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Isolation and characterization of mouse-human somatic cell hybrids which produce a leukemia associated… Lum, Vincent Lawrence 1985

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ISOLATION AND CHARACTERIZATION OF MOUSE-HUMAN SOMATIC CELL HYBRIDS WHICH PRODUCE A LEUKEMIA ASSOCIATED ANTIGEN BY VINCENT LAWRENCE LUM B. Sc., Uni v e r s i t y of B r i t i s h Columbia, 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 of Microbiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Jul y 1985 © Vincent Lawrence Lum, 1985. 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 a n a d v a n c e d d e g r e e 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 a g r e e , t h a t t h e L i b r a r y s h a l l m a k e 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 a n d s t u d y . I f u r t h e r a g r e e 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 p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y 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 b e 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 . . _ MICROBIOLOGY D e p a r t m e n t o f 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 1956 M a i n M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e April 21, 1986 ABSTRACT I n t e r s p e c i f i c somatic c e l l h y b r i d i s a t i o n has the p o t e n t i a l not o n l y o-f producing h y b r i d s e x p r e s s i n g a n t i g e n s o-f two d i f f e r e n t s p e c i e s but a l s o of p r o v i d i n g g e n e t i c i n f o r m a t i o n on thes e f o r e i g n a n t i g e n s . The work presented here, d e s c r i b e s the s u c c e s s f u l i n t e r s p e c i f i c somatic c e l l h y b r i d i s a t i o n of a mouse myeloid c e l l l i n e (WeHi-TG) with p e r i p h e r a l blood l e u k o c y t e s from a p a t i e n t with c h r o n i c g r a n u l o c y t i c leukemia. Hybrid f u s i o n p r o d u c t s were screened f o r t h e i r a b i l i t y t o produce a leukemia a s s o c i a t e d p r o t e i n (CAMAL) as d e t e c t e d by i n d i r e c t immunoperoxidase as w e l l as ELISA u s i n g a monoclonal a n t i b o d y s p e c i f i c f o r CAMAL. P r o d u c t i o n of t h i s antigen was v e r i f i e d by im m u n o p r e c i p i t a t i o n of i n t e r n a l l y l a b e l l e d c e l l l y s a t e s from the s e hybridomas. CAMAL-producing c e l l h y b r i d s were f u r t h e r t e s t e d f o r the prescence of human DNA, using the BLUR 8 " A l u " c o n t a i n i n g DNA probe and a panel of monoclonal a n t i b o d i e s with s p e c i f i c i t i e s t o a v a r i e t y of myeloid a n t i g e n s . These s t u d i e s confirmed the prescence of human DNA and showed some c o r r e l a t i o n between c e l l s producing CAMAL and those e x p r e s s i n g other g r a n u l o c y t e markers. Karyotype a n a l y s i s was c a r r i e d out on hybridoma l i n e s a f t e r long term c u l t u r e . In a l l h y b r i d s examined, no r e c o g n i z a b l e human chromosomes or t r a n s l o c a t i o n s were detected u s i n g e i t h e r the methods of G-banding or d i f f e r e n t i a l G - l l s t a i n i n g . The development o-f an i n v i v o animal model f o r h u m a n leukemia would p r o v i d e a mammalian system f o r the t e s t i n g o-f v a r i o u s cancer t h e r a p i e s . The a b i l i t y o-f our CAMAL e x p r e s s i n g h y b r i d s to grow i n BALB/C mice p r o v i d e d the r a t i o n a l e f o r the development o-f an animal model -for can c e r t h e r a p y . Our p r e l i m i n a r y i.n v i t r o r e s u l t s showed t h a t CAMAL e x p r e s s i n g h y b r i d s c o u l d be s p e c i f i c a l l y e l i m i n a t e d u s i n g hematoporphyrin c o n j u g a t e d t o CAMAL-1. i i i TABLE OF CONTENTS A b s t r a c t . i i Table of Contents i v L i s t o-f Tab l e s v L i s t o-f F i g u r e s v i L i s t o-f A b b r e v i a t i o n s v i i i Acknowl edgements x 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 : I. Evidence -for e x p r e s s i o n of CAMAL (a) I n d i r e c t Immunoperoxidase 33 (b) ELISA 42 (c) Immunoprecipi t a t i o n 42 <d) FACS IV A n a l y s i s 43 I I . Evidence f o r prescence of Human p r o t e i n s and DNA (a) R e a c t i v i t y of h y b r i d s t o Myeloid s p e c i f i c monoclonal a n t i b o d i e s 47 (b) Dot B l o t A n a l y s i s 49 (c> K a r y o t y p i c A n a l y s i s 52 I I I . U t i l i z a t i o n of WC C e l l l i n e s as Models f o r Treatment of Human leukemias using Hp co n j u g a t e s 60 D i s c u s s i o n 68 Appendi x 73 B i b l i o g r a p h y ••• 74 i v LIST OF TABLES . T i t l e R e s u l t s of FACS IV a n a l y s i s of Wehi-d i r e c t l y compared t o h y b r i d f u s i o n p r o d u c t s u s i n g R a n t i CAMAL a n t i s e r a R e a c t i v i t y of v a r i o u s MAbs w i t h s p e c i f i c i t y f o r m y e l o i d markers w i t h s o m a t i c c e l l h y b r i d s . C y t o s p i n p r e p a r a t i o n s were t e s t e d . Normal and marker chromosome number i n s o m a t i c c e l l h y b r i d l i n e s . LIST OF FIGURES ' T i t l e Immunoperoxidase s t a i n i n g o-f c e l l l i n e s using CAMAL-l as the primary a n t i body. R e s u l t s o-f the c e l l u l a r ELISA on WeHi and WC c e l l l i n e s using r a b b i t anti-CAMAL as the primary antibody. R e s u l t s o-f ELISAs u s i n g c e l l l y s a t e s and r a b b i t anti-CAMAL as the primary a n t i body. Immunopreci pi t a t i on o-f WC4, WC6, and WeHi a-fter ^5g methionine i_n vivo l a b e l l i n g , u s i n g e i t h e r CAMAL-l or r a b b i t anti-CAMAL. Dot b l o t a n a l y s i s f o r d e t e c t i o n of human r e p e t i t i v e DNA sequences i n WeHi-TG and WC DNA. v i 6 a) G-banded karyotype o-f the WeHi l i n e . 53 b) G-Banded karyotype o-f WC1. 53 c) G-Banded karyotype o-f WC2. 55 d) G-Banded karyotype of WC4. 55 e) B-Banded karyotype of WC6. 57 T i t r a t i o n of Hematoporphyrin compounds on: a) WeHi-TG c e l l s 62 b) WC 6 c e l l s 64 8 S u r v i v a l of c e l l s of the WeHi, 66 WC2V,WC4 and WC6 c e l l l i n e s f o l l o w i n g treatment with 1 2.0ng/10 6 c e l l s of Hp e i t h e r a lone or conjugated t o v a r i o u s compounds. v i i LIST OF ABBREVIATIONS ALL - A c u t e l y m p h o c y t i c l e u k e m i a AML - A c u t e myelogenous l e u k e m i a ANLL - A c u t e non l y m p h o c y t i c l e u k e m i a BBS - B o r a t e b u f f e r e d s a l i n e BLUR 8 - BamHl u b i q u i t o u s r e p e a t CGL - C h r o n i c g r a n u l o c y t i c l e u k e m i a CML - C h r o n i c myelogenous l e u k e m i a CAMAL - Common a n t i g e n o-f myelogenous a c u t e l e u k e m i a DAB - Diamino B e n z i d i n e DNA - D e o x y r i b o n u c l e i c a c i d DME - D u l b e c c o ' s m o d i f i e d e a g l e ' s medium EDTA - E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ELISA - Enzyme l i n k e d immunoadsorbent a s s a y FACS - F l u o r e s c e n t a c t i v a t e d c e l l s o r t e r FCS - F e t a l c a l f serum FITC - 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 HAT - H y p o x a n t h i n e , A m i n o p t e r i n , t h y m i d i n e Hp - H e m a t o p o r p h y r i n HPRT" - H y p o x a n t h i n e p h o s p h o r i b o s y l t r a n s f e r a s e d e f i c i e n t J/m 2 - j o u l e s per meter squared LAA - Leukemia a s s o c i a t e d a n t i g e n MAbs - M o n o c l o n a l A n t i b o d i e s NaDOC - Sodium d e o x y c h o l a t e RPMI 1640 - Rosewel1 park memorial i n s t i t u t e 1640 medium v i i i SDS-PAGE - Sodium Dodecyl 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 . PBL - P e r i p h e r a l B l o o d L e u k o c y t e s PBS - Phosphate bu-f-fered s a l i n e (137mM NaCl,2.7mM KCl,43mM Na 2HP0 4, 1.5mM K H 2 P 0 4 ) PMSF - Phenyl methyl s u l - f o n y l - f l u o r i d e RIPA - R a d i o i m m u n o p r e c i p i t a t i o n assay (0.1*/. SDS.0.5"/. NaDOC,l'/. NP40,lmM EDTA lOOmM N a C l , lOmM T r i s HC1 pH 7.5) RNA - R i b o n u c l e i c a c i d SDS - Sodium d o d e c y l s u l p h a t e SSPE - S t a n d a r d s a l i n e p h o s p h a t e EDTA TCA - T r i - c a r b o x y l i c a c i d T r i s - T r i s ( h y d r o x y m e t h y l ) aminomethane TX100 - T r i t o n X100 WC - WeHi-CGL -fusion p r o d u c t WeHi-TG - W a l t e r and E l i z a H a l l i n s t i t u t e c e l l l i n e t h i o g u a n i n e r e s i s t a n t . ix A C K N O W L E D G E M E N T S I w o u l d l i k e t o s i n c e r e l y t h a n k my s u p e r v i s o r J u l i a G . L e v y •for h e r s u p p o r t a n d g u i d a n c e t h r o u g h o u t t h i s w o r k , F r e d J . D i l l •for h i s e x p e r t i s e i n g e n e t i c a n a l y s i s a n d h e l p f u l d i s c u s s i o n , C h r i s a n d S h a r o n f o r t h e i r p a t i e n c e , a n d e x p e r i e n c e i n k a r y o t y p i n g a n d c e l l c u l t u r e , S t e p h e n W o o d a n d R a y m o n d P o o n - f o r t h e i r e x p e r t i s e i n m o l e c u l a r b i o l o g y , my - f e l l o w c o l l e a g u e s a n d • f r i e n d s i n t h e l a b f o r t e c h n i c a l a s w e l l a s t h e o r e t i c a l a s s i s t a n c e a n d M a r y f o r u n d e r s t a n d i n g t h e t r i a l s a n d t r i b u l a t i o n s o f a g r a d u a t e s t u d e n t . x INTRODUCTION HISTORY: The Hemopoietic (blood -forming) system i s r e s p o n s i b l e -for the g e n e r a t i o n o-f myeloid c e l l s ( e r y t h r o c y t e s , p l a t e l e t s , g r a n u l o c y t e s , monocytes and macrophages) and lymphoid c e l l s (lymphocytes and plasma c e l l s ) which are produced i n the bone marrow, s p l e e n , and lymph nodes. R a d i a t i o n induced chromosomal markers, as w e l l as isoenzyme s t u d i e s have v e r i f i e d the a b i l i t y of s e l f renewing p l u r i p o t e n t stem c e l l s t o g i v e r i s e t o a l l c e l l s of the hemopoietic system (1). The appendix o u t l i n e s the m u l t i p l e pathways ( p i u r i p o t e n t i a l > t h a t a hemopoietic stem c e l l can f o i l o w . C h r o n i c and a c u t e myelogenous leukemias are 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 s of hemopoietic p r o g e n i t o r c e l l s ( i e . stem c e l l s t h a t are p r e d e s t i n e d f o r e i t h e r a myeloid or lymphoid pathway). Chromosomal markers as w e l l as isoenzyme G6PD a n a l y s i s have p r o v i d e d e v i d e n c e f o r the c l o n a l i t y ( s i n g l e c e l l o r i g i n of t h e s e cancsr t y p e s (2,3). There has been much evidence f o r a g e n e t i c p r e d i s p o s i t i o n ( e i t h e r h e r e d i t a r y or acquired) to cancer development. P a t i e n t s with r a r e h e r e d i t a r y d i s o r d e r s such as Fanconi's Anemia and A t a x i a T e l a n g i e c t a s i a a r e at i n c r e a s e d r i s k t o develop leukemia, lymphomas and e p i t h e l i a l tumors (4). It i s a l s o known th a t i n d i v i d u a l s exposed t o r a d i a t i o n such as b l a s t v i c t i m s of Hiroshima and Nagasaki have an i n c r e a s e d r i s k t o leukemia and have a high i n c i d e n c e of chromosome breakage i n t h e i r p e r i p h e r a l 1 lymphocytes. P a t i e n t s with chromosmal d i s o r d e r s such as Down's syndrome (5) a l s o have an i n c r e a s e d r i s k t o cancer development. These g e n e t i c p r e d i s p o s i t i o n s are d i r e c t l y r e