band#1, seen i n F i g u r e 4, i s a p p r o x i m a t e l y 68,000 d a l t o n s and t h i s i s f u r t h e r c o n f i r m e d s i n c e a f t e r 2D-PAGE, the p r o t e i n m i g r a t e s w i t h an a p p a r e n t m o l e c u l a r w e i g h t o f 68,000 d a l t o n s and a p i o f 7.16. The a p p a r e n t d i s c r e p a n c y i n the m o l e c u l a r w e i g h t o f AML band#1 i s most l i k e l y due t o t h e d i f f e r e n t PAGE c o n d i t i o n s used t o r u n the a b s o r b e d AML c e l l membrane sample i n F i g u r e s 3 and 4. At p r e s e n t , the p r e c i s e n a t u r e and o r i g i n o f t h e AML band #1 a n t i g e n i s not known. I t has r e c e n t l y been shown t o be p r e s e n t on a h i g h p e r c e n t a g e o f b o t h bone marrow c e l l s and PBL of p a t i e n t s w i t h AML or CML ( 2 1 ) . Most i n t e r e s t i n g , has been the f i n d i n g t h a t the AML a n t i g e n i s not o n l y p r e s e n t on the b l a s t c e l l s o f p a t i e n t s w i t h the a c t i v e d i s e a s e but a l s o on a h i g h p e r c e n t a g e of n o r a m a l l y d i f f e r e n t i a t i n g l y m p h o i d and m y e l o i d c e l l s o f p a t i e n t s i n c l i n i c a l r e m i s s i o n . These o b s e r v a t i o n s i m p l y t h a t the e x p r e s s i o n o f t h i s a n t i g e n on the s u r f a c e o f a p l e u r i p o t e n t stem c e l l i s an i n d i c a t i o n o f a 45 p o t e n t i a l l y m a l i g n a n t change i n t h a t c e l l . At t h i s t i m e , we have not been a b l e t o d e m o n s t r a t e t h e p r e s e n c e of t h i s a n t i g e n on c e l l s from normal i n d i v i d u a l s or p a t i e n t s w i t h l y m p h o c y t i c l e u k e m i a or lymphomas. T h e r e a r e a number o f p o s s i b i l i t i e s r e g a r d i n g the o r i g i n o f t h i s a n t i g e n . I t may r e p r e s e n t a v i r a l gene p r o d u c t s i n c e t h e s e have been shown t o be p r e s e n t a t t h e c e l l s u r f a c e and a r e u s u a l l y h i g h l y g l y c o s y l a t e d . I t i s a l s o p o s s i b l e t h a t t h e a n t i g e n r e p r e s e n t s a normal d i f f e r e n t i a t i o n a n t i g e n which i s p r e s e n t a t such low c o n c e n t r a t i o n s and on such a low number o f c e l l s t h a t i t has not been d e t e c t e d i n normal p r e p a r a t i o n s , by t h e t e s t s used h e r e . These p o s s i b i l i t i e s a r e p r e s e n t l y under i n v e s t i g a t i o n . Ongoing s t u d i e s a r e a l s o a d d r e s s i n g t h e number and d i s t r i b u t i o n o f the AML a n t i g e n on v a r i o u s c e l l p o p u l a t i o n s ( b l a s t c e l l s , r e m i s s i o n c e l l s , e t c . ) , the copy number per c e l l , i t s p o s s i b l e f u n c t i o n and whether or not t h e a n t i g e n i s a r e c o m b i n a n t gene p r o d u c t . At p r e s e n t , t h e i n t e n t i o n i s t o c l o n e t h e g e n e ( s ) e n c o d i n g t h i s p u t a t i v e AML a n t i g e n and a d d r e s s t h e q u e s t i o n o f how e x p r e s s i o n o f t h i s g e n e ( s ) p r o d u c t i s r e l a t e d t o t h e o n s e t o f l e u k e m i a or t o t h e dominance o f a p o t e n t i a l l y m a l i g n a n t stem c e l l c l o n e i n t h e bone marrow o f AML or r e m i s s i o n p a t i e n t s . R e g a r d l e s s o f t h e outcome o f t h e s e s t u d i e s , t h e i m p o r t a n c e o f t h i s AML a n t i g e n a p p e a r s t o be l a r g e l y c l i n i c a l due t o i t s a p p a r e n t s p e c i f i c e x p r e s s i o n i n AML and CML c e l l s . 