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An examination of in vitro erythropoiesis by utilizing agents that mimic the in vitro activity of erythropoietin Murthy, Sudish C. 1987

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AN EXAMINATION OF IN VITRO ERYTHROPOIESIS BY UTILIZING AGENTS THAT MIMIC THE IN VITRO ACTIVITY OF ERYTHROPOIETIN By Sudish C. Murthy Sc., University of C a l i f o r n i a at Los Angeles, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology, Faculty of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1987 © Sudish C. Murthy, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/8-n ABSTRACT The major in vivo hormonal regulator of terminal erythropoiesis is erythropoietin (Ep). This 38,000 dalton acidic glycoprotein has been shown to stimulate the formation of hemoglobinizing erythroblasts. Two in vitro assays designed to measure Ep bioactivity were utilized to determine i f other agents could mimic Ep activity in vitro. It was hoped that this approach might yield insights into the mechanism of action of Ep. Several agents have now been identified, and two, dimethyl sulfoxide (DMSO) and sodium orthovanadate had previously been shown (in other systems) to stimulate membrane phosphorylation changes. Accordingly, Ep, DMSO and sodium orthovanadate were assayed with Y-^^P-ATP and plasma membranes purified from Ep-responsive cells to determine i f each could induce significant phosphorylation changes as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. It was found that while both sodium orthovanadate and DMSO effected profound phosphorylation alterations, Ep did not e l i c i t any detectable phosphorylation changes. Specifically, vanadate caused a generalized increase in membrane base-stable phosphoproteins, and DMSO reproducibly stimulated the base resistant phosphorylation of a 35 Kd membrane-associated protein. It is reasonable to postulate that the latter phosphorylation event might be responsible for the stimulatory activity of DMSO on terminally differentiating erythroid cel l s . To understand whether Ep and Ep-mimicking agents were operative on the same target c e l l population, homogeneous, virally-infected, erythroblasts were cultured in vitro and assayed for ^H-thymidine incorporation in the presence of each agent at various intervals during erythroid c e l l differentiation. It was found that Ep greatly stimulated very early, as well i i i as differentiated, erythroblasts to proliferate, while four different Ep-mimicking agents could only effect thymidine incorporation into a more mature erythroid population. From this work it is conceivable that Ep-mimicking agents stimulate in vitro erythropoiesis through specific membrane phosphorylation changes and function primarily on late erythroblasts, while the mechanism of action of Ep on primitive and late erythroblasts remains unresolved. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS i x INTRODUCTION Hemopoietic Growth C o n t r o l 1 Hemopoietic Growth Factors 1 In V i t r o E r y t h r o i d Colony Formation 8 E r y t h r o p o i e t i c Growth Factors 9 Assays of E r y t h r o p o i e t i n A c t i v i t y 12 E r y t h r o p o i e t i n 14 The E r y t h r o p o i e t i n Receptor 15 Mechanism of Ac t i o n of E r y t h r o p o i e t i n 18 Other Molecules A f f e c t i n g In V i t r o E r y t h r o p o i e s i s . . . 1 9 Growth S i g n a l Transduction 22 Thesis Objectives 29 MATERIALS and METHODS E r y t h r o p o i e t i n Bioassays 31 In V i t r o ^H-Thymidine I n c o r p o r a t i o n Assay 31 In V i t r o 59p e i n c o r p o r a t i o n Assay 32 C e l l s and C e l l Lines 33 In V i t r o Phosphorylation Assay 34 Plasma Membrane I s o l a t i o n 34 Membrane Phosphorylation 35 C e l l P u r i f i c a t i o n 36 RESULTS S t i m u l a t i o n of E r y t h r o i d C e l l s By Sodium Orthovanadate 37 Target C e l l Assessment f o r Ep and Ep-Mimicking Agents 41 Membrane-Specific P r o t e i n Phosphorylation... 45 DISCUSSION -55 REFERENCES 62 V L IST OF TABLES Page TABLE I H e m o p o i e t i c Growth F a c t o r s and T h e i r R e c e p t o r s 7 TABLE I I The a b i l i t y o f v a r i o u s a g e n t s to s u b s t i t u t e f o r Ep i n ^ H - t h y m i d i n e and 5 9 p e i n c o r p o r a t i o n a s s a y s 21 TABLE I I I The s t i m u l a t i o n o f 3 H - t h y m i d i n e u p t a k e i n t o PHZ s p l e e n c e l l s by sod ium o r t h o v a n a d a t e 38 TABLE I V The e f f e c t o f sod ium o r t h o v a n a d a t e on 5 9 p e i n _ c o r p o r a t i o n i n t o PHZ s p l e e n c e l l s 40 TABLE V The s t i m u l a t o r y e f f e c t s o f HMBA, b u t y r i c a c i d , DMSO, and sod ium o r t h o v a n a d a t e on 3 H _ t h y m i c U n e u p t a k e i n t o d i f f e r e n t i a t i n g FVA c e l l s 44 v i L I S T OF FIGURES FIGURE 1 A s i m p l e s c h e m a t i c r e p r e s e n t i n g the h e m o p o i e t i c h e i r a r c h y . M a t u r e p rogeny f rom a l l m y e l o i d and l y m p h o i d l i n e a g e s a r e though t to d e s c e n d f r om the s tem c e l l compar tment as i l l u s t r a t e d h e r e . Some h e m o p o i e t i n s o p e r a t i v e on v a r i o u s c e l l l i n -eages a r e i n c l u d e d to d e m o n s t r a t e t he numerous f o c i where h e m o p o i e s i s may be r e g u l a t e d . F o r g r o w t h f a c t o r a b b r e v i a t i o n s p l e a s e r e f e r t o t e x t 2 FIGURE 2 T h i s d i a g r a m , r e p r i n t e d f rom McDona ld and G o l d v a s s e r ( 1 2 2 ) , i l l u s t r a t e s the c o m p u t e r - g e n e r a t e d t e r t i a r y p r o t e i n s t r u c t u r e s o f human, monkey and m u r i n e Ep based on the gene s e q u e n c e o f e a c h . The c y l i n d e r s r e p r e s e n t r e g i o n s o f a h e l i x c o n f o r m a t i o n , w h i l e the a r r o w s i n d i c a t e r e g i o n s o f p o s s i b l e 0 s h e e t s t r u c t u r e . The a u t h o r s s p e c u l a t e t h a t t he a r e a s u r r o u n d i n g the d i s u l f i d e b r i d g e be tween r e s i d u e s 7 and 161 i s the a c t i v e s i t e o f a l l t h r e e Eps 16 FIGURE 3 T h i s s c h e m a t i c r e p r e s e n t s many o f t he h y p o t h e s i z e d mechanisms by w h i c h a c e l l may become a c t i v a t e d i n r e s p o n s e to a g o n i s t b i n d i n g . From t h i s d i a g r a m i t can be s e e n t h a t c e l l a c t i v a t i o n i n v o l v e s a m y r i a d o f r e l a t e d p a t h w a y s , e a c h o f w h i c h i s p o t e n t i a l l y r e g u l a t e d by a s e r i e s o f s e c o n d m e s s e n g e r s .27 FIGURE A R e p r i n t e d f rom a r e c e n t r e v i e w ( 1 6 6 ) , t h i s d i a g r a m i l l u s t r a t e s the c o m p l e x i t y o f the a g o n i s t - r e c e p t o r ( R i ) - G p r o t e i n ( G ) - p h o s p h o l i p a s e C ( P D E ) - p h o s p h a t i -d y l i n o s i t o l r e l a t i o n s h i p . The p r o d u c t s o f t h e pa thway , i n o s i t o l t r i p h o s p h a t e ( I n s P3) and d i a c y l -g l y c e r o l (DG) a r e p o t e n t b i o l o g i c a l e f f e c t o r s f o r many h o r m o n a l s y s t e m s 28 FIGURE 5 A c o m p a r i s o n o f d o s e - r e s p o n s e c u r v e s f o r Ep and sod ium o r t h o v a n a d a t e i n s t i m u l a t i n g 3 H - t h y m i d i n e i n c o r p o r a t i o n i n t o PhZ s p l e e n c e l l s . V a n a d a t e i n d u c e d c e l l t o x i c i t y a t c o n c e n t r a t i o n s above 15 uM 39 vii FIGURE 6 The decline in Ep-responsiveness as FVA cells mature is represented here. When freshly iso-lated, there appeared to be a large proportion of Ep-responsive cells in the cell poplulation. However, when cells were permitted to differen-tiate, the ability of Ep to stimulate ^ -thymi-dine incorporation decreased. It should be noted that cells maintained in culture for 24 hours, but not permitted to differentiate (cul-tured without Ep), remained as responsive to Ep as those cultured with Ep .43 FIGURE 7 Autoradiograph demonstrating the specific phos-phorylation changes induced by DMSO. The arrow indicates the 35 Kd phosphoprotein stimulated by DMSO in the absence or presence of vanadate (VAN). The buffer control lane is also repre-sented (C) . .47 FIGURE 8 Autoradiograph demonstrating the loss of the DMSO-stimulated 35 Kd phosphoprotein when 10 mM manganese chloride was substituted for 30 mM magnesium chloride in the incubation mixture. The arrow indicates the position normally occu-pied by the 35 Kd protein .48 FIGURE 9 Autoradiograph of a 1 M NaOH treated polyacryl-amide gel. Samples electrophoresed were solu-bilized membranes from red cell-free PhZ cell preparations. The arrow indicates the position of the DMSO-stimulated 35 Kd protein. The van-adate lane demonstrates an overall increase in base-stable phosphate linkages .50 FIGURE 10 Autoradiograph demonstrating the specificity of the DMSO stimulatory effect for PhZ cell mem-branes. Normal spleen (NS) and JG-1 (B6SUtA) membranes were incubated with DMSO (20% final) and Y-^P-ATP in an identical fashion to that used with PhZ cell membranes. The 35 Kd phos-phoprotein so evident with PhZ cell membranes is virtually absent from the NS and JG-1 lanes. These later two cell types had been previously shown in in vitro bioassays to be unresponsive to DMSO 52 v i i i FIGURE 11 Autoradiograph shoving the reduced (R) and non-reduced (NR) forms of the DMSO-stimulated phos-phoprotein. The difference in molecular weights between the two is approximately 1.0 Kd (the nonreduced form being 36 Kd) 54 ACKNOWLEDGEMENTS I wou ld g r a t e f u l l y l i k e to a c k n o w l e d g e the S e n i o r S t a f f . P o s t - d o c t o r a l F e l l o w s , and G r a d u a t e S t u d e n t s o f t he T e r r y Fox L a b o r a t o r y , B r i t i s h C o l u m b i a C a n c e r R e s e a r c h C e n t r e , f o r t h e i r g u i d a n c e and h e l p f u l s u g g e s t i o n s d u r i n g t h i s p r o j e c t . I wou ld e s p e c i a l l y l i k e to thank D r . G e r a l d K r y s t a l f o r s u p e r v i s i o n above and beyond the c a l l o f d u t y . A f i n a l t h a n k s to Mom and Dad . 1 HEMOPOIETIC GROWTH CONTROL The g e n e r a l l y a c c e p t e d scheme o f h e m o p o i e s i s , i l l u s t r a t e d i n F i g u r e 1, i n v o l v e s a p o o l o f p l u r i p o t e n t h e m o p o i e t i c s tem c e l l s , l o c a t e d p r i m a r i l y i n the bone mar row, t h a t s e l f - r e n e w to m a i n t a i n t h e i r numbers and a l s o d i f f e r e n t i a t e to g i v e r i s e to a s p e c t r u m o f commi t t ed p r o g e n i t o r c e l l s f rom w h i c h e r y t h r o c y t e s , m o n 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 and B and T l y m p h o c y t e s a r e d e r i v e d . I t i s a l s o now g e n e r a l l y a c c e p t e d t h a t h e m o p o i e t i c c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n a r e c o n t r o l l e d , a t l e a s t i n p a r t , by a v a r i e t y o f s p e c i f i c f a c t o r s , many o f w h i c h a r e s e c r e t e d and h e n c e a v a i l a b l e f o r s t u d y i n s o l u b l e fo rm (1) . These i n c l u d e b o t h s t i m u l a t o r y (1) and i n h i b i t o r y m o l e c u l e s ( 2 - 7 ) . However , b e c a u s e o f d i f f i c u l t i e s i n e s t a b l i s h i n g t a r g e t c e l l s p e c i f i c i t y and i n v i v o s i g n i f i c a n c e o f i n h i b i t o r s , most i n v e s t i g a t o r s have c o n c e n t r a t e d t h e i r e f f o r t s on the c h a r a c t e r i z a t i o n o f m o l e c u l e s t h a t s u s t a i n o r s t i m u l a t e c e l l s . H e m o p o i e t i c G row th F a c t o r s . S e v e r a l h e m o p o i e t i c g r o w t h f a c t o r s have now been p u r i f i e d t o h o m o g e n e i t y f r om b o t h m u r i n e and human s o u r c e s . T h e i r ma jo r s i t e s o f a c t i o n a r e i n d i c a t e d on t he h e m o p o i e t i c l i n e a g e d i a g r a m s shown i n F i g u r e 1. S i x h e m o p o i e t i c g r o w t h f a c t o r s o f m u r i n e o r i g i n have been p u r i f i e d : 1) M u r i n e g r a n u l o c y t e - m a c r o p h a g e c o l o n y s t i m u l a t i n g f a c t o r )GM-CSF) ( a l s o c a l l e d C S F - 2 and m a c r o p h a g e - g r a n u l o c y t e i n d u c e r - 1 (MGI -1)) was f i r s t p u r i f i e d f rom e n d o t o x i n s t i m u l a t e d l u n g c o n d i t i o n e d medium ( 8 ) . I t s u p p o r t s the g r o w t h FIGURE 1. A s i m p l e s c h e m a t i c r e p r e s e n t i n g t he hemo-p o i e t i c h i e r a r c h y . M a t u r e p rogeny f rom a l l m y e l o i d and l y m p h o i d l i n e a g e s a r e though t to d e s c e n d f rom the s tem c e l l compar tment as i l l u s t r a t e d h e r e . Some hemo-p o i e t i n s o p e r a t i v e on v a r i o u s c e l l l i n e a g e s a r e i n c l u d e d to d e m o n s t r a t e the numerous f o c i where h e m o p o i e s i s may be r e g u l a t e d . F o r g r o w t h f a c t o r a b b r e v i a t i o n s p l e a s e r e f e r to the t e x t . - 3 and c o m p l e t e m a t u r a t i o n o f p r o g e n i t o r s commi t t ed t o g r a n u l o c y t e o r macrophage p r o d u c t i o n ( 8 ) . Mouse GM-CSF i s a l s o c a p a b l e o f d i r e c t l y s t i m u l a t i n g some p l u r i p o t e n t s tem c e l l s to unde rgo a few d i v i s i o n s ( 9 ) , t h u s , i m p l y i n g t h a t GM-CSF r e c e p t o r s a p p e a r on s tem c e l l s u r f a c e s b e f o r e r e s t r i c t i o n t o g r a n u l o p o i e t i c d i f f e r e n t i a t i o n o c c u r s . 2 ) Mouse macrophage c o l o n y s t i m u l a t i n g f a c t o r ( M - C S F ) ( a l s o c a l l e d L c e l l C S F , C S F - 1 and MGI-1M) has been p u r i f i e d f rom L c e l l c o n d i t i o n e d medium ( 1 0 ) . I t s u p p o r t s p r o g e n i t o r s commi t t ed to macrophage p r o d u c t i o n and has no known d i r e c t e f f e c t on p l u r i p o t e n t s tem c e l l s ( 1 1 ) . 3) M u r i n e i n t e r l e u k i n - 3 ( I L - 3 ) ( a l s o c a l l e d b u r s t p r o m o t i n g a c t i v i t y (BPA) ( 1 2 ) , h e m o p o i e t i n - 2 ( H - 2 ) ( 1 3 ) , m u l t i - C S F ( 1 4 ) , T h y - 1 i n d u c i n g a c t i v i t y ( 1 5 ) , h e m o p o i e t i c c e l l g r o w t h f a c t o r (HCGF) ( 1 6 ) , mast c e l l g r o w t h f a c t o r (MCGF) ( 1 7 ) , h i s t a m i n e - p r o d u c i n g c e l l s t i m u l a t i n g f a c t o r ( 1 8 ) and p e r s i s t i n g c e l l g r o w t h f a c t o r ( P - c e l l GF ( 1 9 ) ) has been p u r i f i e d f r om medium c o n d i t i o n e d by WEH1-3B c e l l s , a m y e l o m o n o c y t i c l e u k e m i c c e l l l i n e ( 2 0 , 2 1 ) . I t i s a m u l t i l i n e a g e a c t i n g g r o w t h f a c t o r w h i c h s t i m u l a t e s p l u r i p o t e n t p r o g e n i t o r s as w e l l a s c o m m i t t e d p r e c u r s o r s o f e r y t h r o i d c e l l s , m e g a k a r y o c y t e s , n e u t r o p h i l s , m a c r o p h a g e s , mast c e l l s , and B and T l y m p h o c y t e s ( 1 4 , 1 6 , 2 2 - 2 4 ) . 