l a t e d to i n s t a b i l i t y o-f chromosomes in p a t i e n t s with these v a r i o u s g e n e t i c d i s o r d e r s . These u n s t a b l e chromosomes are more s u s c e p t i b l e t o spontaneous or e p i g e n e t i c induced breakage, mutation ( i n s e r t i o n , d e l e t i o n s and s u b s t i t u t i o n s ) , rearrangements, and r e c o m b i n a t i o n s (£>> . Consequently p a t i e n t s with these g e n e t i c a b n o r m a l i t i e s are at a h i g h e r r i s k t o cancer. Thus, i t i s the i n s t a b i l i t y o-f chromosomes and there-fore the s u s c e p t i b i l i t y to chromosomal change that i s thought t o be r e s p o n s i b l e f o r a g e n e t i c p r e d i s p o s i t i o n t o cancer. An oncogene h y p o t h e s i s f o r the development of neoplasms has been a f a v o r e d mechanism f o r t r a n s f o r m a t i o n . Oncogenes are e v o l u t i o n a r i 1 y h i g h l y conserved human c e l l u l a r gene sequences that are thought t o have f u n c t i o n s i n normal c e l l growth and d i f f e r e n t i a t i o n . Oncogenes present w i t h i n t h e i r human host are r e f e r r e d t o as c-onc. These oncogenes have a p o t e n t i a l f o r causing cancer i f s u b j e c t e d to a i n d i r e c t or d i r e c t change i n l o c a t i o n or e x p r e s s i o n . The source of t h e s e malignant changes i n c l u d e p h y s i c a l (eg. r a d i a t i o n ) (7), chemical (eg. Benzene) (8) , and v i r a l (retroviruses)(9,10) agents. These agents are thought to be oncogene a c t i v a t i n g e i t h e r d i r e c t l y or i n d i r e c t l y . Chromosomal t r a n s l o c a t i o n s have r e c e n t l y been i m p l i c a t e d i n oncogene a c t i v a t i o n i n a number of n e o p l a s t i c s y s t e m s ( l l ) . 2 (a) P h y s i c a l and Chemical Agents: P h y s i c a l and c h e m i c a l a g e n t s a r e thought t o e x e r t t h e i r n e o p l a s t i c e f f e c t s by c a u s i n g m u t a t i o n of u n s t a b l e DNA and i n d i r e c t or d i r e c t a c t i v a t i o n of c e l l u l a r oncogenes. These two mechanisms a r e s t r o n g l y i m p l i c a t e d i n c a s e s of l e u k e m i a a s s o c i a t e d w i t h g e n e t i c p r e d i s p o s i t i o n s . (b) V i r a l A g e n t s : A v i r a l l y r e l a t e d t r a n s f o r m a t i o n mechanism has a major theme of r e s e a r c h f o r t h e pa s t s e v e r a l y e a r s . V i r a l gene sequences have l o n g been a s s o c i a t e d w i t h t h e maintenance o r i n d u c t i o n of n e o p l a s t i c t r a n s f o r m a t i o n . T r a n s f e c t i o n s t u d i e s u s i n g t r a n s f o r m a t i o n c a u s i n g DNA from b l a d d e r , l u n g and c o l o n c a r c i n o m a s , n e u r o b l a s t o m a s , s o f t t i s s u e and boney sarcomas, and neoplasms of B & T c e l l l y m p h o c y t e s , have r e v e a l e d homology or extreme s i m i l a r i t y t o t h e s e l o n g e s t a b l i s h e d v i r a l gene sequences ( 1 2 ) . The a b i l i t y of r e t r o v i r u s e s ( t y p e C RNA v i r u s e s ) t o t r a n s f o r m , has been f o u n d t o be t h e r e s u l t of t h e a c q u i s i t i o n of human t r a n s f o r m i n g sequences or oncogenes. The p r e s e n c e of t h e s e oncogenes i s t h e r e s u l t of r e c o m b i n a t i o n of non t r a n s f o r m i n g r e p l i c a t i o n competent RNA v i r u s e s w i t h h o s t c e l l u l a r oncogenes ( t h e s e h y b r i d t r a n s f o r m i n g DNA segments a r e r e f e r r e d t o as v-onc or v i r a l o n c o g e n e s ) . T r a n s f o r m a t i o n has been found t o be d i r e c t l y r e l a t e d t o t h e a c t i v a t i o n of t h e s e oncogenes e i t h e r by i n s e r t i o n of t h e v i r a l oncogene i n t o a hos t genome or a c t i v a t i o n of a v i r a l p r o m o tor. In a d d i t i o n , f u r t h e r 3 examination o-f bladder and c o l o n oncogene sequences have shown small mutations i n c e l l u l a r genes (and consequently p r o d u c t i o n o-f an a l t e r e d gene product) are r e s p o n s i b l e -for the a c q u i s i t i o n o-f trans-forming p r o p e r t i e s (13-15). Thus, n e o p l a s t i c t r a n s f o r m a t i o n v i a oncogene a c t i v a t i o n can be a r e s u l t of oncogene mutation to produce a l t e r e d gene p r o d u c t s ( v i a p h y s i c a l , chemical or v i r a l mechanisms) or e l e v a t i o n of oncogene t r a n s c r i p t i o n by a promotor ( v i r a l or h o s t ) . (c) Chromosomal T r a n s l o c a t i o n s : Nowell and Hungerford (I960) f i r s t i d e n t i f i e d t h a t a s p e c i f i c chromosomal a b n o r m a l i t y was a s s o c i a t e d with a s p e c i f i c neoplasm ( i e . the P h i l a d e l p h i a chromosome ph' i n c h r o n i c myelogenous 1eukemia)(16). The chromosomes i n v o l v e d i n t h i s t r a n s l o c a t i o n were not e l u c i d a t e d u n t i l 1973 when i t was found t h a t the m a j o r i t y of p a t i e n t ' s with CML or C G L c a r r i e d a r e c i p r o c a l t r a n s l o c a t i o n (tC9;22DCq34;911D) i n t h e i r bone marrow (17). In 1982, gene mapping t e c h n i q u e s were r e s p o n s i b l e f o r the d i s c o v e r y t h a t the P h i l a d e l p h i a t r a n s l o c a t i o n of C M L i n v o l v e d the t r a n s l o c a t i o n of the human c e l l u l a r homolog of the t r a n s f o r m i n g sequence of Abelson murine sarcoma v i r u s , c-ABL, (normally on chromosome 9) to chromosome 22 (18). The mapping of the lambda constant r e g i o n on chromosome 22 was performed by somatic c e l l h y b r i d i s a t i o n . The t r a n s l o c a t i o n of the c-ABL oncogene c l o s e t o the lambda gene promotor , presumably al l o w s e l e v a t i o n of the c-ABL gene product and presumably leads to neop1asi a. 4 S p e c i f i c nonrandom t r a n s l o c a t i o n s a s s o c i a t e d with CML and CGL are well documented. U n f o r t u n a t e l y the p i c t u r e i s not as c l e a r with a c u t e m y e l o b l a s t i c leukemia (AMD. The most f r e q u e n t non random chromosomal t r a n s l o c a t i o n i s the (tC8;21]Cq22;q22]) i n a p p r o x i m a t e l y 17 7. of M2 AML c a s e s (19). The human homolog to the murine sarcoma v i r u s c-MOS has been mapped to the b r e a k p o i n t on chromosome 8 of the 8;21 t r a n n s l o c a t i o n . N e o p l a s t i c t r a n s f o r m a t i o n s have been a s s o c i a t e d with chromosomal rearrangement, s p e c i f i c a l l y the r e l o c a t i o n of human oncogenes t o r e g i o n s of a c t i v e gene f u n c t i o n . Chromosomal t r a n s l o c a t i o n s appear t o have some primary f u n c t i o n i n the complex s t e p s i n v o l v e d i n n e o p l a s i a . In the B u r k i t t ' s lymphoma system t h e r e has been s t r o n g e v i d e n c e f o r a number of t r a n s l o c a t i o n , r e s p o n s i b l e f o r promotor and enhancer a m p l i f i c a t i o n of the c-MYC gene with c o e x p r e s s i o n of c-MYC and immunoglobulin p r o t e i n s (20). The s i g n i f i c a n c e of leukemia a s s o c i a t e d p r o t e i n s f o r the u n d e r s t a n d i n g and treatment of 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 has been a major part of cancer r e s e a r c h f o r the past decade. Myeloid leukemia a s s o c i a t e d a n t i g e n s have been d e s c r i b e d by a number of workers: a 75-BOK d a l t o n mol. wt. g l y c o p r o t e i n of p i 7.8 (21) and a g l y c o p r o t e i n of 400K d a l t o n s mol. wt.(22) are two a n t i g e n s i s o l a t e d from m y e l o b l a s t i c leukemia p a t i e n t c e l l s . 5 These a n t i g e n s have been u t i l i z e d i n d i a g n o s t i c assays as well as development o-f immunotherapy and c l a s s i f i c a t i o n o-f myeloid leukemias. A common antigen o-f myelogenous acute leukemia (CAMAL) has been the c e n t e r o-f much r e s e a r c h i n our l a b o r a t o r y . CAMAL, a leukemia a s s o c i a t e d p r o t e i n i s a p o l y p e p t i d e o-f approx i amatel y 68000 d a l t o n s and a p i o-f 7.2 (23). Using i mmuno-f 1 uorescene or i n d i r e c t immunoperoxidase methods, CAMAL has been -found t o occur at high i n c i d e n c e on the membrane and i n the cytoplasm of c e l l s from p a t i e n t s with 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 ( e i t h e r acute or c h r o n i c ) . Immunoprecipi t a t i o n of CAMAL from p a t i e n t s with c h r o n i c g r a n u l o c y t i c leukemia as compared with normal p a t i e n t s r e v e a l one hundred times g r e a t e r a n t i g e n i n the case of the former. Furthermore, CAMAL cannot be d e t e c t e d a s s o c i a t e d with normal g r a n u l o c y t e e n r i c h e d p o p u l a t i o n s u s i n g immunoperoxidase or immunof 1 uor e s c e n t assays (24). CAMAL has been used s u c c e s s f u l l y as the leukemia a s s o c i a t e d t a r g e t a n t i g e n i n immunotherapy of CAMAL b e a r i n g tumor c e l l s (25) §2 m§ti_c_Cel 1 _d¥bri.di.z at i.gn: I n t e r s p e c i f i c somatic c e l l h y b r i d i z a t i o n procedures (26,27) allow the mapping of s p e c i f i c genes t o p a r t i c u l a r chromosomes (28). These procedures used i n c o n j u n c t i o n with monoclonal a n t i b o d i e s s p e c i f i c t o mapped gene p r o d u c t s a l l o w mapping of genes to s e c t i o n s w i t h i n chromosomes. Recent work by Kessel et a l . (29) i n v o l v i n g i n t e r s p e c i f i c i n t r a l i n e a g e f u s i o n s , have demonstrated e x p r e s s i o n of human myeloid p r o t e i n s on mouse tumor 6 p a r e n t s . These methods have allowed the mapping of tumor a s s o c i a t e d a n t i g e n s on s p e c i f i c chromosomes (11). The e x p r e s s i o n o-f human leukemia a s s o c i a t e d a n t i g e n s on somatic c e l l h y b r i d s have i m p l i c a t i o n s -for the study o-f t h e s e a n t i g e n s : (•function, mapping, t h e r a p e u t i c u s e ) . The a b i l i t y of these h y b r i d s t o grow i n t h e i r mouse syngeneic systems w i l l f a c i l i t a t e the development of an i,n v i v o model f o r human leukemia. C l i n i c a l immunotherapy s t u d i e s (immunological methods t o cause tumor r e g r e s s i o n or bone marrow purging t o d e s t r o y a n t i g e n p o s i t i v e c e l l s ) c o u l d be performed u s i n g an i_n vayg mouse model f o r human leukemia. IMMUNOTHERAPY: The present methods of therapy f o r the treatment of leukemias i n v o l v e n o n s p e c i f i c k i l l i n g of a c t i v e l y d i v i d i n g c e l l s . R a d i a t i o n t h e r a p y as well as chemotherapeutic agents are two such t r e a t m e n t s . The n o n s p e c i f i c k i l l i n g of cancer c e l l s as well as normal c e l l s r e s u l t s i n s e v e r e and o f t e n dangerous s i d e e f f e c t s . There has been much e f f o r t i n the past decade to i n c r e a s e the s p e c i f i c i t y of cancer therapy. S e l e c t i v e drugs such as the p h o t o a c t i v a b l e chemical hematoporphyrin have a higher a f f i n i t y f o r tumor t i s s u e than f o r normal t i s s u e (30-32). P h y s i c a l removal of n e o p l a s t i c c e l l s u s i ng g r a d i e n t s (33) or d i f f e r e n t i a l a g g l u t i n a t i o n (34) have a l s o been used. These methods have