46 REFERENCES C o o p e r , G.M., O k e n q u i s t , S. and S i l v e r m a n , L. (1980) N a t u r e 284, 418-421. S h i h , C. , S h i l o , B.Z., G o l d f a r b , M.P., Dannenberg, A. and W e i n b e r g , R.A. (1979) P r o c . N a t l . Acad. S c i . USA 76, 5714 5718. C o o p e r , G.M. and Neiman, P. (1980) N a t u r e 287, 656-659. K r o n t i r i s , T.G. and C o o p e r , S c i . USA 78, 118 1-1184. S h i h , C., Padhy, C , M u r r a y , N a t u r e 290, 261-264. K l e i n , G. (1981) N a t u r e 294, A l e s s a n d r a , E. e t a l . (1982) G.M. (1981) P r o c . N a t l . Acad. M. and W e i n b e r g , R.A. (1981) 3 13-3 18. N a t u r e 295, 116-119. S h i l o , B.Z. and W e i n b e r g , R.A. (1981) N a t u r e 289, 607-609. M u r r a y , M., S h i l o , B.Z., S h i h , C , Cowing, D., Hsu, H.W. and W e i n b e r g , R.A. (1981) C e l l 25, 355-361. Brugge, J.S. and E r i k s o n , R.L. (1977) N a t u r e 289, 346-348. O s k a r s s o n , M., McClements, W.L., B l a i r , D.G., M a i z e l , J.V. and Vande Woude, G.F. (1980) S c i e n c e 207, 1222-1224. B a r r , R.D. and F i a l k o w , P . J . (1973) New E n g l . J . Med. 289, 307-309. Wiggans, R.G., J a c o b s o n , R . J . , F i a l k o w , P . J . , W o o l l e y , P., McDonald, J . S . ,and S c h e i n , P.S. ( 1 978) B l o o d 52 , 659-663 . F i a l k o w , P . J . , S i n g e r , J.W., Adamson, J.W., Berkow, R.L., F r i e d m a n , J.M., J a c o b s o n , R . J . and Moohr, J.W. (1979) New E n g l . J . Med. 301, 1-5. B a k e r , M.A. e t a l . (1981) i n Leukemia M a r k e r s , Knapp, W. (ed.) Academic P r e s s , London, In p r e s s . Taub, R.N., R o n c a r i , D.A.K. .and B a k e r , M.A. (1 978) C a n c e r Res. 38, 4624-4629. 4 7 17. M u l d e r , A., A l e x a n d e r , S., E n g e l f r i e t , C P . , Von dem B o r n e , A.E.G.-and S t r o m i n g e r , J . L . (1981) P r o c . N a t l . Acad. S c i . USA 78, 5091-5095. 18. M u r r a y , M., S h i l o , B.Z., S h i h , C , Cowing, D., Hsu,H.W. and W e i n b e r g , R.a. (1981) C e l l 25, 355-361. 19. Al-Rammahy, A.Kh., Shipman, R.C., J a c k s o n , A., M a l c o l m , A . J . and L e v y , J.G. (1980) Cancer Immunol. Immunother. 9, 181-185. 20. M a l c o l m , A . J . , Shipman, R.C. and L e v y , J.G. (1982) J . Immunol. 128, 2599-2603. 21. Shipman, R.C. and L e v y , J.G. (1982) M a n u s c r i p t i n p r e p a r a t i o n . 22. M a l c o l m , A . J . , Logan, P.M., Shipman, R.C. and L e v y , J.G. (1982) B l o o d , In p r e s s . 23. Lowry, O.M., R o s e n b r o u g h , N.J., F a r r , A.C. and R a n d a l l , R.J. (1951) J . B i o l . Chem. 193, 265-275. 24. K e l l y , B., L e v y , J.G. and S i k o r a , L. (1979) Immunology 37, 45-52. 25. K e l l y , B. and L e v y , J.G. (1977) B r . J . C a n c e r 375, 828-833 . 26. Avremeus, S. and T e r n y n c k , T. (1969) Immunochem. 6, 53-66 . 27. C u a t r e c a s a s , P. (1970) J . B i o l . Chem. 245, 3059-3065. 28. V o l 1 e r , A. , B i d w e l l , D. and B a r t l e t t , A. ( 1 976) i n Rose,. N. and F r i e d m a n , H. ( e d s . ) , Manual o f C l i n i c a l Immunology, A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y , W a s h i n g t o n pp.506-516. 29. Laemmli, U.K. (1970) N a t u r e 227, 680-685. 30. F a i r b a n k s , G., S t e c k , T.L. and W a l l a c h , D.F.H. (1971) B i o c h e m i s t r y 10, 2606-2616. 31. Wray, W. , B o u l i k a s , T. , Wray, V.P. and Hancock, R. (1981) A n a l . Biochem. 118, 197-203. 32. Wofsy, L., Henry, C , K i m u r a , J . and N o r t h , J . (1980) i n M i s h e l l , B.B. and S h i i g i , S.M. ( e d s . ) , S e l e c t e d methods i n c e l l u l a r immunology, W.H. Freeman and Co., San F r a n c i s c o pp.292-297. 48 3 3 . W e l l s , A . F . , M i l l e r , C E . a n d N a d e l , M . K . ( 1 966 ) A p p l . M i c r o . 1 4 , 2 7 1 - 2 7 3 . 3 4 . H e r z e n b e r g , L . A . a n d H e r z e n b e r g , L . A . ( 1 9 7 8 ) i n W e i r , D . V . ( e d . ) , H a n d b o o k o f E x p e r i m e n t a l I m m u n o l o g y , B l a c k w e l l S c i e n t i f i c , O x f o r d p p . 2 2 . 1 - 2 2 . 2 1 . 3 5 . O ' F a r r e l l , P. ( 1 9 7 5 ) J . B i o l . C h e m . 2 5 0 , 4 0 0 7 - 4 0 2 1 . 4 9 "....and so t h e r e a i n ' t n o t h i n g more t o w r i t e a b o u t , and I am r o t t e n g l a d o f i t , b e c a u s e i f I'd knowed what a t r o u b l e i t was to make a book I w o u l d n ' t a t a c k l e d i t and I a i n ' t a g o i n g t o no more. But I r e c k o n I g o t t o l i g h t o u t f o r the T e r r i t o r y ahead o f the r e s t , b e c a u s e Aunt S a l l y s h e ' s g o i n g t o a d o p t me and s i v i l i z e me and I c a n ' t s t a n d i t . I been t h e r e b e f o r e . " - Mark Twain from ' H u c k l e b e r r y F i n n " . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095769/manifest

Comment

Related Items