4 ) M u r i n e g r a n u l o c y t e c o l o n y - s t i m u l a t i n g f a c t o r (mG-CSF) ( a l s o c a l l e d d i f f e r e n t i a t i o n f a c t o r (DF) and MGI -2 ) has been p u r i f i e d f r om e n d o t o x i n s t i m u l a t e d l u n g c o n d i t i o n e d medium ( 2 5 ) . I t shows more r e s t r i c t e d g r a n u l o p o i e t i c s t i m u l a t i n g a c t i v i t y t h a n mGM-CSF and i s f u r t h e r d i s t i n g u i s h e d f rom mGM-CSF by i t s a b i l i t y to i n d u c e WEH1-3B c e l l s to u n d e r g o t e r m i n a l d i f f e r e n t i a t i o n i n v i t r o ( 2 5 ) . 5 ) M u r i n e i n t e r l e u k i n - 1 ( m I L - 1 ) ( f o r m e r l y c a l l e d l y m p h o c y t e a c t i v a t i n g f a c t o r ( L A F ) ) has been p u r i f i e d f rom medium c o n d i t i o n e d by TPA a c t i v a t e d P388D c e l l s , a macrophage c e l l l i n e ( 2 6 ) . I t i s known to p a r t i c i p a t e i n the 4 i n d u c t i o n o f a n t i g e n - and m i t o g e n - i n d u c e d T c e l l p r o l i f e r a t i o n and i n t h e m a t u r a t i o n and f u n c t i o n a l a c t i v a t i o n o f h e l p e r T , c y t o t o x i c T , and B c e l l s ( 2 7 ) . I t a l s o f u n c t i o n s to s t i m u l a t e f i b r o b l a s t s and a v a r i e t y o f o t h e r mesenchymal c e l l s ( 27 ) and i s p r o b a b l y i d e n t i c a l to endogenous p y r o g e n , c o n t r i b u t i n g to t he f e v e r , h e a d a c h e s , and body p a i n s t h a t commonly accompany i n f e c t i o n s ( 2 8 ) . 6 ) M u r i n e i n t e r l e u k i n - 4 ( I L - 4 ) ( a l s o c a l l e d B c e l l s t i m u l a t i n g f a c t o r ( B S F - 1 ) ) has been p u r i f i e d f rom medium c o n d i t i o n e d by t he T c e l l c l o n e , C l . L y l + 2 ~ / 9 ( 2 9 ) . I L - 4 i n d u c e s the e x p r e s s i o n o f I a a n t i g e n s on r e s t i n g B -c e l l s and e n h a n c e s I g E and I g G j p r o d u c t i o n ( 3 0 - 3 2 ) . R e c e n t s t u d i e s have shown t h a t i t c a n a l s o s t i m u l a t e the p r o l i f e r a t i o n o f c e r t a i n T c e l l s and mast c e l l s , and c a n s y n e r g i z e w i t h I L - 3 to s t i m u l a t e the p r o l i f e r a t i o n o f p r i m i t i v e p r o g e n i t o r c e l l s ( 3 3 ) . The f a c t t h a t many o f t h e s e f a c t o r s have s e v e r a l names r e f l e c t s t h e i r r a n g e o f b i o l o g i c a l a c t i v i t i e s and the use o f d i f f e r e n t b i o a s s a y s f o r t h e i r p u r i f i c a t i o n by i n d e p e n d e n t i n v e s t i g a t o r s . W i t h t he e x c e p t i o n o f G - C S F , most o f t h e s e s i x mouse f a c t o r s have l i t t l e a c t i v i t y on e q u i v a l e n t human t a r g e t c e l l s . However , a n a l o g o u s gene p r o d u c t s o f human o r i g i n have now been i d e n t i f i e d f o r a l l o f t h e s e m u r i n e - d e r i v e d g r o w t h f a c t o r s . I n two c a s e s , i . e . human I L - 3 and I L - 4 , t he l i n k h a s been made t h r o u g h a g e n e t i c r a t h e r t han a p r o t e i n p u r i f i c a t i o n s t r a t e g y and t he r e s u l t s have no t y e t a p p e a r e d i n p u b l i s h e d f o r m . H e m o p o i e t i c g r o w t h f a c t o r s p u r i f i e d to h o m o g e n e i t y f r om human s o u r c e s i n c l u d e : 1) Human GM-CSF ( a l s o c a l l e d C S F - a (34 ) and p l u r i p o i e t i n - a ( 3 5 ) ) i s the human a n a l o g o f m u r i n e G M - C S F . I t was f i r s t p u r i f i e d f rom medium c o n d i t i o n e d by Mo c e l l s , a T l y m p h o b l a s t c e l l l i n e ( 3 6 ) . A s i d e f rom i t s e x p e c t e d 5 granulocyte-monocyte colony stimulating activity, hGM-CSF also stimulates the formation of multi-lineage and large erythroid colonies (37), hence the term pluripoietin-ct. Whether or not it acts directly on the progenitors of these latter colony types is s t i l l controversial. Human GM-CSF is also an effective stimulator of many human myeloid leukemic progenitor cells (38,39) but has no effect on mouse hemopoietic progenitors. 2) Human M-CSF (also called CSF-1) is the analog of murine M-CSF and has been purified from urine (40). Because of its species cross-reactivity, most studies with hM-CSF have actually been performed with mouse target cells. 3) Human IL-2 (also called T cell growth factor (TCGF)), was first purified from medium conditioned by the human leukemic T cell line, JURKAT (41). It triggers the proliferation of both mouse and human activated T lymphocytes and may also stimulate B-cells via specific IL-2 receptors (42). 4) Human G-CSF (also called CSF-6 (34) and pluripoietin (43)) was first purified from medium conditioned by 5637 (44), a human bladder carcinoma cell line. Like mG-CSF, hG-CSF stimulates granulocyte colony formation from normal precursors and induces terminal differentiation of leukemic cells in vitro (44). Also, hG-CSF is able to support the growth and maturation of multi-lineage and erythroid colonies, suggesting action at multiple levels of hemopoietic progenitor cell differentiation. 5) Erythropoietin (Ep) has been purified from the urine of aplastic anemic patients (45). Ep is a circulating hormone which stimulates relatively mature erythroid precursors to proliferate and differentiate into red blood cells (46). There is no evidence that erythropoietin can act directly on pluripotent cells in vivo (47), though, in vitro, stimulation of r e l a t i v e l y primitive erythroid cells has been demonstrated (48). Human erythropoietin acts on all mammalian species thus far tested (49). 6 6) E r y t h r o i d p o t e n t i a t i n g a c t i v i t y (EPA) was p u r i f i e d f r om Mo c e l l c o n d i t i o n e d medium ( 5 0 ) . I t a p p e a r s ( f r o m gene s e q u e n c e c o m p a r i s o n s ) to be an i n h i b i t o r o f m e t a l l o p r o t e i n a s e ( 5 1 ) . EPA w a s , h o w e v e r , p u r i f i e d on t he b a s i s o f i t s a p p a r e n t l y s p e c i f i c a b i l i t y to s t i m u l a t e , i n v i t r o , c o m m i t t e d e r y t h r o i d p r o g e n i t o r s o f human and mouse o r i g i n ( 5 2 ) . 7) H e m o p o i e t i n - 1 ( H - l ) i s a v e r y r e c e n t l y i s o l a t e d f a c t o r f r om medium c o n d i t i o n e d by 5637 c e l l s ( 5 3 ) . A l t h o u g h d e r i v e d f rom human c e l l s , i t has been d e t e c t e d and p u r i f i e d by i t s a b i l i t y to s y n e r g i z e w i t h M-CSF t o i n c r e a s e M-CSF r e c e p t o r e x p r e s s i o n i n marrow c e l l s f rom m ice p r e t r e a t e d w i t h 5 -f l u o r o u r a c i l . H e m o p o i e t i n - 1 can a l s o s y n e r g i z e w i t h I L - 3 t o enhance m u l t i -l i n e a g e c o l o n y f o r m a t i o n by marrow c e l l s f rom 5 - f l u o r o u r a c i l t r e a t e d m i c e ( 5 4 ) , and i n t h i s r e s p e c t i s s i m i l a r to mouse I L - 4 . Many a d d i t i o n a l a c t i v i t i e s d i s t i n c t f rom t h o s e known t o be a t t r i b u t a b l e to the m u r i n e and human g r o w t h f a c t o r s d e s c r i b e d above have a l s o been r e p o r t e d ( e g . 5 5 - 5 8 ) bu t t h e s e a w a i t p u r i f i c a t i o n and amino a c i d s e q u e n c i n g t o e s t a b l i s h t h e i r u n i q u e i d e n t i t i e s . I t i s l i k e l y t h a t some as y e t u n c h a r a c t e r i z e d h e m o p o i e t i c r e g u l a t o r s , as w e l l as some a l r e a d y d i s c u s s e d a b o v e , may a c t i n v i v o i n a c e l l s u r f a c e bound f o r m . An o v e r v i e w o f t he v a r i o u s h e m o p o i e t i c g r o w t h f a c t o r s and t h e i r r e c e p t o r s i s p r e s e n t e d i n T a b l e I. U n t i l r e c e n t l y , b i o l o g i c a l and b i o c h e m i c a l s t u d i e s w i t h t h e s e h e m o p o i e t i c g r o w t h f a c t o r s were hampered by t h e i r i n a d e q u a t e a v a i l a b i l i t y i n p u r e f o r m , s i n c e t h e y a r e p r e s e n t a t v e r y low l e v e l s i n serum o r medium c o n d i t i o n e d . I s o l a t i o n o f the genes t h a t code f o r t h e s e g r o w t h f a c t o r s has now made p o s s i b l e t he p r e p a r a t i o n o f l a r g e q u a n t i t i e s o f r e c o m b i n a n t p r o d u c t s . To date t he c l o n i n g o f s i x m u r i n e genes ( I L - 1 ( 5 9 ) , I L - 2 ( 6 0 ) , I L - 3 ( 6 1 ) , I L - 4 ( 6 2 ) , GM-CSF ( 63 ) and Ep ( 6 4 ) ) and s e v e n human genes (GM CSF ( 6 5 ) , G-CSF ( 6 6 ) , M-CSF Table I Hemopoietic Growth F a c t o r s and T h e i r Receptors F a c t o r (Synonyms) F a c t o r Mwt (Kd) Receptor Mwt (Kd) Receptor N o . / C e l l H o u s e GM-CSF ( C S F - 2 , MGI-1) M-CSF ( C S F - 1 , MGI-1M) I L - 3 ( B P A , m u l t i - C S F ) G-CSF ( D F , MGI-2) I L - 1 ( L A F ) I L - 4 ( B S F - 1 ) Human GM-CSF ( C S F a;, p 1 u r i p o i e t i n at) M-CSF ( C S F - 1 ) I L - 2 ( T C G F ) G-CSF ( C S F P , p l u r i p o i e t i n ) Ep EPA H - l 23 (Ref. 8) 70 ( d i a e r ) (Ref. 10) 26-28 (Ref. 1 2 , 1 3 ) 25 (Ref. 25) 13-19 (Ref. 26) 16 & 21 (Ref. 29) 22 (Ref. 36) 45 ( d i n e r ) (Ref. 40) 16 (Ref. 41) 18 (Ref. 4 3 ) , 3 0 (Ref. 34-39 (Ref. 45) 28 (Ref. 50) 17 (Ref. 53) 44 ) 51 (Ref. 72) 165 (Ref. 73) 67 (Ref. 74) 150 (Ref. 75) ND a ND ND ND 55,60 (Ref. 76) ND 100 (Ref. 132) ND ND 7 0 - 3 5 0 (Ref. 72) 3 0 0 0 - 5 0 , 0 0 0 (Ref. 73) 5 0 - 1 1 5 , 0 0 0 (Ref. 74) 50-500 (Ref. 75) ND ND 1 0 0 - 2 0 0 (Ref. 77) ND 2 0 , 0 0 0 - 1 0 0 , 0 0 0 (Ref. 156] ND 6 0 0 - 7 0 0 (Ref. 127) ND ND a N D = n o t d o n e . 8 (78), IL-1 (67), IL-2 (68), Ep (69) and EPA (70)) have been reported. Sequence data has also made it possible to undertake automated peptide synthesis (71). With these techniques, it is now possible to begin detailed studies of the mechanisms of growth factor action on specific target cells, and to infuse these proteins into animals to determine possible effects in vivo. In Vitro Erythroid Colony Formation Much of the current knowledge concerning hemopoietic cell biology has been derived from in vitro colony assays. The earliest studies demonstrated that individual colonies (formed by committed progenitors) of granulocytes and macrophages could be grown in semi-solid medium (79). It was observed that the presence of either a cell 'feeder' layer (80) or conditioned medium from 'feeder' cells (81) was essential for optimal colony formation, and thus, it was postulated that developing hemopoietic progenitor cells required certain soluble colony stimulating factors (CSF's) released by these feeder cells. Later, assays for megakaryocyte (82), lymphoid (83), and mixed colony progenitors (84) were developed. The first in vitro colony assay for erythroid progenitor cells was reported in 1971 (85). In this report it was observed that mouse fetal liver cells plated in a plasma clot culture system (supplemented with the appropriate CSF—i.e. erythropoietin) yielded small erythroid clusters consisting of 8-64 hemoglobin containing cells. The cell g i v i n g r i s e t o s u c h clusters was subsequently termed a colony forming unit-erythroid (CFU-E). Other studies revealed that the CFU-E was a late progenitor (i.e. r e l a t i v e l y 9 m a t u r e ) o f t he e r y t h r o i d pathway ( 8 6 ) , and t h a t a more p r i m i t i v e p r o g e n i t o r f o u n d i n m u r i n e bone marrow c a p a b l e o f f o r m i n g l a r g e m u l t i - c l u s t e r e d e r y t h r o i d c o l o n i e s , the b u r s t f o r m i n g u n i t - e r y t h r o i d ( B F U - E ) , e x i s t e d ( 8 7 ) . C h a r a c t e r i z a t i o n by p r o l i f e r a t i v e c a p a c i t y , s e n s i t i v i t y t o c e l l c y c l e i n h i -b i t o r s , c e l l vo lume and t ime c o u r s e o f c o l o n y f o r m a t i o n h e l p e d d e l i n e a t e t h e s e c l a s s e s o f e r y t h r o i d p r o g e n i t o r s and t h e i r o r d e r i n t he h e m p o i e t i c h i e r a r c h y ( 8 8 - 9 1 ) . P r o o f o f the s i n g l e c e l l o r i g i n o f e r y t h r o i d c o l o n i e s fo rmed i n p l a s m a c l o t c u l t u r e s was p r o v i d e d i n 1976 by Cormack ( 9 2 ) , who emp loyed t i m e - l a p s e p h o t o g r a p h y to d e m o n s t r a t e t he deve lopmen t o f e r y t h r o i d c o l o n i e s f rom one s i n g l e c e l l . Subsequen t s t u d i e s ( w i t h enzyme and c h r o m o -s o m a l m a r k e r s ( 9 3 , 9 4 ) ) showed b o t h human and m u r i n e e r y t h r o i d b u r s t s t o be o f a c l o n a l n a t u r e , and t h e r e f o r e , d e r i v e d f rom a s i n g l e h e m o p o i e t i c p r o g e n i t o r . E r y t h r o p o i e t i c G row th F a c t o r s I n v i t r o c o l o n y a s s a y s have a l l o w e d f o r d e t a i l e d s c r u t i n y and c r i t i c a l e x a m i n a t i o n o f h e m o p o i e s i s . More s p e c i f i c a l l y , many serum and c o n d i t i o n e d m e d i u m - d e r i v e d g r o w t h f a c t o r s ( h e m o p o i e t i n s ) , i s o l a t e d f r om b o t h m u r i n e and human s o u r c e s , have been found to have p r o f o u n d r e g u l a t o r y i n f l u e n c e s on t he i n v i t r o p r o l i f e r a t i o n and d i f f e r e n t i a t i o n o f h e m o p o i e t i c p r o g e n i t o r c e l l s . T h e s e h e m o p o i e t i n s were i n i t i a l l y d e s c r i b e d as a c t i v i t i e s c a p a b l e o f s t i m u l a t i n g t he g r o w t h o f l i n e a g e - s p e c i f i c c e l l c o l o n i e s i n s e m i - s o l i d medium ( 9 5 ) . As r e v i e w e d b e f o r e , s e v e r a l have now been c h a r a c t e r i z e d e x t e n s i v e l y . E v i d e n c e f o r s o l u b l e r e g u l a t o r s o f e r y t h r o p o i e s i s was o b t a i n e d t h i r t y y e a r s a g o , though the e x i s t e n c e o f s u c h f a c t o r s was f i r s t h y p o t h e s i z e d by C a r n o t i n 1906 ( 9 6 ) . I n 1950 R e i s s m a n d e m o n s t r a t e d t h a t i f one r a t of a p a r a b i o t i c p a i r was e x p o s e d to h y p o x i a , bo th became p o l y c y t h e m i c ( 9 7 ) . O t h e r 10 investigators later documented the presence of a factor (i.e. Ep) in the serum of anemic animals, that was capable of stimulating erythropoiesis when reintroduced into normal recipients (as determined by an increase of ^9pe incorporation into maturing blood cell precursors) (98). It has since been shown that the main site of Ep biosynthesis in the adult mouse (and human) is the kidney (99), where Ep production is modulated by fluctuating oxygen tensions. Fetal liver has also been implicated as a primary source of Ep (100) , and Zucali has clearly demonstrated that fetal liver production of Ep is oxygen tension related as well. It is worthy of note that bilaterally nephrectomized adult rats, subsequently exposed to hypoxia, maintain detectable levels of Ep within their serum. An 80% partial hepatectomy in these animals, however, results in a complete decline of detectable serum Ep (101) . Thus, adult liver, like fetal liver, may prove to be an important extrarenal site of Ep biosynthesis. The absolute requirement of Ep by terminally differentiating red cells was shown in 1957 (99). Jacobsen and Goldwasser demonstrated that all morphologically distinguishable erythroid precursors disappeared from the marrow and spleen of mice whose hematocrits were artificially raised by hypertransfusion. Addition of Ep resulted in a wave of newly hemoglobinizing precursors. The CFU-E content of spleen and marrow has more recently been shown to decrease in mice made polycythemic (102,103). In vitro evidence of the absolute requirement for Ep by CFU-E has been provided by delayed addition experiments (104). The dependence of more primitive erythroid cells (BFU-E's) on Ep i s l e s s clear. The absolute numbers of BFU-E, unlike CFU-E, are unchanged during polycythemia (105), and neither hypertransfusion nor anemia drastically a l t e r their cycling state (106). Yet, there is some evidence to suggest that high 11 c o n c e n t r a t i o n s o f Ep i n c r e a s e t he p r o p o r t i o n o f B F U - E i n a c t i v e c e l l c y c l e ( i . e . i n S - p h a s e ) ( 1 0 5 ) . I t a p p e a r s , h o w e v e r , t h a t t he m a j o r s t i m u l a t o r y f a c t o r f o r B F U - E , i n i t i a l l y f ound i n l e u k o c y t e c o n d i t i o n e d - m e d i u m ( 1 0 7 ) , i s I L - 3 ( s e e page 3 ) . R e c e n t s t u d i e s s u g g e s t t h a t t h i s f a c t o r ' s r o l e i n h e m o p o i e s i s i s o f a p e r m i s s i v e n a t u r e ( 1 9 6 ) . A t h i r d p r o t e i n o p e r a t i v e d u r i n g e r y t h r o p o i e s i s i s e r y t h r o b l a s t e n h a n c i n g f a c t o r ( E E F ) . EEF i s a serum p r o t e i n w h i c h a p p e a r s to have a m o l e c u l a r w e i g h t o f 130 Kd (by g e l f i l t r a t i o n ) and an i s o e l e c t r i c p o i n t o f 4 . 