met with l i m i t e d success. Immunotherapy r e f e r s to the s p e c i f i c k i l l i n g of cancer c e l l s v i a the r e c o g n i t i o n by a n t i b o d i e s of n e o p l a s t i c s p e c i f i c 7 or a s s o c i a t e d a n t i g e n s . Two immunotherapeut 1 c m o d a l i t i e s i n c l u d e anti-tumor antibody p l u s complement mediated d e s t r u c t i o n (35> and anti-tumor antibody c o n j u g a t e d to t o x i n s (36). Our l a b o r a t o r y has shown p r e v i o u s l y , that a monoclonal a n t i b o d y with s p e c i f i c i t y to a t r a n s p l a n t a b l e murine tumor c o u l d be c o n j u g a t e d to hematoporphyrin (Hp) and be used t o e f f e c t i v e l y d e l i v e r t o x i c l e v e l s of Hp t o i n s i t u tumors. These Hp c o n j u g a t e s were ab l e to e l i m i n a t e tumor c e l l s i_n yi.yg at l e v e l s of Hp l e s s than 10"/. of that used i n c l i n i c a l t r i a l s (37). S i m i l a r i^n yi.trg s t u d i e s u s i n g a monoclonal a n t i b o d y d i r e c t e d t o CAMAL and conjugated t o Hp (CAMAL-l-Hp) showed s p e c i f i c k i l l i n g of CAMAL b e a r i n g tumor c e l l s when the MAb-Hp c o n j u g a t e s were l a s e r - a c t i v a t e d (38). The p o s s i b i l i t i e s f o r the treatment of human leukemias u s i n g the CAMAL-l-Hp conjugate a l t h o u g h shown e f f e c t i v e i n v i t r o would r e q u i r e i.n y i y g t e s t i n g . Somatic c e l l h y b r i d s e x p r e s s i n g CAMAL and growing i n mice would p r o v i d e a i_n yi_yg model f o r i mmunother apeut i c treatment of human leukemia. The work presented i n t h i s t h e s i s intended t o develop a mouse-human somatic c e l l h y b r i d system that c o n s t i t u t i v e l y e x p r e s s e d the CAMAL a n t i g e n and would u l t i m a t e l y a l l o w the study of human myeloid leukemia i n an animal model system. 8 MATERIALS AND METHODS P a t i e n t C e l l Samples: P e r i p h e r a l blood l e u k o c y t e (PBL) samples from p a t i e n t s with c h r o n i c g r a n u l o c y t i c leukemia (CGL) were o b t a i n e d from the Department of Hematology, Vancouver General h o s p i t a l . D i a g n o s i s of CGL had p r e v i o u s l y been d e t e r m i n i n e d by c e l l u l a r morphology, b i o c h e m i c a l t e s t s (Sudan B l a c k , P e r i o d i c a c i d s t a i n , combined e s t e r a s e s and Ac i d phosphatase t e s t ) and chromosome a n a l y s i s . H e p a r i n i z e d blood was c e n t r i f u g e d at 1500 RPM through F i c o l l Hypaque (Pharmacia, Uppsala Sweden) i n order t o se p a r a t e mononuclear l e u k o c y t e s , g r a n u l o c y t e s and RBC (39,40) a f t e r which they were washed four times i n PBS pH 7.4. V i a b i l i t y counts were made d i r e c t l y u s i n g Trypan b l u e e x c l u s i o n p r i o r t o f u s i o n (41) . Tumor C e l l L i n e s : The mouse myeloid c e l l l i n e used as the f u s i o n parent was WeHi-TG a hypoxanthine p h o s p h o r i b o s y l t r a n s f e r a s e d e f i c i e n t (HPRT -) mutant which was o b t a i n e d a f t e r u l t r a v i o l e t i r r a d i a t i o n (10 J/m 2)(42) of WeHi-3B c e l l s (29) f o l l o w e d by c u l t u r e i n RPMI 1640 medium (GIBC0 l a b o r a t o r i e s , Grand I s l a n d , New York) p l u s 10'/. f e t a l c a l f serum (FCS) and 10 ug of 9 6-thi oguani ne i n a h u m i d i f i e d 10"/. CO, i n c u b a t o r . C e l l l i n e s were k i n d l y p r o v i d e d by Dr. D. Bootsma, Department of C e l l B i o l o gy and G e n e t i c s , Erasmus u n i v e r s i t y , Rotterdam, The Netherlands. C u l t u r e s are p r e s e n t l y m a i n t a i n e d i n RPMI 1640 p l u s 10'/. FCS i n a h u m i d i f i e d 107. CO 2 i n c u b a t o r . The human promyel ocyt i c c e l l l i n e HL60 and the p r e - e r y t h r o i d c e l l l i n e K562 was p r o v i d e d t o us by Dr. Tony Pawson, Dept. of M i c r o b i o l o g y , U n i v e r s i t y of B.C. HL60 c e l l s were maintained i n DME p l u s 107. FCS i n a h u m i d i f i e d CO-? i n c u b a t o r . The K562 c e l l l i n e was maintained i n RPMI 1640 p l u s 107. FCS i n a h u m i d i f i e d 107. C0 2 i n c u b a t o r . Cel1 Fusi on: Somatic c e l l f u s i o n s were performed e s s e n t i a l l y by procedures d e s c r i b e d by Oi and Herzenberg (43). B r i e f l y , 5 x 10 7 WeHi-TG c e l l s were washed three times i n serum f r e e RPMI 1640 and added t o pre-washed 1 >: 1 0 8 p e r i p h e r a l blood l e u k o c y t e s from a CGL p a t i e n t . The m i x t u r e of c e l l s was c e n t r i f u g e d at 1500 RPM f o r 10 min. The c e l l p e l l e t was warmed to 37°C f o r 10 min and then fused by the a d d i t i o n of 1.0ml of warm (37°) 507. p o l y e t h y l e n e g l y c o l (PEG) 4000 (Serva chemicals) i n PBS, with g e n t l e s t i r r i n g over one min. A f t e r one more minute of g e n t l e s t i r r i n g , 2.0ml of serum f r e e RPMI 1640 was added over two min . Then 8.0ml of serum f r e e RPMI 1640 was added over the next 4 min. Fused c e l l s were g e n t l y spun down at 1000 RPM f o r 5 min. Fusion products were p l a t e d i n 96 well L i n b r o ( F i s h e r S c i e n t i f i c ) p l a t e s at 2 >: 10 5 c e l l s per well 10 i n 100 ul o-f RPM I le>40 + 207. FCS + 1 :•: HAT (44) medium. Thymocytes were added as -feeders at a -final c o n c e n t r a t i o n o-f 5 >: 10^ c e l l s per ml i n lOOul. Medium was changed every -four days with the removal o-f lOOul o-f spent medium and the subsequent a d d i t i o n o-f 100 ul o-f -fresh RPMI 1640 c o n t a i n i n g 207. FCS and 257. conA s t i m u l a t e d r a t s p l e e n s upernatants c o n t a i n i n g 50mM alpha-methyl-mannoside (Sigma). V i a b l e c o l o n i e s were t r a n s f e r r e d t o 24 well L i n b r o p l a t e s f o r expansion f o r subsequent t e s t i n g . Monoclonal A n t i b o d i e s and A n t i s e r a : I n i t i a l s c r e e n i n g of h y b r i d c e l l s f o r the p r o d u c t i o n of a leukemia a s s o c i a t e d a n t i g e n i n v o l v e d the use of a monoclonal antibody CAMAL-l and a c o n v e n t i o n a l r a b b i t antiserum, r a b b i t a n t i CAMAL. CAMAL-l was developed i n our lab (45) and was r a i s e d a g a i n s t the leukemia a s s o c i a t e d a n t i g e n , CAMAL. CAMAL, a 68000 d a l t o n p r o t e i n , p i 7.2 was o r i g i n a l l y i s o l a t e d from acute non l y m p h o b l a s t i c leukemia (ANLL) c e l l membrane e x t r a c t s on 7.5 7. Sodium Dodecyl S u l p h a t e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (23). S i m i l a r l y , Rabbit anti-CAMAL a n t i s e r a were produced i n young a d u l t female a l b i n o r a b b i t s by primary immunisation i n t r a m u s c u l a r l y i n t o f o u r p e r i p h e r a l s i t e s with a 1:1 (v:v) mixture of 12.5 ug of CAMAL a n t i g e n i n a f i n a l 507. Freunds complete emulsion, adjuvant and subsequently a secondary immunization four weeks l a t e r . R a b b i t s were bled one week l a t e r from the marginal ear v e i n . The CAMAL-l monoclonal antibody and the Rabbit anti-CAMAL (R&CAMAL) a n t i s e r a have been shown to 11 r e a c t s p e c i f i c a l l y with CAMAL a n t i g e n present i n bone marrow and PBL c e l l s from p a t i e n t s with CGL or ANLL. CAMAL has been shown t o be present at low c o n c e n t r a t i o n s on normal g r a n u l o c y t e s (24). Rabbit anti-Human PBL membrane e x t r a c t antiserum (R&Hu) was produced by the immunization of young a d u l t female a l b i n o r a b b i t s with normal p e r i p h e r a l blood l e u k o c y t e s membrane e x t r a c t s produced from a pooled s o n i c a t e d p r e p a r a t i o n of normal PBLs. Immunization p r o t o c o l s f o r p r o d u c t i o n of r a b b i t a n t i human a n t i s e r a was performed as p r e v i o u s l y d e s c r i b e d (46). Immunoadsorbents: Immunoadsorbents c o n t a i n i n g 2mg/ml of CAMAL-1 were prepared as f o l l o w s : Monoclonal CAMAL-1 antibody obtained from a 507. ammonium s u l f a t e a s c i t e s s a l t c u t , and p u r i f i e d over an DEAE sepharose column was coupled to cyanogen bromide a c t i v a t e d sepharose CL4B (Pharmacia, U p s a l l a Sweden) (47,48). Immunoadsorbent columns were e q u i l i b r a t e d with b o r a t e b u f f e r e d s a l i n e (BBS) pH 8.5. Samples were a p p l i e d to a f f i n i t y column and c y c l e d over s i x times. The columns were washed with BBS pH 8.5 u n t i l the e f f l u e n t had no adsorbance at 0"n-280nm- T n e CAMAL a n t i g e n was e l u t e d u s i n g a 0.1 N HCl s o l u t i o n . One ml f r a c t i o n s were c o l l e c t e d and read on a spectrophotometer at 280nm. and p r o t e i n f r a c t i o n s were n e u t r a l i z e d with 5"/. Na 2C0 3. Mye l o i d S p e c i f i c Monoclonal Antibody Panel: The panel of monoclonal a n t i b o d i e s used f o r t h i s study 12 i n c l u d e the -following: NKHiA was o b t a i n e d -from Dr. Th. Hercend (49) and r e a c t s with an a n t i g e n on NK c e l l s and on some AML c e l l s CRIS-6 , 9 4-3Dl and JOAN-1 were ob t a i n e d from Dr. R. V i l e l l a , S e r v e i d ' immunologia, Casanova, Barcelona. CRIS-6 i s a monocyte r e a c t i v e monoclonal a n t i b o d y which does not r e a c t with lymphocytes, normal g r a n u l o c y t e s , p l a t e l e t s , e r y t h r o c y t e s , R a j i ( B u r k i t t s Lymphoma) , Daudi , but i n our hands r e a c t e d with c e l l s of p a t i e n t s with CGL and some ANLL samples. 94-3D1 i s r e a c t i v e with p l a t e l e t s and monocytes but not with lymphocytes, normal g r a n u l o c y t e s , e r y t h r o c y t e s , R a j i or Daudi. 94-3D1 r e a c t e d i n our t e s t s with some ANLL specimens, a p r o m y e l o c y t i c leukemia HL60 and KG1. JOAN-1 i s a pan r e a c t i v e monoclonal a n t i b o d y , being p o s i t i v e f o r T and B, PBL, monocytes, normal g r a n u l o c y t e s , e r y t h r o c y t e s , p l a t e l e t s and R a j i . In our hands i t r e a c t e d s t r o n g l y with a l l c e l l t y p e s t e s t e d which i n c l u d e d CGL and ANLL p a t i e n t m a t e r i a l as w e l l as the myeloid l i n e s HL60 and KG1 d e r i v e d from ANLL c e l l s . M101 was o b t a i n e d from R.W. Knowles, Sloan K e t t e r i n g , New York, and i s a p a n - r e a c t i v e monoclonal antibody. We found i t to r e a c t i n immunoperoxidase with e s s e n t i a l l y a l l p a t i e n t samples t e s t e d as well as HL60 and KG1. KD3 was o b t a i n e d from Dr. I. Indo, ICRF, U n i v e r s i t y C o l l e g e , London, and r e a c t s with c e l l s of both lymphoid (Daudi, J u r k a t t , RAJI, T and B b l a s t s , thymus) and myeloid (monocytes, CGL, ANLL, HL60, KG1) l i n e a g e s . GA-1 was obtained from Dr. A. H i r a i w a , Nagoya U n i v e r s i t y School of Medicine and appears to r e a c t mainly with g r a n u l o c y t e s GA-1 i s presumed to be d i r e c t e d t o g l y c o s y l a t e d membrane 13 components s i n c e i t does not y i e l d i d e n t i f i a b l e a n t i g e n s i n i m m u n o p r e c i p i t a t i o n t e s t s . Our t e s t r e v e a l e d r e a c t i v i t y to CGL, some AML, PBL and HL60. CLB-LFA-v and CLB gran were ob t a i n e d •from Drs. F. Miedema and A. Von Dem Borne, C e n t r a l l a b o r a t o r y of the Netherlands Red C r o s s Blood T r a n s f u s i o n s e r v i c e , Amsterdam. CLB-LFA 1/1 i n h i b i t s NK a c t i v i t y and CTL a c t i v i t y , r e c o g n i z e s the lymphocyte f u n c t i o n a s s o c i a t e d (LFA) a n t i g e n (50) as well as an a n t i g e n on T c e l l s , B c e l l s , monocytes, normal g r a n u l o c y t e s and some AML c e l l t y p e s . CLB gran r e a c t s with the n e u t r o p h i l a n t i g e n NA1 (present on lymphocytes, monocytes and g r a n u l o c y t e s , some AML c e l l types , HL60 and the pre e r y t h r o i d l i n e K562. CIPAN-Hu was o b t a i n e d from Dr. T. de K r e s t e r , The Cancer I n s t i t u t e , Melbourne, A u s t r a l i a . T h i s monoclonal antibody i s r e a c t i v e with a pan-human c e l l s u r f a c e a n t i g e n i n higher primates as well as some AML c e l l t y p e s and HL60. A l l of the monoclonal a n t i b o d i e s were t e s t e d o r i g i n a l l y as p a r t of the Second I n t e r n a t i o n a l Conference on Human Leucocyte D i f f e r e n t i a t i o n A n t i g e n s i n Boston (1984) (51). Because of t h e i r r e a c t i v i t y t o one of our o r i g i n a l i n t e r s p e c i f i c somatic c e l l h y b r i d s WC-2 , they were s e l e c t e d f o r f u r t h e r t e s t i n g with our other hybridomas. S e l e c t i o n of CAMAL P o s i t i v e Hybridomas: (a) ELISA (enzyme l i n k e d immunoadsorbent assay) on p u t a t i v e CAMAL c o n t a i n i n g h y b r i d s u p e r n a t a n t s . 50 ul of supernatants from f u s i o n p r o d u c t s i n 96 well p l a t e s were t r a n s f e r r e d t o Immulon I m i c r o t i t r e p l a t e s ( F i s h e r S c i e n t i f i c ) c o n t a i n i n g 50 ul 14 o-f Standard c a r b o n a t e / b i c a r b o n a t e ELISA c o a t i n g b u f f e r pH 9.6. The c o n t e n t s of these p l a t e s were mixed and then incubated at 4°C f o r 16 hours. P l a t e s were washed three times with PBS-TWEEN 20 b u f f e r and 100 ul of a 10~ 4 d i l u t i o n of r a b b i t anti-CAMAL was added to each well of t h e p l a t e , f u r t h e r incubated f o r 2 hours at room temperature and a g a i n washed three times with PBS TWEEN 20 b u f f e r . 100 ul of a l k a l i n e phosphastase (Sigma type VII) (52) l a b e l l e d sheep a n t i - r a b b i t Ig was then added (1:2000) f o r a f u r t h e r two hours at room temperature. The p l a t e s were washed t h r e e times i n PBS-TWEEN 20 and once with PBS. P l a t e s were developed with 100 u l p - n i t r o p h e n y l phosphate-Na (Sigma 104) to each well f o r one hour at room temperature. M i c r o t i t r e p l a t e s were read f o r absorbance at 405 nm on a T i t e r t e k M u l t i s k a n p l a t e reader (Flow L a b o r a t o r i e s , Inglewood C a l i f o r n i a ) , a lthough p o s i t i v e w e l l s c o u l d e a s i l y be scored v i s u a l l y . (b) C e l l u l a r ELISA: The c e l l u l a r ELISA i s a m o d i f i c a t i o n of the above procedure. C e l l s t o be t e s t e d were washed t h r e e times i n PBS and su b s e q u e n t l y resuspended at 1 x 10^ c e l l s / m l i n the standard c a r b o n a t e / b i c a r b o n a t e ELISA c o a t i n g b u f f e r pH 9.6. A l i q u o t s of 100 u l were p l a t e d onto Immulon II p l a t e s . The p l a t e s were spun at 750 rpm f o r ten minutes i n a c l i n i c a l DAMON IEC c e n t r i f u g e and c e l l s were incubated f o r 16 hours at 4°C. Car b o n a t e / b i c a r b o n a t e b u f f e r was removed and the c e l l s were u l t i m a t e l y f i x e d by t h e a d d i t i o n of 17. f o r m a l i n i n PBS f o r 15 one hour at 37 degrees C e l s i u s . ELISA's were c a r r i e d out i n the sta n d a r d manner. <c) Cel1 L y s a t e ELISA: 5 x 10 6 c e l l s t o be t e s t e d were washed t h r e e t i m e s i n PBS pH 7.4 p r i o r t o l y s i s . L y s i s was performed on loosened c e l l p e l l e t s by slow a d d i t i o n of 2x l y s i s b u f f e r (27. T r i t o n X100, .27. SDS and 10 mM PMSF - Sigma, 17. Na DOC - BDH) with g e n t l e a g i t a t i o n . L y s a t e s were spun at 1500 rpm t o remove u n l y s e d m a t e r i a l , and subsequently spun at 50,000xg (12,000 rpm i n the SS 34 r o t o r Beckman C e n t r i f u g e ) t o remove membranes. P r o t e i n c o n c e n t r a t i o n s of the su p e r n a t a n t were determined by the Lowry p r o t e i n assay (54). L y s a t e s were coated on m i c r o t i t r e p l a t e s at 1.0 ug/ml i n the c a r b o n a t e / b i c a r b o n a t e ELISA c o a t i n g b u f f e r . ELISAs were c a r r i e d out i n the standard manner as d e s c r i b e d above. Immunoperoxidase S l i d e P r e p a r a t i o n : Test c e l l s were washed t h r e e times i n serum f r e e RPMI 1640 (GIBC0) medium and resuspended with RPMI 1640 t o a c o n c e n t r a t i o n of 2 x 10 6 c e l l s / m l and loaded (3 drops) i n t o Shandon II c y t o s p i n f u n n e l s f o r s l i d e p r e p a r a t i o n . S l i d e s were prepared by c e n t r i f u g a t i o n of the t h r e e drops of t e s t c e l l s at 700 rpm f o r f i v e minutes on a Shandon c y t o s p i n II apparatus. 16 (c) C e l l L y s a t e ELISA: 5 x 10 6 c e l l s t o be t e s t e d were washed t h r e e times i n PBS pH 7.4 p r i o r t o l y s i s . L y s i s was performed on loosened c e l l p e l l e t s by slow a d d i t i o n of 2x l y s i s b u f f e r (27. T r i t o n X100, .27. SDS and 10 mM PMSF - Sigma, 17. Na DOC - BDH) with g e n t l e a g i t a t i o n . L y s a t e s were spun at 1500 rpm t o remove unlysed m a t e r i a l , and s u b s e q u e n t l y spun at 50,000xg (12,000 rpm i n the SS 34 r o t o r Beckman C e n t r i f u g e ) t o remove membranes. P r o t e i n c o n c e n t r a t i o n s of the supernatant were determined by the Lowry p r o t e i n assay (54). L y s a t e s were co a t e d on m i c r o t i t r e p l a t e s at 1.0 ug/ml i n the c a r b o n a t e / b i c a r b o n a t e ELISA c o a t i n g b u f f e r . ELISAs were c a r r i e d out i n the s t a n d a r d manner as d e s c r i b e d above. Immunoperoxidase S l i d e P r e p a r a t i o n : t e s t c e l l s were washed t h r e e t i m e s i n serum f r e e RPMI 1640 (6IBC0) medium and resuspended with RPMI 1640 to a c o n c e n t r a t i o n of 2 x 10 6 c e l l s / m l and loaded (3 drops) i n t o Shandon II c y t o s p i n f u n n e l s f o r s l i d e p r e p a r a t i o n . S l i d e s were prepared by c e n t r i f u g a t i o n of the t h r e e drops of t e s t c e l l s at 700 rpm f o r f i v e minutes on a Shandon c y t o s p i n II apparatus. 17 Immunoperoxidase S t a i n i n g : Prepared s l i d e s were allowed t o age o v e r n i g h t at room temperature be-fore s t a i n i n g was performed as f o l l o w s : Test s l i d e s were f i x e d f o r 30 minutes i n a methanol - 2"/. H202 s o l u t i o n , and s u b s e q u e n t l y washed by s p r a y i n g t h r e e times with PBS pH 7.4. The p r i m a r y antibody (CAMAL-1 or c o n t r o l MA6 at 10 ug/ml, r a b b i t anti-CAMAL-1 or NRS at 1/400 d i l u t i o n i n PBS) was a p p l i e d t o t h e wet c y t o s p i n s l i d e c o n t a i n i n g the c e l l c i r c l e , allowed t o i n c u b a t e f o r 60 minutes at room temperature and washed t h r e e times by s p r a y i n g with PBS pH 7.4. The secondary a n t i b o d i e s were r a b b i t anti-mouse immunoglobulin l i n k e d to horse r a d i s h p e r o x i d a s e (R&MIg-HRP) and swine a n t i - r a b b i t immunoglobulin l i n k e d t o horse r a d i s h p e r o x i d a s e (S&RIg-HRP) (DAKO). R&MIg-HRP was added to s l i d e s t r e a t e d with monoclonal primary a n t i b o d y t r e a t e d s l i d e s at 1/100 d i l u t i o n whereas S&RIg-HRP was added t o s l i d e s t r e a t e d with r a b b i t a n t i s e r a as a primary a n t i b o d y at a 1/200 d i l u t i o n . Both secondary antibody t r e a t e d s l i d e s were i n c u b a t e d 1 hour at 20°C i n darkness. S l i d e s were h i s t o c h e m i c a l 1 y s t a i n e d f o r horse r a d i s h peroxidase for 10 minutes by immersing the s l i d e s i n a c o p l i n j a r c o n t a i n i n g 50 ml PBS, 10 mg diaminobenzene, and 100 ul of 30% H2O2. S l i d e s were t r a n s f e r r e d t o c o p l i n j a r s c o n t a i n i n g 50 ml PBS, washed f o r f i v e minutes under tap water, and c o u n t e r s t a i n e d with hematoxylin. S l i d e s were c o v e r s l i p p e d u s i n g Permount (Fi s c h e r S c i e n t i f i c ) and examined under l i g h t microscopy using t h e o i l immersion lOOx o b j e c t i v e . If the number of p o s i t i v e c e l l s i n a 18 c e l l s p r e a d was l e s s t h a n 1.0*/. of t h e c e l l sample, a t l e a s t 400 c e l l s were s c o r e d on each s l i d e . I f p o s i t i v e r e a c t i v i t y was much l e s s than 10"/., between 1000 t o 3000 c e l l s were examined f o r each s l i d e . A l l s l i d e t e s t s done were c a r r i e d out a t l e a s t t w i c e on s e p a r a t e o c c a s i o n s (24). 3 5 S M e t h i o n i n e In v i v o L a b e l l i n g of C e l l L i n e s : A p p r o x i m a t e l y 5 x 1 0 6 c e l l s were i s o l a t e d and washed t h r e e t i m e s i n s t e r i l e PBS pH 7.4. C e l l s were r e s u s p e n d e d i n 2.0 ml of RPMI 1640 l a c k i n g m e t h i o n i n e (Met-, GIBC0) t r a n s f e r r e d t o 35mm p e t r i d i s h e s (FALCON). C e l l s were s u b s e q u e n t l y i n c u b a t e d f o r 2 h o u r s i n a 37°C, h u m i d i f i e d , 107. C02 i n c u b a t o r t o d e p l e t e i n t e r n a l p o o l s of c o l d m e t h i o n i n e . ^ S m e t h i o n i n e (NEN) was added t o t h e c e l l s t o a f i n a l c o n c e n t r a t i o n of 150 uCi/ml and i n c u b a t i o n was c a r r i e d out o v e r n i g h t i n a 37°C h u m i d i f i e d 107. C02 i n c u b a t o r . C e l l s not a d h e r i n g t o t h e 35mm d i s h e s were removed and t r a n s f e r r e d t o a 10.0 ml F a l c o n t u b e . P l a t e s were washed t w i c e w i t h i c e c o l d PBS pH 7.4 and t h e s e wash s o l u t i o n s were t r a n s f e r r e d t o t h e same f a l c o n tube. Non-adherent c e l l s were spun and washed t w i c e i n i c e c o l d PBS. C e l l s a d h e r i n g t o t h e 35mm p l a t e s were 1ysed u s i n g 2 changes of 0.5 ml RIPA (Sigma: lOOmM Na C l , lOmM T r i s Base, 17. NP40, 0.57. NaDOC, 0.17. SDS, 17. A p r o t i n i n ) b u f f e r . These changes of RIPA b u f f e r s were added t o t h e washed non-adherant c e l l p e l l e t f o r l y s i s . The r e s u l t i n g l y s a t e s were t r a n s f e r r e d t o 15 ml p o l y c a r b o n a t e t u b e s and spun at 15,000 rpm <50,000g) i n a Beckman RC5B w i t h a f i x e d a n g l e SS34 s o r v a l r o t o r f o r 30 minutes 19 to rtmovt insoluble mtmbrant fragments and n u c l e i . TCA p r e c i p i t a b l e counts were determined on the c e l l l y s i s supernatants by the spotting of 5 ul of each supernatant onto separate 2.5 cm Whatman glass micro-fibre f i l t e r s , a i r drying these f i l t e r s and' subsequently washing them on a m i l l i p o r e vacuum f i l t e r apparatus with two 3ml volumes of 207. TCA, two 3ml volumes of 57. TCA and one 3 ml volume of 957. ETOH. F i l t e r s were allowed to a i r dry in p l a s t i c s c i n t i l l a t i o n v i a l s and then baked for 10 minutes at 90°C. 5.0 ml of s c i n t i l l a t i o n c o c k t a i l (4 gm PPO/BIB MSP/1 toluene) was added to the v i a l and capped. F i l t e r s were read on a united technologies Hewlett Packard beta counter to determine cpm/ul. Immunoprecipitations: Prespun ^ S methionine l a b e l l e d c e l l lysates were precleared by the addition of NRS to lys a t e s at a f i n a l d i l u t i o n of 1/250 and the subsequent concentration of these serum treated lysates for 30 minutes on i c e . Ten volumes of a 107. suspension of staphl ococcus aureus s t r a i n Cowan I (IgG sorb; The Enzyme Center) in RIPA buffer was added to lysates for an additional 30 minutes on i c e . The lysates containing the nonspecific immune complex was spun on a microfuge (Eppendorf) for 5 minutes. 