8 ( f r o m c h r o m a t o f o c u s i w g e x p e r i m e n t s ) ( 1 0 8 , 1 0 9 ) . T h i s p r o t e i n i s a v e r y l a t e a c t i n g s t i m u l a t o r o f i n v i v o e r y t h r o p o i e s i s . From i n v i t r o s t u d i e s , EEF has been shown to i n c r e a s e the v i a b i l i t y o f t e r m i n a l l y d i f f e r e n t i a t i n g e r y t h r o b l a s t s . When i n j e c t e d w i t h Ep i n t o p o l y c y t h e m i c m i c e , i t a c t s s y n e r g i s t i c a l l y ( w i t h Ep) i n s t i m u l a t i n g new red c e l l p r o d u c t i o n . I t h a s t hus been h y p o t h e s i z e d t h a t EEF and Ep a r e no t a c t i n g on the same c e l l p o p u l a t i o n , bu t r a t h e r , t h a t EEF a f f e c t s m a i n l y p o s t - m i t o t i c h e m o g l o b i n s y n t h e s i z i n g e r y t h r o b l a s t s , w h i l e Ep s t i m u l a t e s d i v i d i n g C F U - E . T h i s was e v i d e n c e d by i n v i t r o i r o n i n c o r p o r a t i o n s t u d i e s u s i n g t he m i t o t i c s p i n d l e i n h i b i t o r , c o l c e m i d . ( 1 0 9 ) . R e c e n t l y , y e t a n o t h e r b i o l o g i c a l l y r e l e v a n t f a c t o r p a r t i c i p a t i n g i n e r y t h r o p o i e s i s has been e l u c i d a t e d ( 1 1 0 ) . S u p e r n a t a n t s f rom mouse s p l e e n c e l l c u l t u r e s c o n t a i n a p r o t e i n w h i c h a c t s i n a s i m i l a r manner to Ep i n s t i m u l a t i n g t he f o r m a t i o n o f 2 - d a y e r y t h r o i d c o l o n i e s ( C F U - E ) f rom bone marrow o r f e t a l l i v e r c e l l s . However , the e r y t h r o p o i e t i n - l i k e a c t i v i t y ( E p L A ) , w i t h an SDS/PAGE m o l e c u l a r w e i g h t o f 20 K d , has d i s t i n c t biochemical and b i o p h y s i c a l p r o p e r t i e s f rom Ep as a s s e s s e d by m o l e c u l a r s i z e e x c l u s i o n , h y d r o x y a p a t i t e , and a n i o n exchange c h r o m a t o g r a p h y . P r e l i m i n a r y d a t a s u g g e s t t h a t EpLA i s a c t i v e b o t h i n v i t r o and in v i v o on a s u b s e t o f the E p -r e s p o n s i v e C F U - E p o p u l a t i o n i n f e t a l l i v e r . 12 Assays of Erythropoietin A c t i v i t y The e a r l i e s t Ep bioassays were conducted i n vivo and were predicated on the incorporation of radio-iron (^Fe) into hemoglobinizing erythroblasts following addition of the Ep containing sample. These assays are s t i l l used today and i n the most common of these assays (111), mice are f i r s t made pl e t h o r i c ( e i t h e r by twenty days of hypoxia or i n j e c t i o n of isologous packed red c e l l s ) to reduce their endogenous Ep production. Test materials are then injec t e d i n t r a p e r i t o n e a l l y (I.P.), using a single or fractionated dose protocol and, on the following day, ^ F e - c i t r a t e j s i n j e c t e d intravenously (I.V.) or I.P. One or two days l a t e r , iron incorporation into erythrocytes i s determined and related to that obtained with known Ep standards. Because of animal to animal v a r i a t i o n i n response, a minimum of f i v e animals per sample i s e s s e n t i a l for meaningful r e s u l t s . As i s obvious, t h i s assay system becomes a b i t cumbersome and expensive i f several samples are to be tested. More importantly, the lower l i m i t of s e n s i t i v i t y of the assay i s poor (50 m i l l i u n i t s (mU) of Ep/animal), requiring r e l a t i v e l y high amounts of Ep to e l i c i t a s i g n i f i c a n t response. These problems have led to the advent of shorter and simpler i n v i t r o assays for Ep a c t i v i t y . Two such assays for Ep a c t i v i t y have been developed i n this laboratory (112,113). In both assays, spleen c e l l s from mice previously treated with phenylhydrazine-HCl (PhZ) are used. A two day course of i n j e c t i o n of PhZ at 60 mg/kg body weight induces a severe hemolytic anemia, and r e s u l t s four days l a t e r i n a 40% decrease in hematocrit (114). Under this e r y t h r o p o i e t i c s t r e s s , the spleen becomes a primary s i t e of erythropoiesis. Hara and Ogawa (114) have reported that there i s a s i g n i f i c a n t recruitment of early erythroid progenitors from the bone marrow to the spleen under these 13 c o n d i t i o n s . T h e s e r e c r u i t e d p r i m i t i v e c e l l s t hen g i v e r i s e t o E p - r e s p o n s i v e c e l l s , w h i c h by t h r e e to f o u r days a f t e r the PhZ t r e a t m e n t c o n s t i t u t e a p p r o x i m a t e l y 90% o f n u c l e a t e d c e l l s w i t h i n the s p l e e n . T h e s e s p l e e n s s e r v e as an e x c e p t i o n a l l y r i c h s o u r c e o f t e r m i n a l l y d i f f e r e n t i a t i n g E p - r e s p o n s i v e e r y t h r o i d e l e m e n t s . M o r e o v e r , PhZ s p l e e n s c o n t a i n as many as 3 x 10^ t o t a l c e l l s , and a s i m p l e s e d i m e n t a t i o n a t u n i t g r a v i t y f o r one h o u r a t 4 C i n a p h y s i o l o g i c a l l y b a l a n c e d s a l t s o l u t i o n y i e l d s a s e d i m e n t e d c e l l p o p u l a t i o n t h a t i s 95% n u c l e a t e d and 90% b e n z i d i n e p o s i t i v e ( i . e . h e m o g l o b i n c o n t a i n i n g ) . The s e d i m e n t e d s p l e e n c e l l s c a n then be u t i l i z e d i n e i t h e r 5 9 p e o r ^ H - t h y m i d i n e m i c r o a s s a y s , w h i c h m o n i t o r d i f f e r e n t i a t i v e and p r o l i f e r a t i v e r e s p o n s e s to E p , r e s p e c t i v e l y . The a s s a y t h a t K r y s t a l e t a l (112 ) have d e v e l o p e d t o a s s e s s 5 9 p e i n c o r p o r a t i o n i n v o l v e s i n c u b a t i o n o f 4 X 10^ c e l l s / m l a t 37 C i n t h e p r e s e n c e o f 20% f e t a l c a l f s e r u m , 0 . 1 mM 2 - m e r c a p t o e t h a n o l , and t he s a m p l e t o be a s s a y e d f o r Ep a c t i v i t y f o r t w e n t y - f o u r h o u r s . The c e l l s a r e t h e n c e n t r i f u g e d and washed ( t o remove v a r i a b l e amounts o f i r o n and t r a n s f e r r i n w i t h i n the t e s t s a m p l e s ) and i n c u b a t e d w i t h 1 u C i o f 5 9 p e a n < j human t r a n s f e r r i n f o r a n o t h e r 3 h o u r s a t 37 C . The r a d i o l a b e l e d heme i s e x t r a c t e d w i t h aqueous s c i n t i l l a t i o n f l u i d and c o u n t e d i n a b e t a - c o u n t e r ( s e e M a t e r i a l s and Methods f o r d e t a i l s ) . T h i s a s s a y d e t e c t s b i o a c t i v i t i e s be tween 1-300 mil o f E p / 0 . 1 ml c u l t u r e , mak ing i t s u b s t a n t i a l l y more s e n s i t i v e t h a n t he i n v i v o a s s a y p r e v i o u s l y d i s c u s s e d . M o r e o v e r , b e c a u s e o f t he number o f E p - r e s p o n s i v e c e l l s f rom one P h Z - t r e a t e d s p l e e n , o v e r a t housand s a m p l e s can be a s s a y e d ( i n t r i p l i c a t e ) u s i n g o n l y one mouse. A s e c o n d a s s a y d e v e l o p e d by K r y s t a l (113 ) measures ^ H - t h y m i d i n e i n c o r p o r a t i o n i n t o PhZ s p l e e n c e l l s . A l t h o u g h t e r m i n a l l y d i f f e r e n t i a t i n g p r o e r y t h r o b l a s t s and e r y t h r o b l a s t s do no t m a i n t a i n the same p r o l i f e r a t i v e 14 c a p a c i t y as p l u r i p o t e n t s tem c e l l s ( f r o m F i g u r e 1 ) , a l a r g e r p r o p o r t i o n o f them a r e i n a c t i v e c e l l c y c l e and an e a r l y e r y t h r o b l a s t ( i n the p r e s e n c e o f s u f f i c i e n t q u a n t i t i e s o f Ep) may g i v e r i s e to 32 -64 ma tu re e r y t h r o c y t e s . T h i s i s the most s e n s i t i v e o f the t h r e e a s s a y p r o c e d u r e s d i s c u s s e d , d e t e c t i n g b i o a c t i v i t i e s be tween 0 . 1 - 2 0 mU o f E p / 0 . 1 ml c u l t u r e . The p r o c e d u r a l a s p e c t s o f t h i s a s s a y a r e v e r y s i m i l a r to t h a t d e s c r i b e d f o r 5 9 p e i n c o r p o r a t i o n i n t o PhZ s p l e e n c e l l s . Though b o t h o f t h e s e a s s a y s y i e l d r a p i d q u a n t i t a t i v e r e s u l t s , c a r e f u l i n t e r p r e t a t i o n o f the d a t a must be made s i n c e some n o n -s p e c i f i c e l e m e n t s may be p o t e n t i n v i t r o s t i m u l a t o r s . E r y t h r o p o i e t i n U n t i l r e c e n t l y , t h e r e was v e r y l i t t l e known abou t the b i o l o g y o r b i o c h e m i s t r y o f Ep b e c a u s e o f the d i f f i c u l t i e s e n c o u n t e r e d i n t he p u r i f i c a t i o n o f t h i s hormone. However , g i v e n the r e c e n t b i o a s s a y a d v a n c e s and the a v a i l a b i l i t y o f the c l o n e d g e n e , much has now been l e a r n e d . As o f 1 9 8 5 , the s t a n d a r d p r a c t i c e d p u r i f i c a t i o n p r o t o c o l f o r Ep f rom human u r i n e c o n s i s t e d o f a d i f f i c u l t and l e n g t h y 8 - s t e p p r o c e d u r e ( 1 1 5 ) . As one m igh t e x p e c t , t he y i e l d f rom s u c h a p r o t o c o l was f a i r l y p o o r (15%) and t h i s f i n a l y i e l d c o u l d o n l y be a c h i e v e d when enormous amounts o f s t a r t i n g m a t e r i a l were u s e d (2500 l i t e r s o f a p l a s t i c anemia u r i n e ) . W i t h i n the l a s t two y e a r s , t h o u g h , Yanagawa ( a 2 - s t e p p r o c e d u r e i n v o l v i n g i m m u n o a d s o r p t i o n c h r o m a t o g r a -phy ) ( 1 1 6 ) and K r y s t a l ( a 5 - s t e p p r o t o c o l ) (117 ) have made the r a p i d p u r i f i c a t i o n o f Ep p o s s i b l e . W i t h m ino r d i s c r e p a n c i e s , p a r t i a l amino a c i d s e q u e n c e s f rom Ep p u r i f i e d by a l l t h r e e methods a r e e s s e n t i a l l y the same. G i v e n p a r t i a l amino a c i d s e q u e n c e d a t a , s e v e r a l g r o u p s were s u b s e q u e n t l y a b l e to c l o n e the human ( 1 1 8 , 1 1 9 ) monkey ( 1 2 0 ) , and mouse (121) gene e n c o d i n g 15 E p . McDona ld and G o l d w a s s e r (122 ) have compared t he gene s e q u e n c e o f Ep f r om t h e s e t h r e e s o u r c e s and have found r e g i o n s o f s i g n i f i c a n t g e n e t i c d i v e r g e n c e and c i t e d i m m u n o - c r o s s r e a c t i v i t y o f the gene p r o d u c t s , y e t , a s s e r t t h a t t h e r e g i o n s presumed r e s p o n s i b l e f o r the a c t i o n o f Ep on t a r g e t c e l l s a r e a b s o l u t e l y c o n s e r v e d i n the t h r e e s p e c i e s . From the p r e d i c t e d amino a c i d s e q u e n c e o f the E p s , t hey have g e n e r a t e d mode ls f o r the p r o t e i n s t r u c t u r e o f human, monkey, and m u r i n e Ep ( F i g u r e 2 ) . T h e r e s t i l l i s some q u e s t i o n as t o the amount o f 0 s h e e t c o n f o r m a t i o n w i t h i n the m o l e c u l e a s o t h e r d a t a p r e d i c t s t h e s e s t r u c t u r e s t o be a b s e n t f rom Ep ( 1 2 3 ) . O t h e r s t u d i e s w i t h n a t i v e p u r i f i e d human Ep d e m o n s t r a t e t h a t t he p r o t e i n has 166 amino a c i d s , and e x i s t s i n two g l y c o s y l a t e d f o r m s : a Ep w i t h 31% c a r b o h y d r a t e and 6 Ep w i t h 24% c a r b o h y d r a t e ( 1 2 5 ) . T h e r e a r e 3 p o s s i b l e g l y c o s y l a t i o n s i t e s ( A s n - X - S e r / T h r ) a t r e s i d u e s 2 4 , 3 8 , and 8 3 , and s u g a r m o i e t i e s a r e a t t a c h e d o n l y t h r o u g h amino l i n k a g e s ( N - l i n k e d ) as e n d o g l y s i d a s e F t r e a t m e n t r e d u c e s Ep to the m o l e c u l a r w e i g h t p r e d i c t e d s o l e l y by t he amino a c i d s e q u e n c e ( a p p r o x . 2 0 , 0 0 0 Kd) ( 1 2 4 ) . Of the a t t a c h e d c a r b o h y d r a t e , 40% i s s i a l i c a c i d ( 1 2 4 ) . G i v e n the e l a b o r a t e c a r b o h y d r a t e s t r u c t u r e o f t h e m o l e c u l e , i t i s e s p e c i a l l y i n t e r e s t i n g t h a t d e g l y c o s y l a t e d Ep i s as b i o l o g i c a l l y a c t i v e i n v i t r o as n a t i v e Ep ( 1 2 6 ) . A l s o o f n o t e i s the f a c t t h a t the d i s u l f i d e b r i d g e s w i t h i n the m o l e c u l e (be tween Cys 7 -161 and Cys 2 9 -33) a r e n e c e s s a r y f o r a c t i v i t y ( 1 2 3 ) . The E r y t h r o p o i e t i n R e c e p t o r U n t i l r e c e n t l y , Ep b i n d i n g has p r o v e n d i f f i c u l t to s t u d y due to the loss o f b i o l o g i c a l a c t i v i t y upon s t a n d a r d i o d i n a t i o n p r o c e d u r e s . However , i t was f ound t h a t Ep c o u l d r e t a i n i t s b i o l o g i c a l a c t i v i t y f o l l o w i n g p e r i o d a t e 16 MOUSE MONKEY HUMAN FIGURE 2 . T h i s d i a g r a m , r e p r i n t e d f rom McDona ld and G o l d v a s s e r ( 1 2 2 ) , i l l u s t r a t e s the c o m p u t e r - g e n e r a t e d t e r t i a r y p r o t e i n s t r u c t u r e s o f human, monkey and m u r i n e Ep based on the gene sequence o f e a c h . The c y l i n d e r s r e p r e s e n t r e g i o n s o f a h e l i x c o n f o r m a t i o n , w h i l e the a r r o w s i n d i c a t e r e g i o n s o f p o s s i b l e 3 s h e e t s t r u c t u r e . The a u t h o r s s p e c u l a t e t h a t the a r e a s u r r o u n d i n g the d i s u l f i d e b r i d g e between r e s i d u e s 7 and 161 i s the a c t i v e s i t e o f a l l t h r e e E p s . 17 o x i d a t i o n o f the t e r m i n a l s i a l i c a c i d r e s i d u e s and s u b s e q u e n t r e d u c t i o n w i t h ^ H - s o d i u m b o r o h y d r i d e ( 1 2 7 ) . U n f o r t u n a t e l y , the r e s u l t i n g ^ H - E p p o s s e s s e s v e r y l ow l e v e l s o f r a d i o a c t i v i t y and the r e s u l t s o b t a i n e d f o r t he s p e c i f i c b i n d i n g o f t h i s l a b e l l e d hormone to E p - r e s p o n s i v e c e l l s a r e t h u s d i s p u t e d ( 1 2 8 ) . N o n e t h e l e s s , G o l d w a s s e r e t a l (127 ) have o b t a i n e d some p o t e n t i a l l y i m p o r t a n t b i n d i n g d a t a u s i n g t h i s t e c h n i q u e . S p e c i f i c a l l y , t h e y f ound ( u s i n g a homogeneous p o p u l a t i o n o f E p - r e s p o n s i v e c e l l s , i . e . , s p l e n i c e r y t h r o b l a s t s f rom m i c e i n f e c t e d w i t h the anemic s t r a i n o f F r i e n d v i r u s ) t h a t e q u i l i b r i u m b i n d i n g o f ^ H - E p o c c u r r e d by 2 h o u r s a t 37 C , o r be tween 3 . 5 to 4 h o u r s a t 10 C . M o r e o v e r , t h e i r S c a t c h a r d a n a l y s i s d a t a s u g g e s t e d a l i n e a r p l o t ( i . e . one p o p u l a t i o n o f r e c e p t o r s ) and a mean v a l u e o f 660 r e c e p t o r s p e r c e l l . However , Fukamach i e t a l have now been a b l e to i o d i n a t e Ep w i t h f u l l r e t e n t i o n o f b i o a c t i v i t y ( 1 2 9 ) . T h e i r b i n d i n g d a t a showed a n o n - l i n e a r S c a t c h a r d p l o t and s u g g e s t e d the e x i s t e n c e o f two c l a s s e s o f b i n d i n g s i t e s , one o f h i g h a f f i n i t y ( 0 . 4 6 nM) and the o t h e r o f low a f f i n i t y ( 3 . 9 6 n M ) . F u k a m a c h i ' s r e s u l t s have been c o n f i r m e d by o t h e r s ( 1 3 0 ) . The a d d i t i o n o f a f l u o r e s c e n t a d d u c t to Ep d e m o n s t r a t e d the s u r f a c e b i n d i n g o f Ep to t a r g e t c e l l s ( 1 3 1 ) . O n l y 1.5% o f u n s t i m u l a t e d r a t marrow c e l l s d i s p l a y e d p o s i t i v e f l u o r e s c e n c e , but when r a t s were made anem ic w i t h p h e n y l h y d r a z i n e , 4 .5% o f the c e l l s became p o s i t i v e . The s u r f a c e r e c e p t o r has now been i d e n t i f i e d by S a i t o e t a l ( 1 3 2 ) . C o v a l e n t c r o s s l i n k i n g o f i o d i n a t e d Ep t o d i f f e r e n t i a t i n g m u r i n e e r y t h r o l e u k e m i a c e l l s (by d i s u c c i m i d y l s u b e r a t e ) r e s u l t e d i n t he a p p e a r a n c e o f an i o d i n a t e d comp lex o f 135 Kd ( S D S / P A G E ) . A l l o w i n g f o r the 35 Kd w e i g h t o f E p , the r e c e p t o r was a s s i g n e d a m o l e c u l a r w e i g h t o f 100 K d . I n t e r n a l i z a t i o n o f the E p - r e c e p t o r comp lex i s t hough t to f o l l o w the c l a s s i c r e c e p t o r - m e d i a t e d e n d o c y t o t i c pa thway , w i t h d e g r a d a t i o n o f Ep b e g i n n i n g o n l y a few m i n u t e s a f t e r i n t e r n a l i z a t i o n ( 1 3 3 ) . 18 Mechan ism o f A c t i o n o f E r y t h r o p o i e t i n As y e t , v e r y l i t t l e i s known c o n c e r n i n g how h e m o p o i e t i n s s t i m u l a t e p r o l i f e r a t i o n and d i f f e r e n t i a t i o n b e c a u s e o f l i m i t i n g h e m o p o i e t i n q u a n t i t i e s and the d i f f i c u l t y o f p u r i f y i n g t a r g e t c e l l s . However , now t h a t l a r g e amounts o f p u r e Ep can be o b t a i n e d and a d v a n c e s i n d e v e l o p i n g homogeneous E p -r e s p o n s i v e t a r g e t c e l l p o p u l a t i o n s have been made, some i n s i g h t s i n t o t he mechan ism o f a c t i o n o f Ep s h o u l d be f o r t h c o m i n g . C h a r a c t e r i z a t i o n o f E p ' s mechan ism o f a c t i o n may a l l o w f o r the deve lopmen t o f a mode l to d e s c r i b e t he a c t i o n o f o t h e r h e m o p o i e t i n s as w e l l . A c c o r d i n g l y , s t u d i e s i n t o t he mechan ism o f a c t i o n o f Ep a r e c u r r e n t l y under i n v e s t i g a t i o n i n s e v e r a l l a b o r a t o r i e s . Some " l o n g t e r m " c e l l u l a r e f f e c t s o f Ep have been e l u c i d a t e d and a r e c o m s i s t e n t w i t h the p o s t u l a t e d p h y s i o l o g i c a l r o l e o f Ep a s a r e d c e l l r e g u l a t o r y m o i e t y . An i n c r e a s e i n g l o b i n mRNA i s d e t e c t e d i n t he e r y t h r o p o i e t i c s p l e e n w i t h i n 100 h o u r s o f anemia i n d u c t i o n and has been a t t r i b u t e d to Ep ( 1 3 4 ) . T h e r e i s some e v i d e n c e to s u g g e s t t h a t Ep may f a c i l i t a t e h e m o g l o b i n s w i t c h i n g i n sheep ( 1 3 5 ) . A l s o , t he de novo s y n t h e s i s o f band 3 and 4 . 1 p r o t e i n s and an i n c r e a s e i n s p e c t r i n s y n t h e s i s i s i n d u c e d i n v i r a l l y i n f e c t e d e r y t h r o b l a s t s by Ep ( 1 3 6 ) . The c e l l s u t i l i z e d i n t h i s s t u d y r e p r e s e n t a homogeneous p o p u l a t i o n o f e r y t h r o i d c e l l s a t t he d e v e l o p m e n t a l s t a g e s o f C F U - E ' s and p r o e r y t h r o b l a s t s w h i c h s y n c h r o n o u s l y d i f f e r e n t i a t e i n r e s p o n s e to E p . These c e l l s a r e p r e p a r e d f rom the s p l e e n s o f m i c e w i t h a c u t e e r y t h r o b l a s t o s i s r e s u l t i n g f rom i n f e c t i o n o f m ice w i t h a n e m i a - i n d u c i n g F r i e n d v i r u s ( F V A ) . The use o f t h e s e c e l l s has a l l o w e d f o r a c l o s e s c r u t i n y o f some o f the m o l e c u l a r mechanisms u n d e r l y i n g Ep a c t i o n . 19 I n 1984 , Sawyer and K r a n t z (137 ) examined c a l c i u m m o b i l i z a t i o n f o l l o w i n g i n d u c t i o n o f FVA c e l l s w i t h E p . They were a b l e to d e m o n s t r a t e t h a t Ep b o t h s t i m u l a t e d ^5r; a +2 u p t a k e and c a u s e d an i n c r e a s e d s t e a d y s t a t e l e v e l o f c a l c i u m w i t h i n t h e s e c e l l s . The a p p a r e n t i m p o r t a n c e o f c a l c i u m i n Ep a c t i o n has a l s o been d e m o n s t r a t e d w i t h n o r m a l C F U - E s . Bone marrow c e l l s c u l t u r e d w i t h Ep and the c a l c i u m c h e l a t o r EGTA show d e c r e a s e d C F U - E c o l o n y f o r m a t i o n , bu t t h i s i n h i b i t i o n c o u l d be overcome by the a d d i t i o n o f exogenous c a l c i u m ( 1 3 8 ) . S i m i l a r l y , c a l c i u m i o n o p h o r e s (A23187 and R o 2 - 2 9 8 5 / l ) enhance C F U - E c o l o n y f o r m a t i o n ( 1 3 8 ) . O t h e r M o l e c u l e s A f f e c t i n g I n V i t r o E r y t h r o p o i e s i s The r e l a t i v e e a s e o f the 5 9 p e and ^ H - t h y m i d i n e i n v i t r o a s s a y s has a l l o w e d f o r the d i s c o v e r y o f many o t h e r s u b s t a n c e s w h i c h seem c a p a b l e o f s u b s t i t u t i n g f o r Ep i n e l i c i t i n g p r o l i f e r a t i v e a n d / o r d i f f e r e n t i a t i v e r e s p o n s e s i n PhZ c e l l s ( T a b l e I I ) . Many o f t h e s e a g e n t s were f i r s t d i s c o v e r e d b e c a u s e o f t h e i r a b i l i t y to i n d u c e F r i e n d v i r u s - t r a n s f o r m e d m u r i n e e r y t h r o l e u k e m i a (MEL) c e l l s t o d i f f e r e n t i a t e . T h i s c e l l l i n e was o r i g i n a l l y d e r i v e d f rom F r i e n d v i r u s i n f e c t i o n o f m u r i n e bone marrow c e l l s ( 1 4 0 ) and has c l a s s i c a l l y been u s e d to i n v e s t i g a t e e r y t h r o i d d i f f e r e n t i a t i o n ( 1 4 1 ) . MEL c e l l s a r e t hough t to be d e v e l o p m e n t a l l y a r r e s t e d a t the p r o e r y t h r o b l a s t s t a g e and c a n be i n d u c e d to d i f f e r e n t i a t e to l a t e e r y t h r o b l a s t s by a v a r i e t y o f compounds . T h i s d i f f e r e n t i a t i o n i s though t to f o l l o w a n o r m a l c o u r s e ( i . e . a c c u m u l a t i o n o f g l o b i n mRNA, and the a p p e a r a n c e o f the c h a r a c t e r i s t i c r ed c e l l p r o t e i n m a r k e r s s u c h as g l o b i n p e p t i d e c h a i n s , p r o t e i n s o f the e n z y m a t i c pa thway f o r heme m e t a b o l i s m , and r e d c e l l - s p e c i f i c membrane a n t i g e n s ( 1 4 1 ) ) . T h e r e a r e many a d v a n t a g e s to u s i n g MEL c e l l s i n s t u d i e s o f e r y t h r o i d 20 d i f f e r e n t i a t i o n . These c e l l s a r e e a s i l y c l o n e d and c u l t i v a t e d i n v i t r o . A l s o , MEL c e l l s p r o v i d e a homogeneous p o p u l a t i o n o f t r a n s f o r m e d p r o e r y t h r o b l a s t s w h i c h can be i n d u c e d to unde rgo t e r m i n a l e r y t h r o i d d i f f e r e n t i a t i o n by the a d d i t i o n o f a m u l t i t u d e o f a p p a r e n t l y u n r e l a t e d compounds. However , soon a f t e r t h e i r e s t a b l i s h m e n t as a c e l l l i n e , MEL c e l l s l o s e the a b i l i t y to r e s p o n d to Ep ( i . e . Ep i s no t an i n d u c e r o f MEL c e l l d i f f e r e n t i a t i o n ) . T h u s , s t u d i e s c o n c e r n i n g MEL c e l l d i f f e r e n t i a t i o n may no t t r u l y r e f l e c t i n v i v o e r y t h r o p o i e s i s . Many p o t e n t i n d u c e r s o f MEL c e l l s have been e l u c i d a t e d ( 1 4 1 ) . These i n c l u d e b e n z o d i a z a p i n e s , a c t i n o m y c i n D, 6-t h i o g u a n i n e , b u t y r i c a c i d , d i m e t h y l s u l f o x i d e (DMSO), h e x a m e t h y l e n e b i s a c e t a m i d e (HMBA), a z a c y t i d i n e , m e t h y l i s o x a n t h i n e , o u a b a i n , u l t r a v i o l e t l i g h t , X - r a y s , and h e m i n . A number o f t h e s e a g e n t s have been shown , i n o u r l a b o r a t o r y t o s t i m u l a t e PhZ s p l e e n c e l l s to p r o l i f e r a t e and d i f f e r e n t i a t e ( T a b l e I I ) . The s i m i l a r i t y be tween MEL c e l l i n d u c t i o n and PhZ s p l e e n c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i s r e i n f o r c e d by t he f i n d i n g t h a t c o n c e n t r a t i o n s o f many a g e n t s t h a t o p t i m a l l y i n d u c e MEL c e l l d i f f e r e n t i a t i o n a l s o o p t i m a l l y s t i m u l a t e PhZ c e l l s i n ou r Ep b i o a s s a y s ( e . g . DMSO a t a f i n a l c o n c e n t r a t i o n o f 1.25% s t i m u l a t e s b o t h c e l l t y p e s o p t i m a l l y ) . A c c o r d i n g l y , some mechan isms though t o p e r a t i v e d u r i n g MEL c e l l i n d u c t i o n have been p o s t u l a t e d to be o f i m p o r t a n c e i n n o r m a l t e r m i n a l e r y t h r o i d d i f f e r e n t i a t i o n ( 1 4 1 ) . I t i s u n l i k e l y , h o w e v e r , t h a t Ep a l o n e r e g u l a t e s t e r m i n a l e r y t h r o p o i e s i s as a n t i b o d i e s a g a i n s t the t r a n s f e r r i n r e c e p t o r b l o c k c o m m i t t e d t e r m i n a l e r y t h r o i d d i f f e r e n t i a t i o n , and t h i s s u g g e s t s t h a t the c o o r d i n a t e s y n t h e s i s o f e r y t h r o c y t e p r o t e i n s i s a l s o s u b j e c t to r e g u l a t i o n by hemin o r h e m o g l o b i n ( 1 3 9 ) . TABLE I I The a b i l i t y o f v a r i o u s a g e n t s to s u b s t i t u t e f o r Ep i n - t h y m i d i n e and 5 ' F e i n c o r p o r a t i o n a s s a y s Agent H-thymidine Assay 59 Fe Assay Actinomycin 0 (2.5-20 ng/m)) Angiotensinogen (50 ^jg/ml) A-23187 (Ca** lonophore) (0.125-7 .5 ug/ml) ATP (0.25 fif) Butyric acid (1 mfc) Dlbutyryl cAMP (0.1-4 MY) Dibutyryl c G » (0.2-8 mM) Dimethyl sulfoxide (It) Dextran sul fate (50 pg/ml) GTP (0.25 Hexamethylene bisacetamide (2.5 mM) HypoxantMne (3.7 mM) L-isoproterenol (10* 7 M) • GTP Lithium carbonate (1-2 uM) Monensin (Na* ionophore) (0.25-7.5 ug/ml) Ouabain (10 -10 e v M) Prostaglandin Ej (10* 7 H) • GTP • TPA • A 23187 Sodium orthovanadate (10 jH) L-thyroxine (10" 7 M) TPA (10-100 nM) • A 23187 * GTP • L-1soproterenol Zinc chlor ide (1-100 j * ) inh ib i tory* 0 0 0 25 0 0 25-40 0 0 40-60 17 0 0 inh ib i tory 0 0 15-20 0 0 0 0 0 0 0 50-250 0 0 110-400 50 0 0 0 * The numbers Indicate the level of Ep (mu/ml) that would give the same level of incorporat ion. Where stimulation was observed, the concentration of compound shown in brackets 1s that concentration which gave the maximal response. 22 G r o w t h S i g n a l T r a n s d u c t i o n C l a s s i c a l l y , c y c l i c a d e n o s i n e monophosphate (cAMP) has been though t t o p l a y a key r o l e i n h o r m o n a l s i g n a l t r a n s f e r and a c t i v a t i o n ( o r d e a c t i v a t i o n ) o f t a r g e t c e l l s . E v i d e n c e shoved t h a t a g e n t s v h i c h e l e v a t e d cAMP, i n d e p e n d e n t f rom r e c e p t o r mechanisms ( e . g . c h o l e r a t o x i n ) , c o u l d o f t e n m im ic s p e c i f i c h o r m o n a l e f f e c t s . F u r t h e r m o r e , a g r o v t h f a c t o r , p l a t e l e t - d e r i v e d g r o v t h f a c t o r ( P D G F ) , i n d u c e d the r a p i d a c c u m u l a t i o n o f cAMP i n 3T3 c e l l s v h e n c e l l s v e r e p r e i n c u b a t e d w i t h i n h i b i t o r s o f c y c l i c n u c l e o t i d e d e g r a d a t i o n ( 1 4 2 ) . I t i s now r e c o g n i z e d t h a t a s t e a d y - s t a t e i n c r e a s e o f i n t r a c e l l u l a r cAMP i s a g r o v t h s i g n a l f o r q u i e s c e n t f i b r o b l a s t s ( 1 4 3 ) . Howeve r , s i n c e o t h e r hormones ( i n s u l i n , E G F , v a s o p r e s s i n , e t c . ) d i d no t d r a s t i c a l l y a l t e r i n t r a c e l l u l a r cAMP l e v e l s , o t h e r c A M P - i n d e p e n d e n t mechan isms o f c e l l a c t i v a t i o n v e r e i n v e s t i g a t e d . The a p p a r e n t m o b i l i z a t i o n o f cAMP i n d u c e d by PDGF has s i n c e been h y p o t h e s i z e d to be an i n d i r e c t e f f e c t o f t he hormone ( 1 4 4 ) , whereby the e l e v a t i o n o f cAMP l e v e l s can be a t t r i b u t e d to PDGF-s t i m u l a t e d p r o s t a g l a n d i n E s y n t h e s i s and r e l e a s e ( 1 4 5 ) . Upon c r i t i c a l e x a m i n a t i o n o f s e v e r a l p a r a m e t e r s ( i . e . i o n f l u x e s , pH c h a n g e s , s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n , e t c . ) s u b s e q u e n t to the b i n d i n g o f a g r o w t h f a c t o r t o a c t i v a t e the c e l l , a model o f the e a r l y e v e n t s l e a d i n g t o c e l l s t i m u l a t i o n has been f o r w a r d e d . Some o f the r e l e v a n t a s p e c t s o f t h i s mode l a r e r e v i e w e d h e r e . I t was d i s c o v e r e d t h a t the c e l l s u r f a c e r e c e p t o r s f o r a g roup o f g r o w t h f a c t o r s ( i n s u l i n , e p i d e r m a l g r o w t h f a c t o r ( E G F ) , p l a t e l e t - d e r i v