2 x 10^ CPM of resultant supernatants were aliquoted into four separate 1.5 ml microfuge tubes for s p e c i f i c immunoprecipi tation. A l l t e s t l y s a t e s performed together were normalized for volume prior to addition of primary antibodies. Five m i c r o l i t e r s of primary antibodies NRS, rabbit anti-CAMAL, 20 and CAMAL-1 were added separately to each o-f the three lysate a l i q u o t s to give a f i n a l d i l u t i o n of 1/1000 and incubated for 2 hours on i c e . Ten volumes (50ul) of a 10"/. solution of IgG sorb was added to NRS and rabbit anti-CAMAL containing a l i q u o t s and ten volumes of a 10"/. s o l u t i o n of RfcMIg - IgG sorb was added to mouse monoclonal antibody containing al i q u o t s and subsequently allowed to incubate for 1 hour on i c e with vortexing every 13 minutes. The immune complexes formed were spun down at 12,000 rpm on a miocrofuge (Eppendorf) resuspended in 1.0 ml of RIPA buffer, t r a n s f e r r e d to a new microfuge tube, spun on the microfuge and the p e l l e t was washed three additional times with RIPA buffer. The f i n a l c e l l p e l l e t s a f t e r four washes were resuspended in 50 ul of SDS sample buffer (2"/. SDS, 57. beta-mercaptoethanol , lOmM T r i s base pH 6.8, 10V. vol/vol g l y c e r o l ) , incubated in a 37° incubator for ten minutes to release Ag-Ab complexes from Staphlococcus aureus, and spun down on a microfuge for 5 minutes. Resultant antigen antibody containing supernatant was transferred to a new microfuge tube for heating p r i o r to polyacrylamide electrophoresis (55). 21 SDS - Polyacrylamide Gel Electrophoresim (SDS - PAGE) Proteins present i n the immune complex generated by the immunoprecipitation procedure were characterized using a discontinuous 7.3*/. Sodium Dodecyl Sulphate PAGE system. PAGE was performed e s s e n t i a l l y by the method described by Laemmli (56). B r i e f l y , samples were heated for 3 minutes at 100°C in SDS sample buffer and subsequently applied to a 37. stacking, 7.57. running SDS-PAGE system. Protein samples were stacked at 50 v o l t s and separated at 130 v o l t s using a Hoescht S c i e n t i f i c electrophoresis unit and powere pack on constant voltage. Molecular weight standards were coelectrophoresed and included: Bovine serum albumin (67000 daltons), Ovalbumin (43000 daltons), Carbonic Anhydrase (29000 daltons), and Trypsin Inhibitor (20000 daltons). Immunoprecipitation gels were stained with Coomassie blue (57) overnight, destained for four hours using a 41.47. methanol to 5.47. a c e t i c acid s o l u t i o n . Gels were impregnated with a chemical f l u o r by soaking destained gels for 60 minutes at room temperature i n En^Hance (NEN) and p r e c i p i t a t i n g the fl u o r within the gel by soaking the gel in deionized d i s t i l l e d water for 60 minutes. Gels were dried at 60°C on a BIORAD 1125B slab dryer and subsequently exposed to Kodak X0MAT-AR f i l m . 22 L a b e l l i n g of C e l l * for FACS C e l l s were Isolated from suspension and from f l a s k bottoms using 0.1'/. EDTA in PBS washed 3X in serum free RPMI 1640 pH 7.4 and 1 x 10^ c e l l s were placed i n each tube and spun down for antibody l a b e l l i n g . C e l l p e l l e t s recieved 0.2 ml of a 1/50 d i l u t i o n of e i t h e r : no antibody, no primary antibody, NRS, R&CAMAL or R&Hu. Samples were incubated with these primary antibodies on i c e for 1.5 hours with l i g h t shaking at 15 minute i n t e r v a l s . C e l l samples were then washed 3X in serum free RPMI 1640 and 0.2 ml of f l u o r e s c i n isothiocyanate linked to goat anti rabbit F(ab')2 fragment of IgG (Cappel> at a 1/20 d i l u t i o n was added to each c e l l p e l l e t and samples were allowed to incubate on i c e in darkness for 1 hour. In order to detect dead c e l l s in our samples , 1 ul of a 25 mg/ml solution of propidium iodide so l u t i o n (Becton Dickinson) was added to each test tube and the samples were incubated a further 30 minutes. C e l l s were then washed twice in i c e col d PBS pH 7.4 and c e l l p e l l e t s were f i n a l l y suspended in 1.0 ml of PBS pH 7.4 + 27. FCS and placed on ice p r i o r to FACS IV a n a l y s i s . Fluorescence Activated C e l l Sorter Analysis Fluorescent1y l a b e l l e d c e l l s were analyzed on a Becton Dickinson FACS IV using the 448nm wavelength of the Spectra Physics Model 164-05 Argon laser at a power set t i n g of 400mw. The standard f i l t e r s f o r FITC analysis was used (520 long pass f i l t e r ) . The FACS IV was standardized by using glutaraldehyde 23 f i x e d chicken red blood c e l l s (S8) and -fluorescent monodispersed carboxymethy1ated microspheres (d»1.75m + .02sd; Polysciences Inc., Warrington, PA). Ten thousand c e l l s were analyzed per test sample, the e f f e c t of fluorescent dead c e l l membrane fragment background was negated by gating out propidium iodide red fluorescence on c e l l s r e cieving secondary antibody only. The negative control serum (NRS) as well as the hybrid c e l l parent l i n e were used for gating of FACS for determination of r e l a t i v e p o s i t i v i t y to WC c e l l l i n e s . The head drive frequency was set at 36KHz , and 2000V were applied across the e l e c t r o s t a t i c d e f l e c t i o n plates. The FACS was run at f i v e droplets per d e f l e c t i o n pulse. DNA Dot Blot Analysis Plasmid I s o l a t i o n : The human r e p e t i t i v e "Alu " DNA Sequence (59,60) probe was kindly provided by Dr. S. Wood of the Medical Genetics department, UBC, as a plasmid probe referred to as BLUR 8 (Amersham: "a BamHl linked ubiquitous repeat constructed by the l i g a t i o n of a 300 b.p. repeat sequence from human DNA into the BamHl s i t e of pBR322 using BamHl l i n k e r s " ) . Plasmid DNA was prepared by the method as described by Davis et. a l . (1980) (61). B r i e f l y , Blur 8 containing E. Co l i HB101 was inoculated by loop to 10.0ml of L broth in a 37°C shaking incubator containing 50ug/ml of a m p i c i l l i n (an Ampr plasmid allows only HB101 to 24 survive) and allowed to grow for four hour* before addition of chloramphenicol (180ug/ml to amplify plasmid copy number) and further incubation overnight. The following procedures were performed on i c e : c e l l s were spun down at 2000 rpm for 10 min and resuspended in 5.0 ml of ice cold 25"/. sucrose in 50mM T r i s pH 8.0 . C e l l s were 1ysed by the addition of 1.0ml of lysozyme (3mg/ml in .25M EDTA pH 8.0) and 2.0ml of . 25M EDTA pH 8.0. C e l l s were swirled in t h i s s o l u t i o n for 10 min. C e l l s were further lysed by the addition of 1.0ml of 107. Triton X100 (10ml Trit o n X100 in .01M T r i s pH 8.0, 25ml 0.25M EDTA pH 8.0 ,0.5ml 1.0M T r i s pH 8.0 and f i n a l l y d i s t i l l e d H 20 to 100ml f i n a l volume). This solution was mixed (Triton allows c e l l l y s i s but not c e l l u l a r DNA release) and incubated on i c e for 15 min. Insoluble fragments were spun down at 19000rpm at 4°C in a Beckman RC5B centrifuge with an SS34 rotor (Beckman Instruments, C a l i f . ) . Supernatants containing DNA and RNA were separated by ethidium bromide (EtBr) CsCl density c e n t r i f u g a t i o n for 20 hours at 45000rpm. Plasmid DNA covalently closed c i r c l e s (ccc) were i s o l a t e d as a lower uv long wavelength fluorescing band using a 5.0ml syringe with a 21 gauge needle. EtBr was removed by repeated extraction with isobutanol saturated with aqueous 5.0M NaCl,10mM T r i s and ImM Na2EDTA (pH 8.5). Two volumes of d i s t i l l e d H 20 were added plus six volumes of 957. ethanol in order to p r e c i p i t a t e out plasmid DNA. DNA was dried and resuspended i n 1ml of lOmM T r i s JImM EDTA . DNA concentrations were determined spectrophotometrical1y by r a t i o s of 0 D 2 6 0 / 0 D 2 8 0 <ratios over 1.7 were considered DNA > and DNA 25 concentration* were calculated as O.D. 260/280 extinction c o e f f i c i e n t x d i l u t i o n . Preparation of Eucaryotic DNAi The i s o l a t i o n of high molecular weight eukaryotic DNA from c e l l s grown in t i s s u e culture was performed by the method of B l i n and S t a f f o r d (1976) (62). B r i e f l y , 2 x 10 7 c e l l s were washed twice i n PBS pH 7.4 and resuspended in 0.2 ml of ice cold 10 mM T r i s : ImM EDTA. DNA extr a c t i o n was performed by sequential addition of 0.33ml of 107. SDS and .033ml of Proteinase K (lOmg/ml) res p e c t i v e l y , followed by incubation in a 37°C incubator f o r 18 hours. The following day the resultant DNA was phenol extracted, phenol chloroform (CHC1 3 24:1 in isoamyl alcohol) extracted (3:1 then 1:1), chloroform extracted, and f i n a l l y d ialyzed against two changes of T r i s lOmM : ImM EDTA at 4°C. DNA concentrations were determined spectrophotometrical1y by measuring absorbance at 260nm. RNA contamination was absent as evidenced by 0.3V. agarose gel electrophoresis a f t e r treatment of DNA with lOOug/ml Rnase(Si gma). Nick T r a n s l a t i o n of pPD8 Alu Probe: The human r e p e t i t i v e DNA sequence probe pPD8 was r a d i o a c t i v e l y l a b e l l e d on one strand using nick t r a n s l a t i o n . The following mixture was prepared for nick t r a n s l a t i o n : 0.2ug of pPD8 DNA i s o l a t e d previously as described, 2.5ul of 10X nick t r a n s l a t i o n buffer (0.5M T r i s pH 7.2, .1M MgSo4, ImM DTT, 26 •3mg/ml BSA), 2.3ul o-f lOOuM dXTP (A, T, Q) , l.Oul o-f E . Coli Pol I (posesses a 3'to 3' polymerase + a 3' to 3' exonuclease, approximately 5 u n i t s of enzyme) (63) , l.Oul o-f DNase at 1 mg/ml (in 50mM T r i s pH 7.3, lOmM MgCl2,lmM DTT, 50*/. g l y c e r o l ) , 60uCi o-f alpha 3 2 P dCTP (NEN) and d i s t i l l e d water to make a -final reaction volume of 25ul. This reaction mixture was microfuged and allowed to incubate for 2 hours at ""12-14°C. TCA pr e c i p i t a b l e counts were determined p r i o r to stopping t r a n s l a t i o n r e a c t i o n so that s p e c i f i c a c t i v i t y <108 cpm/ug DNA desired) could be determined. The reaction was terminated by the addition of 25ul of nick t r a n s l a t i o n stop buffer (50mM EDTA, 20mM NaCl, 0.17. SDS, 500ug/ml salmon sperm DNA). Dot Blot Hybridization: NEN gene screen plus (6SP) hybridization transfer membranes (NEN, Dupont) were prepared for use by presoaking them in d i s t i l l e d water f o r ten minutes. SSP was then placed concave side up into a 96 well M i l l i p o r e dot blot vacumn apparatus ( M i l l i p o r e , Bedford, Massachusetts)(aa). Duplicate single stranded DNA q u a n t i t i e s (DNA i s boiled for 10 minutes and ra p i d l y cooled on ice) of 500, 250, 125, and 50ng were added to wells of t h i s blot apparatus and allowed to soak for 15 mins under l i g h t vacumn. Single stranded DNA bound to GSP was baked in a vacumn evacuated dessicator for 1 hour at 65°C. GSP with test s i n g l e stranded DNA was soaked i n 5X SSPE (5mM EDTA, 50mM NaH 2P0 4, 40mM NaOH, . 