e d growth f a c t o r , M-CSF) a l l had t y r o s i n e k i n a s e a c t i v i t y ( 1 4 7 - 1 5 0 ) . P h o s p h o t y r o s i n e was f i r s t d e t e c t e d i n v i r a l t r a n s f o r m i n g p r o t e i n s ( 1 5 1 ) . Now, s e v e r a l s u c h p r o t e i n s ( v i r a l oncogene p r o d u c t s ) have been shown to have i n t r i n s i c t y r o s i n e 23 k i n a s e a c t i v i t y ( 1 5 2 ) , and s i t e d i r e c t e d m u t a g e n e s i s s t u d i e s have shown t h i s k i n a s e a c t i v i t y to be e s s e n t i a l f o r r e t r o v i r a l t r a n s f o r m i n g a b i l i t y ( 1 7 9 ) . G r o w t h f a c t o r r e c e p t o r k i n a s e s a r e most l i k e l y a c t i v a t e d f o l l o w i n g a c o n f o r m a t i o n a l change i n d u c e d by l i g a n d b i n d i n g ( 1 5 3 ) . E a r l y s t u d i e s d e m o n s t r a t e d t h a t EGF r e c e p t o r k i n a s e a c t i v i t y was s t i m u l a t e d 2 -4 f o l d f o l l o w i n g a s h o r t e x p o s u r e o f t a r g e t c e l l s to exogenous EGF ( 1 5 4 ) . The s t i m u l a t i o n o f r e c e p t o r k i n a s e a c t i v i t y i s most l i k e l y t he f i r s t o f a s e r i e s o f r a p i d l y o c c u r r i n g e v e n t s i n d u c e d by a g o n i s t ( g r o w t h f a c t o r ) b i n d i n g . The s i g n i f i c a n c e o f r e c e p t o r k i n a s e a c t i v i t y i s unknown, though an a t t r a c t i v e e x p l a n a t i o n m igh t be t h a t s p e c i f i c t y r o s i n e p h o s p h o r y l a t i o n s ( c a t a l y z e d by r e c e p t o r s ) a r e r e s p o n s i b l e f o r c o n t i n u e d g r o w t h f a c t o r s i g n a l t r a n s d u c t i o n . W i t h r e s p e c t to t h i s , s e v e r a l p r o t e i n s u b s t r a t e s f o r r e c e p t o r k i n a s e s have been e x a m i n e d . I n t e r e s t i n g l y , the r e c e p t o r s t h e m s e l v e s s e r v e a s a s u b s t r a t e f o r t h e i r own k i n a s e a c t i v i t y ( i . e . a u t o p h o s p h o r y l a t i o n ) ( 1 5 7 ) . A l s o , m e m b r a n e - a s s o c i a t e d and c y t o s o l i c p r o t e i n s have been i d e n t i f i e d a s s u b s t r a t e s f o r b o t h r e t r o v i r a l and c e l l u l a r t y r o s i n e k i n a s e s ( 1 5 2 , 1 5 6 ) . Most n o t a b l y , p36 and p81 have been shown to be t y r o s i n e p h o s p h o r y l a t e d by r e t r o v i r a l t y r o s i n e k i n a s e s ( 1 5 2 ) . A n o t h e r , p 4 2 , i s t y r o s i n e p h o s p h o r y l a t e d f o l l o w i n g i n d u c t i o n o f f i b r o b l a s t s by PDGF. And f i n a l l y , a 35 Kd membrane-a s s o c i a t e d s u b s t r a t e i s t y r o s i n e p h o s p h o r y l a t e d , i n v i t r o , by the EGF r e c e p t o r i n a c a l c i u m dependen t manner ( 1 6 4 ) . I t has been h y p o t h e s i z e d by s e v e r a l i n v e s t i g a t o r s t h a t the a c t i v i t y o f the r e c e p t o r k i n a s e on one p a r t i c u l a r s u b s t r a t e , the G p r o t e i n c o m p l e x , may a l l o w f o r t he c o n t i n u e d p r o p a g a t i o n o f the g r o w t h s i g n a l i n t o the c e l l . Though the mechan ism by w h i c h G p r o t e i n s a r e c o u p l e d to a c t i v a t e d r e c e p t o r s i s u n c l e a r , t h e r e i s m o u n t i n g e v i d e n c e to s u g g e s t t h a t t h e s e g u a n i n e n u c l e o t i d e - b i n d i n g p r o t e i n s a r e d i r e c t l y i n v o l v e d i n the a c t i v a t i o n 24 o f p h o s p h o l i p a s e C . A c t i v a t e d p h o s p h o l i p a s e C s u b s e q u e n t l y c a t a l y z e s t he p r o d u c t i o n o f the two s e c o n d messenge rs t hough t o p e r a t i v e d u r i n g t h i s scheme o f c e l l u l a r a c t i v a t i o n . The i m p l i c a t i o n o f G p r o t e i n s i n r e c e p t o r - e f f e c t o r c o u p l i n g i s s t r e n g t h e n e d by the a b i l i t y o f b a c t e r i a l t o x i n s ( p e r t u s s i s and c h o l e r a ) t o a l t e r t he a c t i v i t y o f p h o s p h o l i p a s e C by t h e i r a c t i o n on G p r o t e i n s ( 1 5 8 , 1 5 9 ) . T h e s e t o x i n s A D P - r i b o s y l a t e G p r o t e i n s u b u n i t s , and t hus i n many i n s t a n c e s , u n c o u p l e a c t i v a t e d r e c e p t o r s f rom p h o s p h o l i p a s e C . The e f f e c t s o f t h e s e t o x i n s c a n be shown i n a v a r i e t y o f c a l c i u m - m o b i l i z i n g r e c e p t o r s i g n a l t r a n s d u c i n g s y s t e m s ( e . g . v a s o p r e s s i n , a n g i o t e n s i s I I , e p i n e p h r i n e ( 1 5 9 ) and t h y r o t r o p i n - r e l e a s i n g hormone ( 1 6 1 ) ) . The mechan ism by w h i c h G p r o t e i n s m e d i a t e s i g n a l t r a n s d u c t i o n has been h y p o t h e s i z e d a s f o l l o w s ( 1 6 2 ) : Upon s t i m u l a t i o n by a r e c e p t o r p r o t e i n , the G p r o t e i n comp lex r e l e a s e s GDP and s u b s e q u e n t l y b i n d s GTP . I n t h i s c o n f o r m a t i o n , i t i s a b l e t o r e g u l a t e t h e f u n c t i o n o f e f f e c t o r m o l e c u l e s ( i . e . p h o s p h o l i p a s e C ) . The h y d r o l y s i s o f t he bound GTP to GDP t e r m i n a t e s the r e g u l a t o r y f u n c t i o n o f t he p r o t e i n . The h e t e r o t r i m e r i c G p r o t e i n s c o n t a i n 3 d i s t i n c t s u b u n i t s — a n a c h a i n t h a t b i n d s GTP and d e t e r m i n e s t he s p e c i f i c t y o f e f f e c t o r f u n c t i o n s , and a 0Y comp lex t h a t may s e r v e t o a n c h o r the p r o t e i n to the i n n e r l e a f l e t o f t he p l a s m a membrane ( 1 6 2 ) . O c c u p i e d r e c e p t o r s most l i k e l y f u n c t i o n d i r e c t l y on G p r o t e i n s , s i n c e no i n t e r m e d i a t e s between the two have been i s o l a t e d . The mechan ism by w h i c h t h i s o c c u r s may i n v o l v e t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o -r y l a t i o n s ( 1 6 3 ) . N o r t h u p b e l i e v e s t h i s to be a p o s s i b l e mechan ism to e x p l a i n t he a c t i o n o f g r o w t h f a c t o r r e c e p t o r s on G p r o t e i n s . S p e c i f i c a l l y , he p o s t u l a t e s t h a t t he EGF r e c e p t o r (when a p p r o p r i a t e l y s t i m u l a t e d by EGF) f i r s t a u t o p h o s p h o r y l a t e s , t hen p h o s p h o r y l a t e s the 35 Kd 3 s u b u n i t o f t he G p r o t e i n , t h u s a l l o w i n g the G p r o t e i n to a c t on the n e x t e f f e c t o r i n t he p a t h w a y , 25 p h o s p h o l i p a s e C . I n d e e d , o t h e r s as v e i l have documented t he E G F - s t i m u l a t e d t y r o s i n e - s p e c l f l c p h o s p h o r y l a t i o n o f a 35 Kd m e m b r a n e - a s s o c i a t e d p r o t e i n ( 1 6 4 ) . The a c t u a l mechanism by w h i c h the G p r o t e i n s t i m u l a t e s p h o s p h o l i p a s e C a c t i v i t y r e m a i n s l a r g e l y u n r e s o l v e d , though G p r o t e i n a c t i v i t y g r e a t l y r e d u c e s the c a l c i u m r e q u i r e m e n t o f p h o s p h o l i p a s e C ( 1 6 5 ) . I n g e n e r a l , s i g n a l l i n g s y s t e m s t r a n s l a t e i n f o r m a t i o n f rom e x t e r n a l s o u r c e s i n t o a l i m i t e d number o f i n t e r n a l s i g n a l s , t he s e c o n d m e s s e n g e r s . A t t he c e l l s u r f a c e , the g r o w t h f a c t o r r e c e p t o r f u n c t i o n s as t he a n t e n n a , s p e c i f i c o n l y f o r c e r t a i n t y p e s o f e x t e r n a l s t i m u l i . T h i s i n f o r m a t i o n i s t r a n s d u c e d and a m p l i f i e d by s e c o n d m e s s e n g e r s , t hus e f f e c t i n g t he c o n t r o l o f a v a r i e t y o f c e l l u l a r g r o w t h p r o c e s s e s . P h o s p h o l i p a s e C , once a c t i v a t e d , f u n c t i o n s t o p r o d u c e two p o t e n t s e c o n d m e s s e n g e r s , i n o s i t o l t r i s p h o s p h a t e ( I n s P3) and d i a c y l g l y c e r o l ( D G ) . P h o s p h o l i p a s e C a c t s d i r e c t l y on p h o s p h a t i d y l i n o s i t o l 4 , 5 - b i s p h o s p h a t e ( P t d I n s ( 4 , 5 ) P 2 ) i n r e s p o n s e t o a t ransmembrane s i g n a l . T h i s s u b s t r a t e r e s u l t s f rom the 2 - s t e p p h o s p h o r y l a t i o n o f p h o s p h a t i d y l i n o s i t o l a t b o t h the 4 and 5 p o s i t i o n s o f t he i n o s i t o l head g r o u p . The a c t i o n o f v a r i o u s k i n a s e s and p h o s p h o d i e s t e r a s e s c r e a t e a f u t i l e c y c l e , whereby p h o s p h a t e g r o u p s a r e c o n s t a n t l y b e i n g added and removed f r om the 4 - and 5 - p o s i t i o n s o f the head g r o u p ( 1 6 6 ) . Howeve r , t he o c c u p a t i o n o f a r e c e p t o r by i t s s p e c i f i c a g o n i s t d i v e r t s ( P t d I n s ( 4 , 5 ) P 2 ) ou t o f t h i s c y c l e by s t i m u l a t i n g ( p o s s i b l y v i a G p r o t e i n s ) the p h o s p h o d i e s t e r a s e , p h o s p h o l i p a s e C , t o e n z y m i c a l l y c l e a v e ( P t d I n s ( 4 , 5 ) P 2 ) i n t o DG and I n s P3 ( 1 6 6 ) . P r o t e i n k i n a s e C a c t i v a t i o n must c e r t a i n l y be c o n s i d e r e d t he c e n t r a l hub o f t h i s m i c r o c a s c a d e o f e v e n t s r a p i d l y f o l l o w i n g r e c e p t o r - g r o w t h f a c t o r i n t e r a c t i o n . B e s i d e s i t s C a + 2 and p h o s p h o l i p i d r e q u i r e m e n t , p r o t e i n k i n a s e C r e q u i r e s DG f o r i t s t r a n s l o c a t i o n f rom c y t o s o l t o c e l l membrane, where i t becomes a c t i v a t e d . N i s h i z u k a (167) a r g u e s t h a t DG does no t a n c h o r p r o t e i n 26 k i n a s e C t o the membrane, but t h a t DG i n c r e a s e s the a f f i n i t y o f p r o t e i n k i n a s e C f o r C a + 2 and f o r p h o s p h a t i d y l - s e r i n e , and a l l o w s t he k i n a s e to be a c t i v a t e d w i t h o u t a d r a s t i c change i n the i n t r a c e l l u l a r c a l c i u m l e v e l . The o t h e r e f f e c t o r p r o d u c e d by p h o s p h o l i p a s e C , I n s P3, i s b e l i e v e d t o f u n c t i o n i n i n t r a c e l l u l a r c a l c i u m m o b i l i z a t i o n , p r e s u m a b l y t h r o u g h i t s i n t e r a c t i o n w i t h I n s P3 r e c e p t o r s l o c a t e d on the e n d o p l a s m i c r e t i c u l u m ( 1 6 6 ) . B o t h I n s P3 and DG e x i s t o n l y t r a n s i e n t l y and soon a f t e r t h e i r f o r m a t i o n a r e r a p i d l y d e g r a d e d and t h u s , c o n s t a n t r e c e p t o r a c t i v a t i o n i s r e q u i r e d f o r a p r o l o n g e d g r o w t h s t i m u l u s . Vhen p r o t e i n k i n a s e C i s a p p r o p r i a t e l y a c t i v a t e d , i t f u n c t i o n s t o p h o s p h o r y l a t e ( a t s e r i n e and t h r e o n i n e r e s i d u e s ) a b r o a d s p e c t r u m o f c e l l u l a r p r o t e i n s , t h e r e b y t r a n s d u c i n g and a m p l i f y i n g g r o w t h s i g n a l s . One s u b s t r a t e a p p e a r s to be o f p a r t i c u l a r i n t e r e s t . R e c e n t l y ( r e v i e w e d i n 1 4 5 ) , a r a p i d i n c r e a s e i n the p h o s p h o r y l a t i o n o f an a c i d i c 8 0 , 0 0 0 d a l t o n c e l l u l a r p r o t e i n (80K) was f o u n d upon p r o t e i n k i n a s e C a c t i v a t i o n i n f i b r o b l a s t s . The p h o s p h o r y l a t i o n o f 80K was s e e n when c e l l s were s t i m u l a t e d w i t h p h o r b o l e s t e r s , s y n t h e t i c d i a c y l g l y c e r o l ( i . e an a n a l o g o f D G ) , o r exogenous p r o t e i n k i n a s e C . T h e r e i s e v i d e n c e t h a t g r o w t h f a c t o r a c t i v a t i o n o f c e l l s c a n a l s o s t i m u l a t e p r o t e i n k i n a s e C to p h o s p h o r y l a t e 80K ( 1 6 8 ) . A c o m p r e h e n s i v e o v e r v i e w o f t h i s p r o p o s e d mechan ism o f h o r m o n a l s i g n a l t r a n s d u c t i o n i s d i a g r a m a t i c a l l y r e p r e s e n t e d i n F i g u r e s 3 and 4. F i g u r e 3 r e p r e s e n t s an o v e r v i e w o f p o s s i b l e b i o c h e m i c a l pa thways u l t i m a t e l y l e a d i n g t o h o r m o n e - m e d i a t e d c e l l a c t i v a t i o n . F i g u r e 4 has been r e p r i n t e d f rom a r e c e n t r e v i e w by B e r r i d g e and I r v i n e (166), and f o c u s s e s d i r e c t l y on the m o l e c u l a r mechan isms o f c e l l u l a r a c t i v a t i o n i n v o l v i n g DG and I n s P3 t h a t a r e d i s c u s s e d h e r e . 27 FIGURE 3 . T h i s s c h e m a t i c r e p r e s e n t s many o f t he h y p o t h e s i z e d mechanisms by w h i c h a c e l l may become a c t i v a t e d i n r e s p o n s e to a g o n i s t b i n d i n g . From t h i s d i a g r a m i t c a n be s e e n t h a t c e l l a c t i v a t i o n i n v o l v e s a m y r i a d o f r e l a t e d p a t h w a y s , e a c h o f w h i c h i s p o t e n t i a l l y r e g u l a t e d by a s e r i e s o f s e c o n d m e s s e n g e r s . FIGURE 4 . R e p r i n t e d f rom a r e c e n t r e v i e w ( 1 6 6 ) , t h i s d i a g r a m i l l u s t r a t e s the c o m p l e x i t y o f the a g o n i s t -r e c e p t o r ( R i ) - G p r o t e i n ( G ) - p h o s p h o l i p a s e C ( P D E ) -p h o s p h a t i d y l i n o s i t o l r e l a t i o n s h i p . The p r o d u c t s o f t he p a t h w a y , i n o s i t o l t r i p h o s p h a t e ( I n s P3) and d i a c y l g l y c e r o l (DG) a r e p o t e n t b i o l o g i c a l e f f e c t o r s f o r many h o r m o n a l s y s t e m s . 