9M NaCl) + 0.37. SDS for 5 minutes to 27 remove non-specific binding and then changed to a pre-hybridization mix (5ml of formamide, 2ml of 5M NaCl, 2ml salmon sperm DNA) and allowed to incubate for two hours at 62°C in a heat-sealable bag. 3 x JO 6 TCA p r e c i p i t a b l e cpm of nick translated pPD8 probe was added and allowed to incubate overnight at 62°C in a shaking water bath. The following day hybridized DNA on GSP was washed free of non hybridized r a d i o a c t i v e DNA by four changes of 5X SSPE + 0.3'/. SDS and subsequently dried under a heat lamp and exposed to Kodak XOMAT-AR f i l m on Dupont Lightning-Plus screens. Karyotype Analysis Culture Treatment and S l i d e Preparation: Fresh cultures of WeHi-TG and WC hybridomas were prepared the day before karyotype a n a l y s i s by passing the c u l t u r e s in RPMI 1640 + 107. FCS at a f i n a l concentration of 2 x 105/ml . Colcemid (GIBCO) was added to c u l t u r e s to a f i n a l concentration of 250ug/ml and allowed to incubate for 25 minutes at 37°C in order to arrest c e l l s i n metaphase by a mechanism of spindle f i b e r formation i n h i b i t i o n . C e l l s were spun at lOOOrpm for 10 minutes i n a v bottom glass tube. C e l l s were brought up in 10 ml of .075N KC1 (for hypotonic l y s i s ) and allowed to incubate for 25 minutes at 37°C. C e l l s were again spun down at lOOOrpm for ten minutes and then brought up in 10 ml of 3:1 methanol: Acetic acid f i x a t i v e , mixed and allowed to stand for 30 minutes 2 8 at room temperature. C e l l s were washed 3X with t h i s same -fixative s o l u t i o n and s l i d e s prepared (65). Giemsa Stain i n g ! Chromosomes were analyzed using a modi-fication of the G-banding method of Seabright (1971)(66). B r i e f l y , s l i d e s prepared with chromosomes were heated for 2 hours at 65°C and t r y p s i n i z e d (2.5 ug/ml solution) for 1 minute, washed i n 0. 1 M CaCl2 and allowed to s i t in 0.1M CaCl2 for 1 minute. S l i d e s were washed in d i s t i l l e d water and stained in Harleco Giemsa s t a i n <3ml s t a i n in 40ml dH20> for 1 minute and subsequently washed i n d i s t i l l e d H2O and examined microscopically. D i f f e r e n t i a l Staining: G - l l d i f f e r e n t i a l s t a i n i n g of human and rodent chromosome was done by the method of Bobrow (1974)(67). B r i e f l y , s l i d e s prepared with chromosomes were heat fi x e d for 2 hour at 65°C, wetted with d i s t i l l e d H2O and placed in freshly prepared (no more than 20 minutes) Harleco Giemsa s t a i n (1/50 d i l u t i o n in d i s t i l l e d H 20, pH 11.5) for 8 minutes in a 37°C water bath. S l i d e s were immediately washed in d i s t i l l e d H2O and heated for 2 minutes at 65°C prior to microscopic analysis. 29 P r e p a r a t i o n o-f Hematoporphyr i n (Hp )-Anti body Conjugates: Linkage of Hematoporphyrin to monoclonal a n t i b o d i e s as well as a n t i s e r a has been d e s c r i b e d and c a r r i e d out by Mew et a l . (1784)(25). B r i e f l y , Hp was conjugated t o p r o t e i n s by the c a r b o d i i m i d e l i n k a g e method: to 20mg of Haematoporphyrin di hydrochl or i de (95'/.) (Sigma) i n 1.25ml water and 0.8ml N,N-dimethylformamide, was added 20mg l - e t h y l - 3 -(3-dimethyl a m i n o p r o p y l ) - c a r b o d i i m i d e HC1 i n 0.6ml d i s t i l l e d H2O. A f t e r 30 minutes, t h i s s o l u t i o n was mixed with 15mg of antibody (monoclonals CAMAL-l and anti-L1210=L12, R&CAMAL and R8<Hu) i n 5ml d i s t i l l e d H 20 f o r 5 hours. During t h i s p e r i o d the pH of t h i s s o l u t i o n was c o n t i n u o u s l y a d j u s t e d t o 6-7. A f t e r the 5 hour i n c u b a t i o n p e r i o d , 50ul of monoethanolamine was added and the s o l u t i o n was l e f t o v e r n i g h t at room temperature. Hp conjugates were d i a l y z e d e x h a u s t i v e l y a g a i n s t PBS, f o l l o w i n g which t h e i r Hp c o n c e n t r a t i o n was determined s p e c t r o p h -o t o m e t r i c a l l y by t a k i n g the absorbance at 505nm (1.0 O.D. i s 124ug Hp/ml). P r o t e i n c o n c e n t r a t i o n s were determined by measuring absorbance at 280nm using a c o n v e r s i o n f a c o t o r of 1.4 O.D. = 1.0 mg/ml. Cross absorbance contamination was negated by measuring the absorbance of f r e e Hp at 2S0nm as well as the absorbance of p r o t e i n at 505nm and c a l c u l a t i n g the "/. c o n t r i b u t i o n of each f o r subsequent s u b t r a c t i o n . Hp conjugates were t e s t e d f o r antibody a c t i v i t y u s ing the ELISA assay d i r e c t e d a g a i n s t t h e i r s p e c i f i c a n t i g e n s and a Hp c o n t r o l . Hp c o n j u g a t e s were a l s o t e s t e d f o r Hp a c t i v i t y by measurement of sheep e r y t h r o c y t e hemolysis upon exposure to 30 l i g h t in the prescence or absence of Hp, Hp conjugates or PBS. Hp conjugates were f i l t e r s t e r i l i z e d p r i o r to use in tis s u e culture systems. Hp concentrations as well as protein concentrations were reevaluated at t h i s time. In V i t r o C y t o t o x i c i t y Assays for Hp Conjugate: S p e c i f i c Hp conjugate c y t o t o x i c i t y was determined using a 0.27. trypan blue dye exclusion v i a b i l i t y t e s t . 5 x 10^ prewashed t e s t c e l l s were dispensed in lOOul to 17 x lOOmm Falcon test tubes for each test antibody assayed. Primary antibodies not conjugated to hematoporphyrin were added to c e l l suspensions f o r 1 hour at 4°C pr i o r to exposure to Hp conjugated antibody (Rabbit anti MIg-Hp as the photoactivatable agent). Hematoporphyrin conjugates were added under minimal l i g h t conditions to c e l l s and allowed to incubate in the dark for two hours i n a humidified 107. CO2 incubator. C e l l s were washed three times in serum fre e RPMI 1640 and f i n a l l y resuspended i n 13 ml of RPMI 1640 and exposed to an 100W GE 125V incandescent l i g h t of int e n s i t y 22.5mW/cm2, (as measured by a YSI-Kettering model 65 radiometer) at 30cm for 1 hour at room temperature. C e l l s were then centrifuged, resuspended in 5ml of RPMI 1640 + 107. FCS and incubated in a humidified 107. C0 2 incubator overnight. Relative v i a b i l i t i e s were determined as a percentage of c e l l samples processed in an i d e n t i c a l manner but recieving only PBS(25>. 31 ^H-Thymidine incorporation Assay Viable c e l l s were assessed by incorporation of -Thymidine after treatment as above. After removal of c e l l s for 0.2% Trypan blue dye exclusion t e s t , 5 x 10 4 c e l l s in lOOul was plated in six wells of a Linbro m i c r o t i t r e plate (according to o r i g i n a l c e l l numbers ) and 50ul of RPMI 1640 + 10X FCS containing 2uCi of 3H-Thymidine (NEN) was added to each well and allowed to incubate 24 hours in a 37°C 10*/. CO2 incubator. The c e l l s were then c o l l e c t e d on a MASH harvestor as described previously (68) and 3H Thymidine incorporation was measured as cpm on a Hewlett Packard Tri-carb 4550 s c i n t i l l a t i o n counter. 32 RESULTS I. - Evidence for the Presence of the Common Antigen of dY§L99§Q2y8.-££yt§_Leuke JDi*i. (a) R e a c t i v i t y o-f Hybrids to Rabbit anti-CAMAL and CAMAL-l in the Indirect Immunoperoxidase assay: I n t e r s p e c i f i c somatic c e l l h y b r i dization of rodent WeHi-TG (HPRT-) tumor myeloid c e l l s to mononuclear leukocytes and granulocytes from CGL patients resulted in the i s o l a t i o n of 150 hybrids selected after f i v e weeks in culture which showed weak r e a c t i v i t y i n ELISA associated with hybrid supernatants. Of these 150 hybrids 6 reacted 100"/. p o s i t i v e l y with CAMAL-l and Rabbit anti-CAMAL antibodies in the i n d i r e c t immunoperoxidase. These r e s u l t s suggest that these clones arose from a s i n g l e hybrid c e l l . Representative photomicrographs are shown in Figure 1. The CAMAL antigen i s located on the membrane, nucleus and within the cytoplasm of the hybrid c e l l s in varying degrees. No r e a c t i v i t y was demonstrated with the WeHi control c e l l s . It was also noted that WC hybrid c e l l s demonstrated d i f f e r e n c e s i n growth c h a r a c t e r i s t i c s as compared to the WeHi parent l i n e . WC l i n e s were capable of growth at higher c e l l d e n s i t i e s and shorter c e l l cycels (13 hrs vs WeHi 16 hrs) aft e r the i n i t i a l slow 5 week post fusion growth rate. 33 Figure 1. Immunoperoxidase Staining of c e l l l i n e s using CAMAL-1 as the primary antibody, a, WeHi-TG; b, WC 1; c, WC 2 ; d, WC 4; e, WC 6. 34 35 c. F i g u r e 2. R e s u l t s o-f the c e l l u l a r ELISA on WeHi and WC c e l l l i n e s u s i n g R a b b i t anti-CAMAL as the p r i m a r y a n t i b o d y . • WC 2; A—A , WC 6; A A , WC 1; • — • , WC3; O — o , WC 4; • — • , WeHi-TG. 38 39 Figure 3. Results of ELISA's using c e l l l y sates and Rabbit anti-CAMAL as the primary antibody. • WC 2; A — A , WC 6: • •, WC 4j A — A , WeHi -TG. 40 I I I 1/20000 1/200000 Ab Dilution 41 (b) R e a c t i v i t y of H y b r i d s t o Rabbit anti-CAMAL and CAMAL-1 i n the ELISA a s s a y t Immunoperoxidase p o s i t i v e c l o n e d (by l i m i t i n g d i l u t i o n ) hybridomas were f u r t h e r expanded and t e s t e d f o r t h e prescence of CAMAL u s i n g an ELISA procedure with e i t h e r c e l l l y s a t e s or f i x e d c e l l s as the s o l i d phase a n t i g e n . R e s u l t s u s i n g the c e l l u l a r ELISA and Rabbit anti-CAMAL a n t i s e r a are shown i n F i g u r e 2. The anti s e r u m r e a c t e d s t r o n g l y with a l l the s e l e c t e d h y b r i d s and showed o n l y minimal (background) r e a c t i v i t y with WeHi. S i m i l a r l y , c e l l e x t r a c t s of h y b r i d s e x h i b i t i n g good growth c h a r a c t e r i s t i c s were t e s t e d f o r the prescence of CAMAL. The ELISA r e s u l t s r e v e a l e d s t r o n g r e a c t i v i t y as shown i n F i g u r e 3. In t h e s e t i t r a t i o n s q u a n t i t a t i v e d i f f e r e n c e s e x i s t between the c e l l l i n e s i n terms of o v e r a l l r e a c t i v i t y with the r a b b i t anti-CAMAL p o l y c l o n a l antibody. I t appears t h a t the amounts of CAMAL expressed by the c e l l l i n e s can be a s s i g n e d as WC2 > WC6 > WC1 > WC3 > WC4 >> WeHi-TG. (c> Immunoprecipi t a t i o n of CAMAL from H y b r i d s with Rabbit anti-CAMAL and CAMAL-1.