29 T h e s i s O b j e c t i v e s G i v e n the r e p o r t e d l i n k , be tween c a l c i u m m o b i l i z a t i o n and Ep a c t i v i t y , and a t h e o r y to e x p l a i n how c a l c i u m migh t p a r t i c i p a t e i n h o r m o n a l s i g n a l t r a n s d u c t i o n , we p o s t u l a t e d t h a t Ep ( a s w e l l as a g e n t s t h a t m im ic Ep a c t i v i t y ) may f u n c t i o n t h r o u g h t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n e v e n t s i n s t i m u l a t i n g PhZ s p l e e n c e l l s . I t was known f r om o t h e r c a l c i u m -m o b i l i z i n g r e c e p t o r s y s t e m s (PDGF, E G F , b o m b e s i n , e t c . ( 1 4 5 ) ) , t h a t p o t e n t m i t o g e n i c hormones a l l p o s s e s s e d r e c e p t o r s w h i c h had t y r o s i n e k i n a s e a c t i v i t y . T h u s , i t was h y p o t h e s i z e d t h a t Ep and o t h e r e r y t h r o p o i e t i c s t i m u l a t o r s m igh t i n d u c e the t y r o s i n e - s p e c i f i c p h o s p h o r y l a t i o n o f t he Ep r e c e p t o r , o r t he p h o s p h o r y l a t i o n o f m e m b r a n e - a s s o c i a t e d p r o t e i n s u b s t r a t e s by the r e c e p t o r . F u r t h e r m o r e , i t was h y p o t h e s i z e d t h a t an a g e n t w h i c h c o u l d i n c r e a s e the b a s a l l e v e l o f p h o s p h o t y r o s i n e w i t h i n d i f f e r e n t i a t i n g e r y t h r o i d c e l l s m igh t have p r o f o u n d Ep m i m i c k i n g a c t i v i t y i n s t a n d a r d Ep b i o a s s a y s . Howeve r , to h e l p r a t i o n a l i z e p o t e n t i a l d e s c r e p a n c i e s be tween r e s u l t s o b t a i n e d w i t h Ep v e r s u s t h o s e o b t a i n e d w i t h o t h e r Ep m i m i c k i n g a g e n t s , i t was a l s o n e c e s s a r y t o d e m o n s t r a t e whe the r Ep and E p - m i m i c k i n g a g e n t s o p e r a t e d on t he same c e l l p o p u l a t i o n . T h e r e f o r e t h r e e d i s t i n c t s t u d i e s were c o n d u c t e d . F i r s t , a p o t e n t p h o s p h o t y r o s i n e p h o s p h a t a s e i n h i b i t o r ( i . e . p h o s p h o t y r o s i n e s t i m u l a t o r ) , s o d i u m o r t h o v a n a d a t e , was examined f o r p o s s i b l e m i t o g e n i c and d i f f e r e n t i a t i v e e f f e c t s on E p - r e s p o n s i v e c e l l s . S e c o n d , ^ H - t h y m i d i n e i n c o r p o r a t i o n a s s a y s were p e r f o r m e d a t t w e n t y - f o u r hou r i n t e r v a l s w h i l e homogeneous p o p u l a t i o n s o f e a r l y e r y t h r o b l a s t s were a l l o w e d to d i f f e r e n t i a t e to examine the p o s s i b i l i t y t h a t Ep and Ep m i m i c k i n g a g e n t s migh t no t be f u n c t i o n i n g on the same c e l l 30 population. Finally, cell membranes from Ep-responsive cells vere isolated, incubated with erythropoietic stimulators, exposed to gamma-labelled-32p_ATP, and then examined by SDS-gel electrophoresis and autoradiography to determine if any specific phosphorylations could be detected. The results from these studies are presented within this thesis. 31 MATERIALS and METHODS E r y t h r o p o i e t i n B i o a s s a y s I n V i t r o ^ - T h y m i d i n e I n c o r p o r a t i o n A s s a y T h i s p r o c e d u r e has been d e s c r i b e d e l s e w h e r e ( 1 1 3 ) . B r i e f l y , ( C 5 7 B 1 / 6 J x C3H/HeN) h y b r i d f e m a l e m ice ( B 6 C 3 F i ) were i n j e c t e d on two c o n s e c u t i v e d a y s ( I . P . ) w i t h 60 mg/kg p h e n y l h y d r a z i n e - H C l . T h r e e days l a t e r , t he a n i m a l s were s a c r i f i c e d , t he s p l e e n s removed , and the Ep r e s p o n s i v e c e l l s i s o l a t e d . PhZ s p l e e n c e l l s v e r e d i l u t e d i n RPMI 1640 medium c o n t a i n i n g 20% PCS and 0 . 1 mM 2 - m e r c a p t o e t h a n o l to y i e l d a f i n a l c e l l c o n c e n t r a t i o n o f 4 X 1 0 6 c e l l s / m l . T h i s c e l l m i x t u r e was a l i q u o t e d i n t o L i n b r o U -shaped m i c r o t i t r e p l a t e w e l l s and t e s t s a m p l e s (DMSO, v a n a d a t e , e t c . ) o r Ep were added t o g i v e f i n a l v o l u m e s o f 0 . 1 m l / w e l l . C u l t u r e s were i n c u b a t e d f o r 22 h o u r s a t 37 C i n a h u m i d i f i e d a t m o s p h e r e o f 5% CO2 and 95% a i r . To e a c h w e l l was t h e n added 20 u l o f a 3 H - t h y m i d i n e s t o c k s o l u t i o n (50 u C i / m l i n RPMI) t o g i v e a f i n a l 3 H -t h y m i d i n e c o n c e n t r a t i o n o f 1 u C i / w e l l . The a s s a y p l a t e s were r e t u r n e d t o t he 37 C i n c u b a t o r f o r an a d d i t i o n a l 2 h o u r s , and then the w e l l c o n t e n t s were h a r v e s t e d o n t o g l a s s f i b e r f i l t e r s u s i n g a m i c r o h a r v e s t e r ( R i c h t e r S c i e n t i f i c , V a n c o u v e r C a n a d a ) . F o r h a r v e s t i n g , the w e l l s were r i n s e d w i t h d i s t i l l e d w a t e r , the c e l l c o n t e n t s c a p t u r e d on g l a s s f i b e r f i l t e r s , and t h e f i l t e r s s u b s e q u e n t l y washed w i t h m e t h a n o l . The f i b e r d i s c s , a l l o w e d t o d r y f o r one h o u r , were p l a c e d i n s c i n t i l l a t i o n v i a l s ( w i t h 2 ml o f non-aqueous s c i n t i l l a t i o n f l u i d ) and c o u n t e d i n a Beckman LS7500 l i q u i d s c i n t i l l a t i o n c o u n t e r . 32 I n V i t r o 5 9 p e i n c o r p o r a t i o n A s s a y < As d e s c r i b e d by K r y s t a l e t a l ( 1 1 2 ) , the PhZ s p l e e n c e l l a s s a y s u s p e n s i o n was p r e p a r e d i n an a n a l o g o u s f a s h i o n t o t h a t d e t a i l e d a b o v e . Howeve r , the f i n a l 0 . 1 ml c e l l m i x t u r e was i n c u b a t e d i n d i s p o s a b l e f l i n t g l a s s s c r e w cap t u b e s (16 X 100 mm, F i s h e r #14 -959 -25B) c o v e r e d w i t h l o o s e -f i t t i n g a luminum c a p s (16 mm), r a t h e r t han i n m i c r o t i t r e p l a t e w e l l s , s i n c e t he b u t a n o n e u s e d to e x t r a c t the 5 9 p e _ n e m e ( s e e b e l o w ) w o u l d d i s s o l v e t he p l a s t i c m i c r o t i t r e p l a t e s . The t u b e s were p l a c e d i n a 37 C i n c u b a t o r (5% CO2) a n d , a f t e r 24 h o u r s , the c e l l s were washed t w i c e w i t h 5 m l RPMI medium, r e s u s p e n d e d i n the same medium c o n t a i n i n g 1 u C i [ ^ F e ] - f e r r o u s c i t r a t e and 25 ug human t r a n s f e r r i n , and p l a c e d back, a t 37 C f o r 3 h o u r s . The c e l l s were t h e n washed t w i c e w i t h 5 ml o f PBS a n d , a f t e r the s e c o n d w a s h i n g , 2 . 7 5 m l o f a f r e s h l y p r e p a r e d m i x t u r e o f d i s t i l l e d w a t e r , D r a b k i n ' s s o l u t i o n , and 0 . 2 N HC1 ( i n a v o l u m e t r i c r a t i o o f 1 : 1 : 0 . 7 5 ) were added t o t he c e l l p e l l e t s . A f t e r e a c h tube was v o r t e x e d , 3 ml b u t a n e - 2 - o n e were a d d e d , the t u b e s s e a l e d w i t h s c r e w c a p s , t hen v o r t e x e d v i g o r o u s l y , and c e n t r i f u g e d a t 600 x g f o r 5 m i n . A l i q u o t s ( 1 . 0 m l ) o f t he bu tanone ( t o p ) l a y e r were t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s , 10 ml o f aqueous s c i n t i l l a t i o n f l u i d were a d d e d , and 5^Fe l e v e l s d e t e r m i n e d i n a Beckman LS7500 l i q u i d s c i n t i l l a t i o n c o u n t e r . 33 C e l l s and C e l l L i n e s B e s i d e s PhZ s p l e e n c e l l s , n o r m a l s p l e e n c e l l s t a k e n f rom ^(,C^i f e m a l e m i c e , and B6SUtA c e l l s ( a mur i ne h e m o p o i e t i c c e l l l i n e t h a t i s E p -u n r e s p o n s i v e , bu t m u l t i - C S F d e p e n d e n t ) (168 ) were u s e d i n p h o s p h o r y l a t i o n e x p e r i m e n t s . To o b t a i n r e l a t i v e l y homogeneous e r y t h r o i d p r o g e n i t o r c e l l p o p u l a t i o n s a t d i f f e r e n t s t a g e s o f d i f f e r e n t i a t i o n , B a l b C m ice v e r e i n j e c t e d ( I . V . ) w i t h 0 . 5 ml o f a 1 :50 d i l u t i o n o f the 24 hou r s u p e r n a t e f rom 5 - 2 0 RF c e l l s (NIH 3T3 c e l l s i n f e c t e d w i t h S F F V A - L and F -MuLV, i . e . , FVA i n f e c t e d ) g rown t o c o n f l u e n c e i n D u l b e c o ' s M o d i f i e d E s s e n t i a l Medium w i t h 10% f e t a l c a l f s e r u m . T w e n t y - o n e d a y s l a t e r t h e i r s p l e e n s were removed and the c e l l s d i s p e r s e d i n RPMI c o n t a i n i n g 20 mM MOPS b u f f e r (pH 7 . 4 ) u s i n g g e n t l e a g i t a t i o n . The r e s u l t i n g c e l l s u s p e n s i o n was l a y e r e d o n t o a 60 ml 1-2% b o v i n e se rum a l b u m i n g r a d i e n t a t a c e l l c o n c e n t r a t i o n o f 1 X 10** c e l l s / m l . A f t e r t he c e l l s had s e d i m e n t e d f o r 4 . 5 h o u r s a t u n i t g r a v i t y and 4 C , t he b o t t o m 30 ml o f t he g r a d i e n t were c o l l e c t e d and the c e l l s c o n t a i n e d i n t h i s f r a c t i o n were washed t h o r o u g h l y i n a l p h a medium and p l a t e d a t 1 X 10^ c e l l s / m l i n an a l p h a - b a s e d s e m i - s o l i d medium c o n t a i n i n g m e t h y l c e l l u l o s e (0 .88% f i n a l ) , f e t a l c a l f se rum (30% f i n a l ) , b o v i n e serum a l b u m i n (1% f i n a l ) , 2 - m e r c a p t o e t a n o l ( 0 . 1 mM f i n a l ) , g l u t a m i n e (2 mM f i n a l ) , 1 u n i t / m l o f p e n i c i l l i n and 1 ng / ra l o f s t r e p t o m y c i n , + / - Ep ( 0 . 2 U /ml f i n a l ) , and i n c u b a t e d a t 37 C i n 5% C O 2 and 95% a i r . A t c h o s e n t ime i n t e r v a l s , the c u l t u r e d c e l l s were h a r v e s t e d by d i l u t i o n o f t he s e m i - s o l i d medium w i t h RPMI ( 1 : 1 ) f o l l o w e d by l ow s p e e d c e n t r i f u g a t i o n (400 x g f o r 5 m i n . ) . C e l l p e l l e t s were washed two a d d i t i o n a l t i m e s i n RPMI , and s u b s e q u e n t l y d i l u t e d to a p p r o p r i a t e c e l l c o n c e n t r a t i o n s ( i n RPMI) f o r use i n 3 { j _ t n y m i d i n e i n c o r p o r a t i o n a s s a y s d e s c r i b e d a b o v e . I n V i t r o P h o s p h o r y l a t i o n A s s a y 34 P l a s m a Membrane I s o l a t i o n A p p r o x i m a t e l y 5 X 10^ c e l l s ( e i t h e r PhZ s p l e e n , n o r m a l s p l e e n , o r B6SUtA ) were washed i n RPMI and c e n t r i f u g e d a t 1 ,100 rpm (400 x g ) f o r 5 m i n . i n a Beckman mode l T J - 6 c e n t r i f u g e . The p e l l e t e d c e l l s were r e s u s p e n d e d i n 10 ml o f i c e c o l d 10 mM T r i s - H C l b u f f e r (pH 7 . 4 ) and v o r t e x e d a t top speed w i t h a K - 5 5 0 - G V o r t e x - G e n i e f o r 1 m i n . The c e l l s u s p e n s i o n was then s u b j e c t e d to h o m o g e n i z a t i o n by hand (30 s t r o k e s ) i n a 55 ml c o r e x h o m o g e n i z e r w i t h a t i g h t f i t t i n g t e f l o n p e s t l e , and f o r c e d t h r o u g h a s y r i n g e w i t h a 26 gauge n e e d l e t h r e e t i m e s . The c e l l n u c l e i were removed by c e n t r i f u g a t i o n a t 2 , 0 0 0 rpm (1100 x g ) f o r 5 m i n . ( T J - 6 c e n t r i f u g e ) and t he s u p e r n a t e p l a c e d on a 3 ml 40% s u c r o s e c u s h i o n ( i n dH20) and c e n t r i f u g e d a t 2 0 , 0 0 0 rpm f o r 60 m i n . u s i n g a Beckman L8-80M u t r a c e n t r i f u g e and an SW 27 r o t o r . The p r e c i p i t a n t a t the i n t e r f a c e was c o l l e c t e d and b r o u g h t up t o a f i n a l vo lume o f 17 ml by the a d d i t i o n o f i c e c o l d 10 mM T r i s - H C l b u f f e r (pH 7 . 4 ) . The membrane s u s p e n s i o n was then c e n t r i f u g e d a g a i n a t 2 0 , 0 0 0 rpm f o r 60 m i n . (SW 27 r o t o r ) to p e l l e t the p l a s m a membranes. The p e l l e t o b t a i n e d f r om the f i n a l c e n t r i f u g a t i o n was suspended i n 1-3 ml o f a 20 mM P i p e s b u f f e r (pH 7 . 0 ) s u c h t h a t t he f i n a l p r o t e i n c o n c e n t r a t i o n was a p p r o x i m a t e l y (by OD280) 10 m g / m l . 3 5 Membrane P h o s p h o r y l a t i o n A l i q u o t s (50 u l ) o f the membrane s u s p e n s i o n (500 ug) were p i p e t t e d i n t o 1 .5 m l E p p e n d o r f m i c r o f u g e tubes and p l a c e d i n a 37 C w a t e r b a t h . The membrane p r e p a r a t i o n s were then s u p p l e m e n t e d w i t h 20 u l o f e i t h e r 100% DMSO (20% f i n a l ) , 50 uM v a n a d a t e ( p r e p a r e d and s t o r e d as a 1 mM s o l u t i o n i n a 1 mM s o d i u m b i c a r b o n a t e b u f f e r , pH 8 . 3 ) (10 uM f i n a l ) , 1000 U / m l p u r e ( i . e . 8 0 , 0 0 0 U/mg ( 1 9 8 ) ) Ep (200 U /m l f i n a l ) , o r a c o n t r o l b u f f e r (100 u g / m l b a c i t r a c i n and 0 .1% b o v i n e serum a l b u m i n i n PBS) and i n c u b a t e d a t 37 C f o r one h o u r ( i t s h o u l d be n o t e d t h a t v a n a d a t e and Ep were d i l u t e d i n t h e c o n t r o l b u f f e r ) . G a m m a - 3 2 P - A T P ( 1 - 3 C i / m m o l ) and MgCl2 ( o r MnCl2 ) were added to f i n a l c o n c e n t r a t i o n s o f 10 pM and 30 mM ( o r 10 mM f o r MnC l2 ) r e s p e c t i v e l y , and a 3 m in i n c u b a t i o n ensued a t room temp. The r e a c t i o n was s t o p p e d by t he a d d i t i o n o f 20 u l 20% SDS, 5 u l 100% 2 - m e r c a p t o e t h a n o l , and 10 u l 100% g l y c e r o l , f o l l o w e d i m m e d i a t e l y by b o i l i n g f o r 2 m i n . An a l i q u o t (50 u l ) o f t he r e s u l t i n g m i x t u r e was a p p l i e d to a p o l y a c r y l a m i d e g r a d i e n t g e l (8-18%) and e l e c t r o p h o r e s e d as d e s c r i b e d by L a e m m l l i ( 1 6 9 ) . S o m e . g e l s were t h e n f i x e d i n 10% a c e t i c a c i d / 10% i s o p r o p a n o l (30 m i n . ) , washed i n w a t e r f o r 15 m i n , and d r i e d f o r a u t o r a d i o g r a p h y . To q u a l i t a t e the amount o f b a s e - s t a b l e p h o s p h o r y l a t i o n s s t i m u l a t e d by e a c h agen t o t h e r g e l s were p l a c e d d i r e c t l y i n t o 1 M NaOH f o r 15 min ( room temp) , t hen 1 M NaOH f o r 1 hou r a t 42 C , and f i n a l l y i n t o 10% a c e t i c a c i d / 10% i s o p r o p a n o l f o r two h o u r s ( 1 4 6 ) . A f t e r a w a t e r wash (10 m i n . ) , g e l s were d r i e d and exposed to Kodak XAR X - R a y f i l m f o r 4 h o u r s to 6 d a y s , and then d e v e l o p e d u s i n g a Kodak RP X-0MAT P r o c e s s o r . 36 C e l l P u r i f i c a t i o n I n some p h o s p h o r y l a t i o n e x p e r i m e n t s , E p - r e s p o n s i v e PhZ s p l e e n c e l l s were s e p a r a t e d f rom matu re r e d c e l l s by two me thods . F i r s t , c e l l s , once removed f r om the s p l e e n , were s e d i m e n t e d a t u n i t g r a v i t y , i n R P M I , a t 4 C . One h o u r l a t e r , t he p e l l e t was r e c o v e r e d and found to be e s s e n t i a l l y f r e e o f ma tu re e r y t h r o c y t e s ( t h e p e l l e t c o n t a i n e d 95% n u c l e a t e d c e l l s ) . I n t he s e c o n d me thod , f r e s h l y i s o l a t e d PhZ c e l l s (1 X 1 0 8 c e l l s i n 2 m is RPMI) were l a y e r e d o n t o 3 ml o f M o n o - P o l y R e s o l v i n g Medium ( d e n s i t y 1 .114 g / c m 3 , F i s h e r ) and c e n t r i f u g e d a t 800 x g f o r 40 min a t 16 C . C e l l s c o l l e c t e d f rom the i n t e r f a c e were f ound to be a l m o s t c o m p l e t e l y d e v o i d o f ma tu re r e d c e l l s . 37 RESULTS S t i m u l a t i o n o f E r y t h r o i d C e l l s by Sod ium O r t h o v a n a d a t e From ^ H - t h y m i d i n e and 5 9 p e i n c o r p o r a t i o n a s s a y s , K r y s t a l ( 170 ) had f o u n d t h a t s e v e r a l MEL c e l l i n d u c i n g a g e n t s c o u l d s t i m u l a t e the i n v i t r o e r y t h r o p o i e s i s o f n o r m a l e r y t h r o i d p r e c u r s o r c e l l s . T a b l e I I (See I n t r o d u c t i o n ) summar i zes h i s r e s u l t s . HMBA and DMSO a p p e a r e d to be t he most p o t e n t , w i t h s t i m u l a t o r y a c t i v i t i e s c o m p a r a b l e to 5 0 - 7 5 mU/ml o f Ep a c t i v i t y i n t he ^ H - t h y m i d i n e a s s a y . I n the ^ F e i n c o r p o r a t i o n a s s a y , t h e s e a g e n t s c o n s i s t e n t l y showed a g r e a t e r s t i m u l a t o r y e f f e c t w i t h r e s p e c t t o t he amount o f Ep a c t i v i t y t h e y m i m i c k e d . I n an a t t e m p t to u n d e r s t a n d the mechanism o f a c t i o n o f E p , a p h o s p h o t y r o s i n e p h o s p h a t a s e i n h i b i t o r , sod ium o r t h o v a n a d a t e ( 1 7 1 ) , was exam ined f o r i t s a b i l i t y to s u b s t i t u t e f o r Ep i n ^ H - t h y m i d i n e and 5 9 p e i n c o r p o r a t i o n b i o a s s a y s . I t was though t t h a t i f t y r o s i n e p h o s p h o r y l a t i o n was an i m p o r t a n t e a r l y e v e n t i n the i n d u c t i o n o f t e r m i n a l e r y t h r o p o i e s i s , t h a t v a n a d a t e s h o u l d be a b l e to m im ic Ep a c t i v i t y . T e s t e d a t c o n c e n t r a t i o n s be tween 1 uM-30 uM i n t he ^ H - t h y m i d i n e a s s a y , i t was f ound t h a t a 10 uM f i n a l c o n c e n t r a t i o n o f v a n a d a t e d i d i n d e e d s t i m u l a t e s p l e e n c e l l s t o i n c o r p o r a t e t h y m i d i n e ( T a b l e I I I ) . T h y m i d i n e i n c o r p o r a t i o n d e c l i n e d above 15 uM v a n a d a t e , s u g g e s t i n g t o x i c i t y . The d a t a i s d i a g r a m a t i c a l l y r e p r e s e n t e d i n F i g u r e 5 . The s t i m u l a t o r y a c t i v i t y o f v a n a d a t e was c o n t r a s t e d w i t h a s t a n d a r d Ep d o s e - r e s p o n s e c u r v e and i n d i c a t e d a maximum s t i m u l a t o r y e f f e c t e q u i v a l e n t to 20 mU/ml E p . However , v a n a d a t e a t 10 uM had no e f f e c t i n the 59Fe i n c o r p o r a t i o n a s s a y ( T a b l e I V ) ; a l t h o u g h , g i v e n t he i n s e n s i t i v i t y o f t h i s a s s a y to E p , i t i s d i f f i c u l t to a s s i g n any s i g n i f i c a n c e to t h i s f i n d i n g . 38 TABLE I I I The s t i m u l a t i o n o f ^ H - t h y m i d i n e u p t a k e i n t o PhZ s p l e e n c e l l s by sod ium o r t h o v a n a d a t e V a n a d a t e C o n c e n t r a t i o n C o u n t s p e r M i n u t e 0 uM ( c o n t r o l ) 3559 + 2 2 5 * 1 uM 4377 + 112 5 uM 18887 + 500 10 uM 31665 + 834 15 uM 29121 + 756 20 uM 12273 + 307 30 uM 5327 + 132 * E a c h v a l u e i s the mean o f t h r e e d e t e r m i n a t i o n s + S . E . M . 39 F i g u r e 5 . A c o m p a r i s o n o f d o s e - r e s p o n s e c u r v e s f o r Ep and sod ium o r t h o v a n a d a t e i n s t i m u l a t i n g ^ H - t h y m i d i n e i n c o r p o r a t i o n i n t o PhZ s p l e e n c e l l s . V a n a d a t e i n d u c e d c e l l t o x i c i t y a t c o n c e n t r a t i o n s above 15 uM. 40 TABLE IV The e f f e c t o f sod ium o r t h o v a n a d a t e on 5 9 p e i n c o r p o r a t i o n i n t o PhZ s p l e e n c e l l s V a n a d a t e C o n c e n t r a t i o n C o u n t s p e r M i n u t e 0 uM ( c o n t r o l ) 1122 + 2 8 6 * 5 uM 1229 + 385 10 uM 1218 + 369 20 uM 1154 + 302 30 uM 987 + 211 * E a c h v a l u e i s t he mean o f t h r e e d e t e r m i n a t i o n s + S . E . M . 41 To i n s u r e t h a t v a n a d a t e was no t s t i m u l a t i n g the endogenous p r o d u c t i o n o f Ep by a s u b s e t o f c e l l s w i t h i n the PhZ c e l l p o p u l a t i o n , w h i c h c o u l d i n t u r n s t i m u l a t e ^ - t h y m i d i n e i n c o r p o r a t i o n i n t o E p - r e s p o n s i v e c e l l s , an e x p e r i m e n t was p e r f o r m e d whereby e x c e s s p o l y c l o n a l r a b b i t a n t i s e r a d i r e c t e d a g a i n s t Ep was i n c u b a t e d i n the p r e s e n c e o f 10 uM v a n a d a t e i n the 3 H - t h y m i d i n e a s s a y . I t had p r e v i o u s l y been d e m o n s t r a t e d t h a t t h i s a n t i - E p a n t i s e r a c o u l d c o m p l e t e l y n e u t r a l i z e 1000 mU/ml o f Ep i n the 3 H - t h y m i d i n e a s s a y . Howeve r , no d i m i n u t i o n on t he v a n a d a t e - s t i m u l a t e d 3 H - t h y m i d i n e i n c o r p o r a t i o n was o b s e r v e d . T a r g e t C e l l A s s e s s m e n t f o r Ep and E p - M i m i c k i n g A g e n t s To o b t a i n f u r t h e r i n s i g h t i n t o whe the r E p - m i m i c k i n g a g e n t s c o u l d s u b s t i t u t e d i r e c t l y f o r Ep and f u n c t i o n e d on the same c e l l p o p u l a t i o n , 3 H -t h y m i d i n e i n c o r p o r a t i o n s t u d i e s were p e r f o r m e d w i t h r e l a t i v e l y homogeneous e r y t h r o i d p r e c u r s o r s a t d i f f e r e n t s t a g e s o f m a t u r i t y . The s o u r c e o f c e l l s f o r t h i s s t u d y was s p l e e n s f rom m ice p r e v i o u s l y i n f e c t e d w i t h t he a n e m i c s t r a i n o f the F r i e n d v i r u s (FVA c e l l s ) ( 1 7 2 ) . Upon r e m o v a l o f c e l l s f r o m i n f e c t e d s p l e e n s , t h e y were found to be u n i f o r m l y u n h e m o g l o b i n i z e d (85%) , and c o u l d , i n t he p r e s e n c e o f Ep and a s e m i s o l i d s u p p o r t , d i f f e r e n t i a t e i n t o f u l l y h e m o g l o b i n i z e d c e l l s (>90%) w i t h i n s i x t y h o u r s o f c u l t u r e . FVA c e l l s , i n c u b a t e d i n m e t h y l c e l l u l o s e w i t h E p , were removed f r om s e m i -s o l i d medium c u l t u r e a t v a r i o u s i n c u b a t i o n t i m e s and a s s a y e d f o r 3 H - t h y m i d i n e u p t a k e i n the p r e s e n c e o f E p , HMBA, b u t y r i c a c i d , DMSO, o r v a n a d a t e . F i g u r e 6 shows the r e s p o n s i v e n e s s o f FVA c e l l s to Ep as a f u n c t i o n o f d i f f e r e n t i a t i o n . When f r e s h l y removed f rom m ice ( t i m e z e r o ) t h i s c e l l p o p u l a t i o n d i f f e r e d m a r k e d l y f rom a s t a n d a r d PhZ s p l e e n c e l l p o p u l a t i o n i n 42 i t s d o s e - r e s p o n s e w i t h r e s p e c t to E p . S p e c i f i c a l l y , f rom F i g u r e 6 , no s a t u r a t i o n o f b i o a c t i v i t y was o b s e r v e d w i t h f r e s h l y i s o l a t e d FVA c e l l s even when a s s a y e d up t o 1000 m i l / m l , w h e r e a s , the same number o f PhZ c e l l s p l a t e a u e d i n t h e i r r e s p o n s e a t 100 mi l /ml Ep ( F i g u r e 5 ) . T h i s s u g e s t e d t h a t t he FVA c e l l p o p u l a t i o n was l e s s s e n s i t i v e to Ep than PhZ s p l e e n c e l l s . Howeve r , a f t e r 24 h o u r s o f m e t h y l c e l l u l o s e c u l t u r e , t he h a r v e s t e d FVA c e l l s d i s p l a y e d s a t u r a t i o n k i n e t i c s more l i k e t h a t o f PhZ s p l e e n c e l l s , p l a t e a u i n g a t a p p r o x i m a t e l y 100 m i l /m l . F o l l o w i n g 48 h o u r s o f c u l t u r e , t he FVA c e l l s were e s s e n t i a l l y u n r e s p o n s i v e to E p , s u g g e s t i n g t h a t v e r y few m i t o t i c a l l y . a c t i v e FVA c e l l s r e m a i n e d ( F i g u r e 6 ) . T a b l e I V d e m o n s t r a t e s the s t i m u l a t o r y a c t i v i t i e s o f HMBA, b u t y r i c a c i d , DMSO, and v a n a d a t e when a s s a y e d w i t h d i f f e r e n t i a t i n g FVA c e l l s . W i t h f r e s h l y i s o l a t e d FVA c e l l s , t h e s e a g e n t s were found to have v e r y l i t t l e , i f a n y , e f f e c t i n i n c r e a s i n g ^ H - t h y m i d i n e u p t a k e . When the c e l l s were p e r m i t t e d t o d i f f e r e n t i a t e f o r 24 h o u r s , h o w e v e r , a l l t he a g e n t s became s t i m u l a t o r y i n t he 3 H - t h y m i d i n e i n c o r p o r a t i o n a s s a y . T h e i r a c t i v i t i e s on one day o l d FVA c e l l s were f o u n d to be v e r y s i m i l a r t o t h e i r a c t i v i t i e s on PhZ c e l l s ( f r o m T a b l e I ) . S p e c i f i c a l l y HMBA, b u t y r i c a c i d , DMSO, and v a n a d a t e m i m i c k e d 7 5 , 1 3 , 4 5 , and 13 mi l /ml o f Ep a c t i v i t y ( T a b l e V ) . No m i m i c k i n g a g e n t was a b l e t o i n v o k e a s t i m u l a t o r y r e s p o n s e i n the 3 H - t h y m i d i n e a s s a y w i t h FVA c e l l s c u l t u r e d f o r 48 h o u r s i n m e t h y l c e l l u l o s e . To be c e r t a i n t h a t the FVA c e l l s were a c t u a l l y d i f f e r e n t i a t i n g when c u l t u r e d i n v i t r o , t he c e l l s were r o u t i n e l y a s s a y e d f o r t he p r e s e n c e o f h e m o g l o b i n by b e n z i d i n e s t a i n i n g . When f r e s h l y i s o l a t e d , o n l y 15% of the c e l l s were p o s i t i v e f o r t h i s s t a i n . A f t e r 24 h o u r s , 59% o f the c e l l s were b e n z i d i n e p o s i t i v e , and a t 48 h o u r s , 70% s t a i n e d p o s i t i v e l y . I n c o m p a r i s o n , n u c l e a t e d PhZ s p l e e n c e l l s were 90% h e m o g l o b i n p o s i t i v e . 10 1 00 1000 mU/ml Ep F i g u r e 6 . The d e c l i n e i n E p - r e s p o n s i v e n e s s as FVA c e l l s ma tu re i s r e p r e s e n t e d h e r e . When f r e s h l y i s o l a t e d , t h e r e a p p e a r e d to be a l a r g e p r o p o r t i o n o f E p - r e s p o n s i v e c e l l s i n t he c e l l p o p u l a t i o n . Howeve r , when c e l l s were p e r m i t t e d to d i f f e r e n t i a t e , t he a b i l i t y o f Ep to s t i m u l a t e ^ H - t h y m i d i n e i n c o r p o r a t i o n d e c r e a s e d . I t s h o u l d be n o t e d t h a t c e l l s m a i n t a i n e d i n c u l t u r e f o r 24 h o u r s , bu t no t p e r m i t t e d t o d i f f e r e n t i a t e ( c u l t u r e d w i t h o u t E p ) , r e m a i n e d as r e s p o n s i v e to Ep as t h o s e c u l t u r e d w i t h E p . TABLE V The s t i m u l a t o r y e f f e c t s o f HMBA, b u t y r i c a c i d , DMSO, and s o d i u m o r t h o v a n a d a t e on ^ H - t h y m i d i n e u p t a k e i n t o d i f f e r e n t i a t i n g FVA c e l l s Agen t E q u i v a l e n c e i n Ep A c t i v i t y (mU/ml) A t t i m e z e r o : H 1.5 B T o x i c D T o x i c V 1.5 A t 24 h o u r s : H 70 B 13 D 45 V 13 A t 48 h o u r s : H 0 B 0 D 0 V 0 A b b r e v i a t i o n s : H - HMBA, B - b u t y r i c a c i d , D - DMSO, V - v a n a d a t e . E q u i v a l e n c e o f Ep a c t i v i t y was computed by c o m p a r i n g the c . p . m . o b t a i n e d f o r e a c h agen t ( a t e a c h t ime p o i n t ) to s t a n d a r d Ep d o s e -r e s p o n s e c u r v e s p e r f o r m e d a t e a c h t ime p o i n t ( F i g u r e 5) . 45 M e m b r a n e - S p e c i f i c P r o t e i n P h o s p h o r y l a t i o n Of s p e c i a l i n t e r e s t to us was t h a t two a g e n t s shown i n o u r l a b o r a t o r y t o s t i m u l a t e PhZ s p l e e n c e l l s i n v i t r o (DMSO and v a n a d a t e ) had been d e m o n s t r a t e d p r e v i o u s l y t o e f f e c t p r o t e i n p h o s p h o r y l a t i o n e v e n t s i n o t h e r s y s t e m s . F o r e x a m p l e , i n 1 9 8 3 , E a r p (173) found t h a t DMSO i n c r e a s e d t he amount o f p h o s p h o p r o t e i n s c o n t a i n i n g p h o s p h o t y r o s i n e i n MEL c e l l s ( s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n changes were no t i n v e s t i g a t e d ( 1 7 4 ) ) . DMSO has r e c e n t l y been shown t o i n d u c e t he p h o s p h o r y l a t i o n o f the EGF r e c e p t o r a t t y r o s i n e r e s i d u e s i n l i v e r c e l l s ( 1 7 5 ) . V a n a d a t e has a l s o been s t r o n g l y c o r r e l a t e d w i t h i n c r e a s e d t y r o s i n e p h o s p h o r y l a t i o n v i a i t s a b i l i t y to i n h i b i t an enzyme ( p h o s p h o t y r o s i n e p h o s p h a t a s e ) t h a t b r e a k s p h o s p h a t e - t y r o s i n e b o n d s . I t h a s been p o s t u l a t e d t h a t i t i s t h r o u g h t h i s mechanism t h a t v a n a d a t e p o t e n t i a t e s t he g r o w t h p r o m o t i n g e f f e c t s o f EGF and i n s u l i n ( 1 7 6 ) . T r a c e y and G r e s s e r ( 1 7 7 ) a r g u e t h a t t he i n s u l i n m i m e t i c e f f e c t o f vanad ium i s due t o the e s t e r i f i c a t i o n o f t y r o s i n e m o i e t i e s by v a n a d a t e and c o r r e s p o n d i n g a c t i v a t i o n o f t he i n s u l i n r e c e p t o r k i n a s e . T h u s , t h e s e two a g e n t s found to s t i m u l a t e PhZ c e l l s and Ep ( t h e i n v i v o r e g u l a t o r ) were t e s t e d to d e t e r m i n e i f t hey c o u l d i n d u c e s i g n i f i c a n t c h a n g e s i n p l a s m a membrane p h o s p h o r y l a t i o n . S a m p l e s , once the p h o s p h o r y l a t i o n e x p e r i m e n t s were c o m p l e t e d , were e l e c t r o p h o r e s e d u s i n g p o l y a c r y l a m i d e g r a d i e n t g e l s f r om 8% to 20% i n o r d e r to a l l o w f o r t he r e s o l u t i o n o f b o t h l a r g e and s m a l l p r o t e i n s p e c i e s . I t s h o u l d be n o t e d t h a t a l l t he f i n d i n g s p r e s e n t e d h e r e r e p r e s e n t e x p e r i m e n t s a t l e a s t c o n d u c t e d i n t r i p l i c a t e . The r e s u l t s o f t h e s e i n v i t r o p h o s p h o r y l a t i o n e x p e r i m e n t s when c a r r i e d ou t in the p r e s e n c e o f magnesium c h l o r i d e (30 mM f i n a l ) and when t o t a l membrane p h o s p h o r y l a t i o n was e x a m i n e d , r e v e a l e d no s i g n i f i c a n t p h o s p h o r y l a t i o n changes 46 due t o Ep (200 U / m l ) o r v a n a d a t e (10 uM) when compared to t he c o n t r o l b u f f e r ( F i g u r e 7 ) . However , DMSO c o n s i s t e n t l y s t i m u l a t e d s i g n i f i c a n t p h o s p h o r y l a t i o n c h a n g e s . S p e c i f i c a l l y , DMSO i n d u c e d t he p h o s p h o r y l a t i o n o f a 35 Kd membrane a s s o c i a t e d p r o t e i n . V a n a d a t e , when i n c u b a t e d i n c o n j u n c t i o n w i t h DMSO n e i t h e r a b r o g a t e d n o r enhanced the p h o p h o r y l a t i o n o f t h i s 35 Kd p r o t e i n ( F i g u r e 7 ) . The p r e s e n c e o f t he D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n a p p e a r s t o be dependen t on t he p r e s e n c e o f the d i v a l e n t c a t i o n M g + 2 . F i g u r e 8 r e p r e s e n t s an a u t o r a d i o g r a m where manganese c h l o r i d e (10 mM f i n a l ) was s u b s t i t u t e d f o r magnesium c h l o r i d e . As i n d i c a t e d by t he a r r o w , t he p h o s p h o r y l a t i o n o f t he 35 Kd p r o t e i n i s no t s e e n . A P r o l o n g e d e x p o s u r e o f the g e l ( n o t shown) p r o v i d e d f u r t h e r e v i d e n c e t h a t t h i s s p e c i f i c p h o s p h o r y l a t i o n e v e n t d i d no t o c c u r when Mn + 2 was s u b s t i t u t e d f o r M g + 2 . One p r o b l e m w i t h PhZ s p l e e n c e l l s i s t h a t t h e y a r e a v e r y h e t e r o g e n e o u s p o p u l a t i o n o f c e l l s w i t h r e s p e c t to r e d c e l l d i f f e r e n t i a t i o n , t h o u g h , v e r y homogeneous i n b e i n g o f t he e r y t h r o i d l i n e a g e . To e n s u r e t h a t p r e v i o u s r e s u l t s were no t due to r e d c e l l c o n t a m i n a t i o n , i n v i t r o p h o s p h o r y l a t i o n e x p e r i m e n t s were p e r f o r m e d u s i n g f r a c t i o n a t e d PhZ s p l e e n c e l l s . PhZ s p l e e n c e l l s were p u r i f i e d f r e e o f r e d c e l l s e i t h e r by c e n t r i f u g a t i o n o n t o a 1 .114 g m / c m 3 F i c o l l - 4 0 0 Hypaque-85 c u s h i o n ( t h e r e d c e l l s p a s s t h r o u g h the c u s h i o n w h i l e more p r i m i t i v e e r y t h r o i d p r e c u r s o r s r e m a i n on t o p ) , o r by u n i t g r a v i t y s e d i m e n t a t i o n a t 4 C ( E p - r e s p o n s i v e c e l l s p e l l e t and r e d c e l l s s t a y i n s u s p e n s i o n ) . PhZ s p l e e n c e l l s p u r i f i e d by e i t h e r method r e m a i n e d as E p -r e s p o n s i v e as u n p u r i f i e d c e l l s when a s s a y e d by ^ H - t h y m i d i n e i n c o r p o r a t i o n . R e s u l t s f rom i n v i t r o p h o s p h o r y l a t i o n e x p e r i m e n t s w i t h t h e s e p u r i f i e d P h Z s p l e e n c e l l s were s i m i l a r to t h o s e o b t a i n e d w i t h u n f r a c t i o n a t e d c e l l s ( i . e . no s p e c i f i c e f f e c t s were n o t e d f o r Ep o r v a n a d a t e , and DMSO c o n t i n u e d to o m < > o o + < > 47 o FIGURE 7. A u t o r a d i o g r a p h d e m o n s t r a t i n g the s p e c i f i c p h o s p h o r y l a t i o n changes i n d u c e d by DMSO. The a r r o w i n d i c a t e s the 35 Kd p h o s p h o p r o t e i n s t i m u l a t e d by DMSO i n the a b s e n c e o r p r e s e n c e o f v a n a d a t e ( V A N ) . The Ep l a n e and b u f f e r c o n t r o l l a n e a r e a l s o r e p r e s e n t e d (C ) o o 21.5 FIGURE 8 . A u t o r a d i o g r a p h d e m o n s t r a t i n g the l o s s o f the D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n when 10 mM manganese c h l o r i d e was s u b s t i t u t e d f o r 30 mM magnesium c h l o r i d e i n t he i n c u b a t i o n m i x t u r e . The a r r o w i n d i c a t e s the p o s i t i o n n o r m a l l y o c c u p i e d by the 35 Kd p r o t e i n . 