• To determine whether apparent e x p r e s s i o n of CAMAL as d e t e c t e d by i n d i r e c t immunoperoxidase and ELISA c o r r e l a t e d with the p r o d u c t i o n of the 68000 d a l t o n CAMAL p r o t e i n , immunoprecipi t a t i o n s were c a r r i e d out on 3 ^ S - M e t h i o n i n e l a b e l l e d h y b r i d s WC4 , WC6 (these h y b r i d s showed the most 42 c o n s i s t e n t growth p a t t e r n s ) and on the parent l i n e WeHi, using both the r a b b i t anti-CAMAL antibody as well as the monoclonal CAMAL-l. The r e s u l t s shown on F i g u r e 4. c l e a r l y demonstrate the prescence of a p r o t e i n of appro*iamately 68000 d a l t o n s p r e c i p i t a t e d from both WC4 and WC6 3 5 s -methionine l a b e l l e d c e l l l y s a t e s u s i n g r a b b i t anti-CAMAL as well as CAMAL-l, whereas the n e g a t i v e c o n t r o l antibody (NRS), does not show a 68000 d a l t o n s p r o t e i n . In a d d i t i o n , no e q u i v a l e n t 68000 da l t o n p r o t e i n was immunoprecipitated from ^ S - M e t h i o n i n e l a b e l l e d WeHi c e l l l y s a t e s by any of the a n t i b o d i e s used. (d) FACS IV a n a l y s i s f o r i d e n t i f i c a t i o n of c e l l s u r f a c e CAMAL: The p r e v i o u s experiments showing the prescence of CAMAL ant i g e n a s s o c i a t e d with our h y b r i d c e l l l i n e s do not d i f f e r e n t i a t e between an outer membrane or i n t r a c e l l u l a r l o c a t i o n . In order t o determine whether t h e r e was CAMAL on the outer membrane of our h y b r i d s WC4 and WC6 we performed FACS IV a n a l y s i s using the r a b b i t anti-CAMAL a n t i s e r a . R e s u l t s i n TABLE I show d i r e c t comparisons between WeHi and the h y b r i d s and r e v e a l a l a r g e number of h i g h l y f l u o r e s c i n g c e l l s on h y b r i d s WC4 (72.67.) and WC6 (63.77.) using r a b b i t anti-CAMAL a n t i s e r a as compared t o the WEHI (8.47.) parent or the non-CAMAL e x p r e s s i n g v a r i a n t of WC2, WC2V (15.97.) l i n e . These f i n d i n g s suggest t h a t t h e r e are the prescence of CAMAL molecules on the outer membrane of our c e l l h y b r i d s WC4 and WC6. 43 Figure 4. Immunoprecipi t a t i o n of WC 4, WC 6 , and WeHi-TG after 3 5S-Methionine in vivo l a b e l l i n g , using e i t h e r CAMAL-l or Rabbit anti-CAMAL. Lane a, NRS; lane b, Rabbit anti-CAMAL; lane c, CAMAL-l. 44 A B C A B C A B C 45 T A B L E I R e s u l t s o-f F A C S I V a n a l y s i s ( u s i n g R a b b i t a n t i - C A M A L a n t i s e r u m ) o-f W e H i - T G d i r e c t l y - c o m p a r e d t o h y b r i d f u s i o n p r o d u c t s . A n t i s e r u m C e l l L i n e N u m b e r o f C e l l s F l u o r e s . A v g . o f 2 t r i a l s . '/. C e l l s F l u o r e s c i n g - * 1 ) N o r m a l W e H i - T G 1 1 0 7 1 1 . 1 R a b b i t W C 2 V 5 5 2 5 . 5 S e r u m WC4 2 6 4 3 2 6 . 4 WC6 1 2 5 2 1 2 . 5 2 ) R a b b i t W e H i - T G 8 3 7 8 . 4 a n t i - W C 2 V 1 5 8 7 1 5 . 9 C A M A L WC4 7 2 6 0 7 2 . 6 s e r u m WC6 6 3 7 4 6 3 . 7 * 7. o f 1 0 0 0 0 c e l l s a n a l y z e d p e r s a m p l e . 46 1 1 • §yA°!§Q£§_i9c_the_Pc5*SQ<=^ ( a ) . R e a c t i v i t y o-f H y b r i d s t o M y e l o i d M o n o c l o n a l A n t i b o d i e s : T h e p r e s e n c e o-f t h e C A M A L a n t i g e n o n h y b r i d c e l l s a n d i t s a p p a r e n t a b s e n c e o n t h e t u m o r p a r e n t l i n e W e H i - T G s u g g e s t e d t o u s t h a t i t w a s a p r o t e i n o b t a i n e d - f r o m t h e C G L - f u s i o n p a r t n e r . B e c a u s e t h e h u m a n f u s i o n p a r t n e r w a s o f m y e l o i d o r i g i n a n d we w e r e a b l e t o d e m o n s t r a t e t h e p r e s c e n c e o f t h e m y e l o i d s p e c i f i c a n t i g e n C A M A L o n h y b r i d c e l l s , we d e c i d e d t o e x a m i n e o u r h y b r i d s f o r o t h e r m y e l o i d s p e c i f i c h u m a n a n t i g e n s . A g r o u p o f m o n o c l o n a l a n t i b o d i e s , r e a c t i n g w i t h a v a r i e t y o f m y e l o i d c e l l m a r k e r s , w e r e t e s t e d w i t h t h e h y b r i d s a n d W e H i u s i n g t h e i n d i r e c t i m m u n o p e r o x i d a s e m e t h o d . O n e o f t h e h y b r i d c e l l l i n e s WC2 w a s p a r t o f a s t u d y o n m o n o c l o n a l r e a c t i v i t y t o h u m a n d i f f e r e n t i a t i o n a n t i g e n s , a n d t h e m o n o c l o n a l a n t i b o d i e s u s e d t o t e s t t h e o t h e r WC h y b r i d s w e r e s e l e c t e d b e c a u s e o f t h e i r s t r o n g r e a c t i v i t y w i t h WC2 i n t h i s s t u d y . T h e r e a c t i v i t y o f t h e s e m o n o c l o n a l a n t i b o d i e s o n o u r h y b r i d c e l l s i s s h o w n i n T a b l e I . A n u m b e r o f m o n o c l o n a l a n t i b o d i e s r e a c t e d n o t o n l y w i t h W C 2 , a g a i n s t w h i c h t h e y w e r e o r i g n a l l y s e l e c t e d , b u t w i t h a n u m b e r o f t h e o t h e r C A M A L p r o d u c i n g h y b r i d s . I n p a r t i c u l a r , t h e m o n o c y t e r e a c t i v e C R I S - 6 s h o w e d r e a c t i v i t y t o a l l h y b r i d s a n d n o t t o t h e p a r e n t t u m o r l i n e W e H i - T G . N K H 1 A , t h e NK r e a c t i v e m o n o c l o n a l s h o w e d v e r y s t r o n g r e a c t i v i t y t o t h e h y b r i d l i n e s WC2 a n d WC6 w h i c h e x p r e s s t h e l a r g e s t a m o u n t s o f C A M A L a s s u g g e s t e d b y E L I S A r e s u l t s . C L B g r a n , a n N A 1 r e a c t i v e m o n o c l o n a l , s h o w s r e a c t i v i t y 47 Table D. R e a c t i v i t y of various MAba with r e a c t i v i t y f o r myeloid markers with the somatic ce11•hybrids. Cy t o s p i n p r e p a r a t i o n s were t e s t e d . C e l l Line Monoclonal A n t i b o d y ^  NKH1A1 CRIS-6 2 94-3D1 3 JOAN-1 4 M101 5 KD3* GA-1 7 CLB LFA l / l 8 C1PAN 9 CLB g r a n 1 0 WEHI - - -WC-1 - ++ WC-2 •••• + ++• UC-3 - •+ ++• VC-4 - • WC-6 •••• * 1. NK c e l l r e a c t i v e . 2. Monocyte r e a c t i v e , not r e a c t i v e w i t h lymphocytes, p l a t e l e t s or e r y t h r o c y t e s . J . Monocyte and p l a t e l e t r e a c t i v e . 4. Fan r e a c t i v e (myeloid • lymphoid). 5. F a n r e a c t i v e (myeloid • lymphoid). 6. Fanreactive (myeloid + lymphoid). 7. Myeloid marker (mainly g r a n u l o c y t e s ) . 8. NK c e l l r e a c t i v e , recognizes LFA 1/1. 9. Panreactive (human and other primate c e l l s ) -10. NA1 antigen r e a c t i v e . 11. • signs i n d i c a t e i n t e n s i t y of inmunoperoxidase s t a i n i n g going from ± to +•++. - - ++ - -•f++ +++ ++++ ++++ ++ •+ +• + ++ - + + - • + + t + +++ ++ ++•+ + + •••+ o n l y t o t h e h i g h C A M A L e x p r e s s i n g h y b r i d WC2 ( a s s h o w n b y E L I S A ) a n d n o t t o a n y o f t h e o t h e r h y b r i d s o r W e H i - T G . T h u s , w h i l e a h i g h f r e q u e n c y o f r e a c t i v i t y w a s o b s e r v e d w i t h a n u m b e r o f m y e l o i d a s s o c i a t e d m o n o c l o n a l a n t i b o d i e s ( C R I S - 6 , J O A N - 1 , C L B L F A 1 / 1 ) , n o a b s o l u t e p a t t e r n o f l i n k a g e c o u l d b e c o n s t r u e d f r o m t h e s e e x p e r i m e n t a l f i n d i n g s . ( b ) D o t B l o t A n a l y s i s f o r i d e n t i f i c a t i o n o f " A l u " S e q u e n c e s : I m m u n o l o g i c a l a n d b i o c h e m i c a l e v i d e n c e f o r h u m a n m y e l o i d a n t i g e n s a s w e l l a s t h e 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 C A M A L s u g g e s t e d t h a t h u m a n D N A w a s p r e s e n t i n o u r c e l l h y b r i d s . I n o r d e r t o v e r i f y t h i s c o n t e n t i o n we p e r f o r m e d D o t B l o t a n a l y s i s o f h y b r i d D N A u s i n g t h e B L U R 8 p l a s m i d p r o b e w h i c h p o s s e s s e s t h e h u m a n r e p e t i t i v e A l u s e q u e n c e . R e s u l t s a r e s h o w n i n F i g u r e 5, p o s i t i v e c o n t r o l s p P D 8 ( B L U R 8) h y b r i d i z e d t o i t s e l f a n d 8 3 4 8 h y b r i d i z e d t o A l u s e q u e n c e s w i t h i n t h e p P D 8 p r o b e s v e r i f y t h e a b i l i t y o f t h i s n i c k - t r a n s l a t e d p r o b e t o h y b r i d i z e h u m a n DNA s e q u e n c e s a s w e l l a s p B R 3 2 2 . S i n c e t h e d o t t e d v o l u m e s o f s p e c i f i c D N A a m o u n t s w e r e n o r m a l i z e d a c r o s s s a m p l e s , t h e r e l a t i v e d o t d i a m e t e r s a n d r e s p e c t i v e i n t e n s i t i e s o f h y b r i d i s e d DNA d o t s , w e r e a b l e t o p r o v i d e s o m e i n f o r m a t i o n o n h u m a n DNA q u a n t i t i e s w i t h i n h y b r i d c e l l s . R e l a t i v e D N A a m o u n t s p r e s e n t i n h y b r i d s c a n b e o r d e r e d i n t e r m s o f d e c r e a s i n g q u a n t i t i e s a s f o l l o w s : WC 6 > WC 4 > WC 3 > WC 1 > WC 5 > W e H i . 49 F i g u r e 5. Dot b l o t a n a l y s i s f o r d e t e c t i o n of human r e p e t i t i v e DNA sequences i n WeHi-TG and WC DNA. DNA i s s p o t t e d i n d u p l i c a t e at 500, 250, 125 and 50 ng. pPD8 r e f e r s to the A l u c o n t a i n i n g plasmid probe and 8348 i s a human DNA p o s i t i v e c o n t r o l . 50 (c) A n a l y s i s of Hybrid C e l l K a r y o t y p e s i Karyotype a n a l y s i s was c a r r i e d out on a l l h y b r i d l i n e s t o determine whether any of the h y b r i d s c o n t a i n e d whole human chromosomes or i d e n t i f i a b l e human t r a n s l o c a t i o n s t o mouse chromosomes. Karyotype r e s u l t s a r e shown i n F i g u r e 6 , WeHi-TG the parent l i n e , e x h i b i t e d 32 normal mouse chromosomes and 6 d i s t i n c t marker chromosomes. A l l h y b r i d c e l l s karyotyped r e v e a l e d an i n c r e a s e i n chromosome number as well as s e v e r a l new marker chromosomes as l i s t e d i n T a b l e I I I . D e s p i t e the i n c r e a s e i n chromosome number as w e l l as the emergence of new marker chromosomes, no ob v i o u s chromosomes or segments of human chromosomes were demonstrated u s i n g Giemsa banding or d i f f e r e n t i a l s t a i n i n g p r o c e d u r e s . Thus, our r e s u l t s suggest t h a t t r a n s l o c a t i o n of human DNA to murine chromosomes has taken p l a c e (as evidenced by HAT r e s i s t a n c e which i s a t r a i t mapped t o the long arm of the X chromosome, CAMAL p r o d u c t i o n and e x p r e s s i o n of other human myeloid a s s o c i a t e d markers) i n the absence of i d e n t i f i a b l e human chromosome m a t e r i a l . 52 F i g u r e b. a) G-Banded k a r y o t y p e o-f the WeHi-TG c e l l l i n e . M1-M6 re-fer t o c h a r a c t e r i s t i c markers o-f t h i s c e l l l i n e . b) G-Banded k a r y o t y p e of the WC1 c e l l l i n e . M5' i s an isochromosome d e r i v a t i v e of a segment of M5. M7-M10 are new markers c h a r a c t e r i s t i c of WC1. No i d e n t i f i a b l e human chromosomes or segments a r e pres e n t . 53 J i U fi S f a* OA M 1 I 5 M ' M 2 M 3 M 4 M S M 6 a. _ _ tit nil un n i t m i i i i m w,i uu Mt SSfttf IH 86 88SS ftHH 81 fid S3£?> §ft* iAASe C « \ ft 8 / "1 M 2 M 3 M 4 M 5' M 7 M 8 M 9 M 10 54 F i g u r e 6. c) G- Banded k a r y o t y p e o-f t h e WC 2 e e l 1 l i n e d) G-Banded k a r y o t y p e o-f t h e WC 4 c e l l l i n e . 55 IU H'tj UH MM MM M2 i l l Un II M 8 t SaS*. uses lift* It 33* III* | . a n «»«« M M » 8 | • t i « Ml M 5 1 M9 M10 M13 C. 169 edit » I T fillr i l l ! i l l ! III! bi Hit mm sift •&* nisi iiic itus *t«ft » u u i I f l iS U t ». • « ! • M3 M6 M7 M8 M13 M M d 56 Figure 6. e) G-Banded karyotype o-f the WC 6 c e l l l i n e . 57 m am 88011 fiftOfi ansa M O&M o a t I 1 f i r A f t ft • M2 M3 M5 1 M9 M i l M12 M 13 58 TABLE I I I CELL LINE TOTAL CHROMOSOME « WeHi MARKER NEW MARKER CHROMOSOME CHROMOSOME WeHi WC1 WC2 WC3 WC4 WC6 38 80 78 7? 82 59 6 4 1 2 2 2 5 4 5 5 59 I I I . Ut i 1 i z at ion of WC_Cel 1 _1 LQes_as_Mgdel s_f or Treatment of The development o-f an i n t e r s p e c i e s somatic c e l l h y b r i d e x p r e s s i n g human leukemia a s s o c i a t e d a n t i g e n on i t s s u r f a c e (as shown by FACS a n a l y s i s ) yet b e i n g o-f murine o r i g i n had i m p l i c a t i o n s t o us -for the development of an i n v i v o model f o r cancer t h e r a p y . The a b i l i t y of WC c l o n e s t o grow i n H-2 d Balb/c mice as well as t h e c e l l s u r f a c e e x p r e s s i o n of CAMAL p r o v i d e d us with a r a t i o n a l e f o r the use of our h y b r i d s f o r human leukemia models. Work i n our l a b o r a t o r y by Daphne Mew(37) on antibody-Hp conjugated tumor s p e c i f i c d e s t r u c t i o n i n an i n v i v o mouse sytem, p r o v i d e d us w i t h the background f o r our p r e l i m i n a r y work. De t e r m i n a t i o n of Hp c o n j u g a t e l e v e l s f o r an i n i t i a l i n v i t r o k i l l i n g assay was performed by t i t r a t i o n of Hp-conjugates ( F i g u r e 7) on a f i x e d number of c e l l s (1 x 1 0 6 ) , e i t h e r WeHi or WC 6. The hematoporphyrin l e v e l chosen was t h a t which maximized s p e c i f i c k i l l i n g and minimized t o x i c e f f e c t s of hematoporphyrin by i t s e l f . T i t r a t i o n r e v e a l e d t h a t 12 ng Hp/10 6 c e l l s was the optimal l e v e l f o r our h y b r i d system. Consequently a l l experiments performed i n v o l v e d the use of Hp-conjugate c o n c e n t r a t i o n s of 12 ng/10 6 c e l l s . P r e l i m i n a r y r e s u l t s demonstrating a n t i g e n - s p e c i f i c tumor d e s t r u c t i o n a r e shown i n F i g u r e 8. High l e v e l s of k i l l i n g o c c u r r e d i n t h e WC 4 and WC 6> c e l l l i n e s with both Hp conjugated CAMAL-l and r a b b i t anti-Hu whereas none o c c u r r e d with the other 60 preparations. The antibody preparations f a i l e d to demonstrate any s p e c i f i c k i l l i n g when used to treat the parent c e l l l i n e WeHi-TG. In a d d i t i o n , the WC2V c e l l l i n e , ( a non CAMAL expressing variant of WC2) was un-affected by either Hp conjugated CAMAL-l or rabbit anti-Hu a n t i s e r a , suggesting complete loss of CAMAL-l and other human myeloid surface ant igens. 61 F i g u r e 7 a) T i t r a t i o n of hematoporphyrin compounds on WeHi-TG at -fixed c e l l numbers o-f 1 x 1 0 6 were t r e a t e d w i t h v a r i o u s amounts o-f d i f f e r e n t Hp c o n j u g a t e s . V i a b i l i t y was determined by e o s i n y e x c l u s i o n . ( • # ), Rabbit a n t i Human-Hp; ( • — • ), MAb CAMAL-Hp; ( A — • ), MAb a n t i L1210-Hp; ( A A ), Hematoporphyrin alone Hp; 62 W I H t H»0 T 8» 60 £ 40 65 F i g u r e 7. b> T i t r a t i o n o-f hematoporphyrin compounds on WC 6 at •fixed c e l l numbers o-f 1 x 10^ t r e a t e d as per a ) . (• • >, MAb CAMAL-Hp; (O O ) , Rabbit a n t i Human-Hp; (• • ), MAb a n t i L1210-Hp; ( £i A ) , Hematoporphyr i n alone Hp; 64 Figure 8. Survival o-f c e l l s o-f the WeHi-TG, WC 2 V, WC 4 and WC 6 c e l l l i n e s , following treatment with 12.0 ng/10 6 c e l l s of Hp eithe r alone ( U M M > conjugated to Rabbit anti-Hu Ab (EZZ2Z7J), MAb CAMAL-l < CZ1 > or MAb anti L1210 ( ) . PBS (!==_§) control was shown in each case. 66 DISCUSSION The e x p r e s s i o n of human p r o t e i n s i n i n t e r s p e c i f i c (Human-Mouse) h y b r i d s depends not o n l y on t h e p r e s e n c e of t h e genes f o r t h e s e p r o t e i n s but on a p p r o p r i a t e i n t r i n s i c and/or e x t r i n s i c r e g u l a t o r y f a c t o r s . I n t r a l i n e a g e f u s i o n p a r t n e r s p r e s u m a b l y p r o v i d e f o r t h i s r e g u l a t o r y environment and t h u s a l l o w e x p r e s s i o n of c e r t a i n l i n e a g e a s s o c i a t e d p r o t e i n s o v e r o t h e r s ( 2 7 ) . In t h i s s t u d y we p r e s e n t e v i d e n c e f o r t h e s u c c e s f u l f u s i o n of a mouse m y e l o i d c e l l l i n e and human m y e l o i d c e l l s (from a p a t i e n t w i t h CGL), and t h e subsequent e x p r e s s i o n of human m y e l o i d p r o t e i n s as w e l l as a l e u k e m i a - a s s o c i a t e d p r o t e i n . The e x p r e s s i o n of t h i s 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 , CAMAL, as w e l l a s o t h e r m y e l o i d a n t i g e n s i n our h y b r i d s has been v e r i f i e d u s i n g b i o c h e m i c a l <immunoprecipi t a t i o n s ) , c y t o l o g i c a l (immunoperoxidase) and i m m u n o l o g i c a l (ELISA, FACS a n a l y s i s , Hp CAMAL-1 s p e c i f i c c e l l d e s t r u c t i o n ) methodology. The a p p a r e n t m o l e c u l a r w e i g h t of CAMAL e x p r e s s e d o n / i n h y b r i d c e l l s a p p e a r s t o have a v a l u e of a p p r o x i m a t e l y 68000 d a l t o n s . The c o n c u r r e n t p r e s e n c e of a v a r i e t y of human m y e l o i d markers and t h e CAMAL a n t i g e n s u g g e s t e d t o us t h e p o s s i b i l i t y of l i n k a g e w i t h some of t h e s e genes but f u r t h e r s t u d y u s i n g s p e c i f i c DNA p r o b e s must be done t o v e r i f y t h i s ( m y e l o i d as w e l l as CAMAL a n t i g e n s p e c i f i c p r o b e s ) . 68 Somatic c e l l h y b r i d c l o n e s WC 4 and WC 6 were chosen f o r t h e m a j o r i t y of our s t u d i e s due t o t h e i r growth s t a b i l i t y as w e l l as t h e i r c o n s i s t e n t e x p r e s s i o n of CAMAL over t h e c o u r s e of t h e i r one year i n c o n t i n u o u s c u l t u r e . E x p r e s s i o n of CAMAL by WC h y b r i d s was shown t o be v a r i a b l e by ELISA, i m m u n o p r e c i p i t a t i o n (WC 6 showing more o v e r a l l CAMAL as compared w i t h WeHi) as w e l l as FACS IV a n a l y s i s (WC 4 showing more c e l l s u r f a c e CAMALthan WeHi). These r e s u l t s were c o n f i r m e d when c e l l s u r f a c e a n t i g e n dependent Hp-CAMAL k i l l i n g showed WC 4 t o be more s u s c e p t i b l e t o c e l l d e s t r u c t i o n as compared w i t h WC 6 . A l t h o u g h we have no d a t a on c e l l s u r f a c e e x p r e s s i o n of human m y e l o i d a n t i g e n s on WC 4 , r e a c t i v i t y t o human m y e l o i d a n t i g e n s p e c i f i c monoclonal a n t i b o d i e s u s i n g immunoperoxidase, s u g g e s t s a l a r g e v a r i e t y of m y e l o i d a n t i g e n s a r e p r e s e n t . Presumably t h i s would p r o v i d e f o r g r e a t e r k i l l i n g of WC4 as compared w i t h WC6 when t r e a t e d w i t h t h e p o s i t i v e c o n t r o l R&Hu-Hp c o n j u g a t e s . Some of t h e h y b r i d s d e s c r i b e d h e r e (WC 6 f o r e x a m p l e ) , produce c o n s i d e r a b l y more CAMAL tha n do human c e l l l i n e s d e r i v e d from human m y e l o i d l e u k e m i a s , t h u s p r o v i d i n g a s u i t a b l e c e l l l i n e f o r i n y i . y g 1 abeling,CAMAL f u n c t i o n a l s t u d i e s and p o s s i b l e c l o n i n g of t h e CAMAL gene. Dot b l o t a n a l y s i s as w e l l as a m i n o p t e r i n r e s i s t a n c e and e x p r e s s i o n of o t h e r human m y e l o i d a n t i g e n s p r o v i d e d s t r o n g e v i d e n c e t h a t human DNA m a t e r i a l was p r e s e n t and b e i n g e x p r e s s e d i n WC h y b r i d s . C o n t r a r y t o Dot b l o t f i n d i n g s , k a r y o t y p i c a n a l y s i s by Giemsa and d i f f e r e n t i a l s t a i n i n g f a i l e d t o r e v e a l t h e p r e s c e n c e of whole human chromosomes or v i s i b l e t r a n s l o c a t i ons. 6 9 Thus i t was not p o s s i b l e t o d e t e r m i n e u n e q u i v o c a l l y whether or n o t t h e r e were p r e s e n t s h o r t segments o-f human DNA c o d i n g -for s p e c i - f i c gene p r o d u c t s w i t h i n h y b r i d c e l l s . These r e s u l t s s u g g e s t s m a l l k a r y o t y p i c a l 1 y u n d e t e c t a b l e t r a n s l o c a t i o n s have o c c u r e d w i t h i n our h y b r i d l i n e s . C o n s e q u e n t l y , we were u n a b l e t o a s s o c i a t e or a s s i g n t h e CAMAL gene t o a p a r t i c u l a r chromosome(s). Somatic c e l l h y b r i d i s a t i o n t e c h n i q u e s have been - f r e q u e n t l y used f o r t h e p u r p o s e of a s s i g n i n g v a r i o u s gene p r o d u c t s t o s p e c i f i c chromosomes. R e c e n t work by Von Dem B o r n e has r e v e a l e d t h a t e x p r e s s i o n of a n e u t r o p h i l a n t i g e n NA1 p r e v i o u s l y mapped t o chromosome 11 can o c c u r i n t h e absence of k a r y o t y p i c a l 1 y d e t e c t a b l e chromosome 11 ( p e r s o n a l c o m m u n i c a t i o n ) . These f i n d i n g s , s u g g e s t t h a t one must e x e r c i s e c a u t i o n when a s s i g n i n g s p e c i f i c p r o t e i n s t o s p e c i f i c chromosomes i n s o m a t i c c e l l h y b r i d systems. The e x p r e s s i o n of CAMAL a n t i g e n i n or on h y b r i d c e l l l i n e s t h a t a p p a r e n t l y l a c k k a r y o t y p i c a l 1 y d e t e c t a b l e human chromosome m a t e r i a l s u ggest t h a t g enes c o d i n g f o r CAMAL a n t i g e n may p r o v i d e a s e l e c t i v e advantage t o s o m a t i c c e l l f u s i o n p r o d u c t s . T h i s c o n t e n t i o n i s s u p p o r t e d by changes i n growth c h a r a c t e r i s t i c s ( l o s s of anchorage dependant growth i e . g r o w t h a t h i g h e r d e n s i t i e s and l e s s f i b r o b l a s t i c i n a p p e a r a n c e ) , f a s t e r growth r a t e s compared w i t h WeHi (13 vs 16 h r s / d o u b l i n g ) . Presumably t h e o r i g i n a l f u s i o n p r o d u c t s p o s s e s s e d d e t e c t a b l e 70 human chromosome* and throughout the hybrids one year in cu l t u r e were l o s t . Surviving c e l l s must have retained that portion of the X chromosome coding for the HPRT enzyme in order for i t s continued growth in s e l e c t i v e medium HAT. The retention of other portions of human chromosomes would l o g i c a l l y provide for greater s u r v i v a l . Although the function of the CAMAL protein i s at present unknown, one can speculate as to the function of t h i s leukemia associated antigen. CAMAL i s present on a major population of CGL c e l l s thus i t i s concievable that i t may have a p o s i t i v e function i n the growth and survival of leukemic c e l l s . The o r i g i n and function of the CAMAL gene product i s cur r e n t l y being investigated. In addition, analysis of DNA from our hybrid c e l l l i n e s using DNA probes from a human chromosome l i b r a r y i s presently underway, in an attempt to elucidate which human gene sequences are present. Isoenzyme analysis of our hybrids w i l l further v e r i f y the prescence of human gene segments. The successful antibody mediated hematoporphyrin destruction of hybrid c e l l s expressing s p e c i f i c antigens has v e r i f i e d the potential use of MAb-Hp conjugate for "magic b u l l e t type" cancer immunotherapy. The s p e c i f i c homing a b i l i t y of antibodies coupled with the destructive e f f e c t s of hematoporphyrin, provides for a p o t e n t i a l l y powerful tool for the treatment of cancer victims. These in yi.tro r e s u l t s 71 demonstrated that WC hybrid c e l l s posess c h a r a c t e r i s t i c s of human leukemia s u i t a b l e -for development into an in vivo mouse model for human leukemia study. 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