49 s t i m u l a t e the p h o s p h o r y l a t i o n o f the 35 Kd p r o t e i n ) . T h e r e w e r e , h o w e v e r , changes i n t he o v e r a l l p r o f i l e o f membrane p h o s p h o p r o t e i n s ( p r e s u m a b l y due t o t he r e m o v a l o f n o n - s p e c i f i c k i n a s e a c t i v i t y a s s o c i a t e d w i t h r e d c e l l membranes) . I t was d e c i d e d t h a t a l l s u b s e q u e n t e x p e r i m e n t s be p e r f o r m e d w i t h f r a c t i o n a t e d s p l e e n c e l l s . I n an a t t e m p t to f u r t h e r c h a r a c t e r i z e the D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n , some p o l y a c r y l a m i d e g e l s were t r e a t e d w i t h 1 N NaOH ( s e e M a t e r i a l s and Me thods ) j u s t p r i o r to f i x i n g and d r y i n g f o r a u t o r a d i o g r a p h y . The p u r p o s e o f t h i s t r e a t m e n t was to remove p h o s p h a t e g r o u p s p r e f e r e n t i a l l y f r om s e r i n e and t h r e o n i n e r e s i d u e s ( b a s e l a b i l e p h o s p h a t e l i n k a g e s ) s u c h t h a t p h o s p h o t y r o s i n e c o n t e n t w i t h i n a p r o t e i n migh t b e t t e r be exam ined ( 1 0 4 ) . F i g u r e 9 i l l u s t r a t e s the f i n d i n g s o b t a i n e d f rom base t r e a t m e n t o f g e l s ( i t s h o u l d be n o t e d t h a t the f i l m was exposed f i v e t i m e s l o n g e r t h a n n o r m a l , s i n c e r e m o v a l o f p h o s p h a t e f rom s e r i n e and t h r e o n i n e r e s i d u e s r e s u l t s i n f a r l e s s r a d i o a c t i v e g e l s ) . Membranes f rom p u r i f i e d PhZ s p l e e n c e l l s i n c u b a t e d w i t h Ep p r o v e d to be no d i f f e r e n t f rom the c o n t r o l w i t h r e s p e c t to t y r o s i r i e -s p e c i f i c p h o s p h o r y l a t i o n s . V a n a d a t e d e m o n s t r a t e d an o v e r a l l i n c r e a s e i n b a s e s t a b l e p h o s p h o r y l a t i o n , though no s p e c i f i c p h o s p h o r y l a t i o n changes c o u l d be d e t e c t e d . The D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n r e m a i n e d i n t a c t a f t e r b a s e t r e a t m e n t , s u g g e s t i n g t h a t t h i s p r o t e i n was p a r t i a l l y o r c o m p l e t e l y p h o s p h o r y l a t e d a t t y r o s i n e r e s i d u e s . D M S O - s t i m u l a t e d d e p h o s p h o r y l a t i o n ( o r n o n p h o s p h o r y l a t i o n ) e v e n t s were a l s o o b s e r v a b l e on b a s e t r e a t e d g e l s . Of p o s s i b l e i m p o r t a n c e a r e the a b s e n c e o f l o w e r m o l e c u l a r w e i g h t p h o s p h o p r o t e i n s p e c i e s i n DMSO s a m p l e s . I t s h o u l d be n o t e d t h a t t h e s e base l a b i l i t y s t u d i e s c a n no t be t a k e n as a b s o l u t e p r o o f f o r t y r o s i n e - s p e c i f i c p h o s p h o r y l a t i o n s i n c e , d e p e n d i n g upon the amino a c i d s e q u e n c e , some p h o s p h o s e r i n e , and e s p e c i a l l y , p h o s p h o t h r e o n i n e l i n k a g e s may r e m a i n i n t a c t f o l l o w i n g the base m cn > O 73 O 2 3 5 K d FIGURE 9. Autoradiograph of a 1 M NaOH treated polyacrylamide gel. Samples electrophoresed vere solubilized membranes from fractionated PhZ c e l l preparations. The arrov indicates the position of DMSO-stimulated 35 Kd protein. The vanadate lane demonstrates an overall increase in base-stable phosphate linkages. 51 t r e a t m e n t . When s a m p l e s a r e m o n i t o r e d f o r t o t a l r a d i o a c t i v i t y b e f o r e and a f t e r b a s e t r e a t m e n t , a p p r o x i m a t e l y 80% o f the 0 c o u n t s a r e f o u n d removed a f t e r t r e a t m e n t . I f p h o s p h a t e g r o u p s f rom a l l p h o s p h o r y l a t e d s e r i n e and t h r e o n i n e r e s i d u e s were to be removed , we wou ld e x p e c t l e s s t h a n 1% o f the o r i g i n a l c o u n t s t o be r e t a i n e d . Thus i t i s c o n c e i v a b l e t h a n t he r e l a t i v e r e s i s t a n c e o f t he 35 Kd p h o s p h o p r o t e i n to base t r e a t m e n t may be a r e s u l t o f i n c o m p l e t e b a s e h y d r o l y s i s o f e i t h e r s e r i n e o r t h r e o n i n e phosphoamino a c i d s . F o r t he D M S O - s t i m u l a t e d p h o s p h o r y l a t i o n changes on PhZ s p l e e n c e l l s t o be i m p l i c a t e d a s a mechanism o f i n v i t r o e r y t h r o i d d i f f e r e n t i a t i o n , i t was n e c e s s a r y t o d e m o n s t r a t e t h a t o t h e r n o n - t a r g e t c e l l t y p e s were no t s u b j e c t t o s i m i l a r membrane p h o s p h o r y l a t i o n changes upon DMSO i n d u c t i o n . T h u s , two n o n -t a r g e t c e l l t y p e s were a s s a y e d . PhZ s p l e e n c e l l s , though g r e a t l y e n r i c h e d i n E p - r e s p o n s i v e t e r m i n a l l y d i f f e r e n t i a t i n g e r y t h r o i d p r e c u r s o r s , c o n t a i n a s u b s e t o f n o r m a l s p l e n i c l y m p h o c y t e s (5-10% o f the n u c l e a t e d c e l l c o u n t ) . I t was c o n c e i v a b l e t h a t t h i s l y m p h o i d e lemen t was r e s p o n s i b l e f o r t he DMSO-s t i m u l a t e d p h o s p h o r y l a t i o n changes p r e v i o u s l y a s c r i b e d to t he E p - r e s p o n s i v e c e l l s . A c c o r d i n g l y , n o r m a l m u r i n e s p l e e n ( f o u n d by K r y s t a l to be n o n - E p -r e s p o n s i v e ) s e r v e d as one c o n t r o l . The B6SUtA c e l l l i n e (JG -1 c e l l s ) p r o v i d e d c e l l s f o r t he o t h e r n o n - t a r g e t c o n t r o l . These c e l l s , d e r i v e d f r om the n o n - a d h e r e n t l a y e r o f a m u r i n e bone marrow c u l t u r e ( 1 6 8 ) , a r e m u r i n e i n t e r l u e k i n - 3 - d e p e n d e n t and u n r e s p o n s i v e to b o t h Ep and DMSO ( a s a s s a y e d by 3 H - t h y m i d i n e i n c o r p o r a t i o n ) . The i n v i t r o p h o s p h o r y l a t i o n a s s a y o f membranes f r om t h e s e n o n - t a r g e t c e l l s ( F i g u r e 10) r e v e a l e d t h a t e a c h c e l l t y p e y i e l d e d a d i f f e r e n t p o p u l a t i o n o f p h o s p h o p r o t e i n s , and more i m p o r t a n t l y , the DMSO-s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n , so e v i d e n t w i t h p u r i f i e d PhZ s p l e e n c e l l s , was v i r t u a l l y a b s e n t f rom b o t h the n o r m a l s p l e e n and JG -1 c e l l s a m p l e s . A l t h o u g h no t shown, i t s h o u l d , be n o t e d t h a t DMSO c a u s e d no s i g n i f i c a n t 52 FIGURE 10. Autoradiograph demonstrating the s p e c i f i c i t y of the DMSO st i m u l a t o r y e f f e c t f o r PhZ c e l l membranes. Normal spleen (NS) and JG-1 (B6SUtA) membranes were incubated with DMSO (20% f i n a l ) and Y-32p_ATP i n an i d e n t i c a l fashion to that used with PhZ c e l l membranes. The 35 kd phosphoprotein so evident with PhZ c e l l membranes i s v i r t u a l l y a b s e n t from the NS and JG-1 lanes. These l a t e r two c e l l t y p e s had been p r e v i o u s l y shown i n i n v i t r o b i o a s s a y s t o be unresponsive to DMSO. 53 changes i n e i t h e r n o r m a l s p l e e n o r J G - 1 membrane p h o s p h o r y l a t i o n when compared to c o n t r o l b u f f e r s f o r e a c h c e l l t y p e . I n a d d i t i o n , DMSO (PhZ s p l e e n c e l l membrane) p h o s p h o r y l a t i o n s a m p l e s were e l e c t r o p h o r e s e d unde r r e d u c i n g v e r s u s n o n - r e d u c i n g c o n d i t i o n s . I t was f o u n d ( F i g u r e 11) t h a t unde r n o n - r e d u c i n g c o n d i t i o n s , the 35 Kd p h o s p h o p r o t e i n c o n s i s t e n t l y m i g r a t e d s l o w e r , s u g g e s t i n g a s l i g h t l y h i g h e r m o l e c u l a r w e i g h t ( a p p r o x i m a t e l y 1 .0 Kd h e a v i e r ) o f t he u n r e d u c e d s p e c i e s . T h i s m e m b r a n e - a s s o c i a t e d p r o t e i n c o u l d t h e r e f o r e be a h e t e r o d i m e r , t h a t u n d e r r e d u c i n g c o n d i t o n s d i s s o c i a t e s f rom a s m a l l p e p t i d e . I t s h o u l d a l s o be n o t e d t h a t t h e r e were f a r f ewe r p h o s p h o p r o t e i n s o b s e r v a b l e u n d e r n o n - r e d u c i n g c o n d i t i o n s ( w h i c h p r o b a b l y r e s u l t s f rom many o f t he n o n - r e d u c e d m a c r o m o l e c u l e s b e i n g too l a r g e to e n t e r the g e l ) . L a s t l y , t he r e s u l t s o b t a i n e d above were c o n s i s t e n t w i t h t he p o s s i b i l i t y t h a t t he p h o s p h o r y l a t e d 35 Kd p r o t e i n m igh t be a s u b u n i t o f t he G T P - b i n d i n g p r o t e i n c o m p l e x . From o t h e r s t u d i e s , the & s u b u n i t o f t he G T P - b i n d i n g p r o t e i n comp lex had been shown to be a 35 Kd p h o s p h o t y r o s i n e - c o n t a i n i n g p r o t e i n t h a t c o u l d be r e l e a s e d f rom a human p l a c e n t a c r u d e membrane p r e p a r a t i o n by t he c a l c i u m c h e l a t o r EDTA ( 1 7 8 ) . I n an a t t e m p t t o d e t e r m i n e i f t he 35 Kd p h o s p h o p r o t e i n o b s e r v e d h e r e was t h i s 6 s u b u n i t , an i n v i t r o p h o s p h o r y l a t i o n e x p e r i m e n t was c o n d u c t e d whereby membranes s t i m u l a t e d w i t h DMSO were i n c u b a t e d i n t he p r e s e n c e o f 5 mM EDTA, and c e n t r i f u g e d ( 1 5 , 0 0 0 x g f o r 15 m i n . ) p r i o r to e l e c t r o p h o r e s i s . I t was hoped t h a t t h i s t r e a t m e n t w o u l d remove o u r 35 Kd p r o t e i n f rom a f a r more p u r e membrane p r e p a r a t i o n , h o w e v e r , o u r r e s u l t s ( no t shown) s u g g e s t t h a t i t c o u l d no t be removed by t h i s t r e a t m e n t . FIGURE 1 1 . A u t o r a d i o g r a p h s h o w i n g the r e d u c e d (R) and n o n r e d u c e d (NR) fo rms o f the D M S O - s t i m u l a t e d p h o s p h o p r o t e i n . The d i f f e r e n c e i n m o l e c u l a r w e i g h t s be tween the two i s a p p r o x i m a t e l y 1 . 0 Kd ( t h e n o n r e d u c e d form b e i n g 36 K d ) . 55 DISCUSSION I t i s becoming a p p a r e n t t h a t c e l l u l a r g r o w t h and d i f f e r e n t i a t i o n a r e r e g u l a t e d a t t h r e e d i s t i n c t s i t e s : the c e l l membrane, the c y t o s o l , and t he n u c l e u s . The ne t g r o w t h s t i m u l u s a c e l l r e c e i v e s r e s u l t s f rom a comp lex i n t e r a c t i o n o f t he r e g u l a t o r y mechanisms o p e r a t i v e a t e a c h l o c u s . A t t he c e l l membrane, t he p r e d o m i n a t i n g mechanism o f g r o w t h f a c t o r s i g n a l t r a n s f e r - a p p e a r s to be t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n . I t i s now a c c e p t e d t h a t many g r o w t h f a c t o r r e c e p t o r s a r e t y r o s i n e k i n a s e s , and i t i s p resumed t h a t t h e s e r e c e p t o r s r e l a y h o r m o n a l s i g n a l s t h r o u g h t y r o s i n e - p h o s p h o r y l a t e d i n t e r m e d i a t e s . M o r e o v e r , many o n c o g e n e s , whose gene p r o d u c t s when a b e r r a n t l y e x p r e s s e d i n d u c e m a l i g n a n t t r a n s f o r m a t i o n , encode p r o t e i n s d e m o n s t r a t i n g t y r o s i n e k i n a s e a c t i v i t y . I n many c a s e s , d e c r e a s e d t r a n s f o r m i n g c a p a b i l i t i e s o f mu ta ted oncogenes have been c o r r e l a t e d w i t h an i m p a i r e d t y r o s i n e k i n a s e a b i l i t y o f t he gene p r o d u c t ( 1 7 9 ) . T h u s , a s t r o n g a s s o c i a t i o n be tween t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n and c e l l g r o w t h has been e s t a b l i s h e d . I t has been d e m o n s t r a t e d h e r e t h a t sod ium o r t h o v a n a d a t e p romo tes 3 H -t h y m i d i n e i n c o r p o r a t i o n i n t o t e r m i n a l l y d i f f e r e n t i a t i n g e r y t h r o i d p r e c u r s o r s . T h i s i s no t s u r p r i s i n g as v a n a d a t e has been shown to enhance DNA s y n t h e s i s i n a number o f d i f f e r e n t s y s t e m s ( i n c l u d i n g MEL c e l l s , where v a n a d a t e c a u s e s a two f o l d i n c r e a s e i n c e l l d i v i s i o n ) ( 1 8 0 , 1 8 1 ) . V a n a d a t e , a p h y s i o l o g i c a l l y r e l e v a n t t r a n s i t i o n m e t a l i o n ( 1 8 2 ) , has been a s s o c i a t e d w i t h a w i d e v a r i e t y o f f u n c t i o n s ( 1 8 3 - 1 8 6 ) . The most n o t a b l e o f t h e s e f u n c t i o n s i s i t s a b i l i t y t o a c t s y n e r g i s t i c a l l y (180) o r a d d i t i v e l y (176) w i t h g r o w t h f a c t o r s to i n c r e a s e ^ H - t h y m i d i n e i n c o r p o r a t i o n i n t o r e s t i n g f i b r o b l a s t s . I n a d d i t i o n , i t has r e c e n t l y been shown t h a t v a n a d a t e may p a r t i a l l y s u p p l a n t the i n s u l i n 56 r e q u i r e m e n t i n d i a b e t i c r a t s ( 1 8 7 ) . I t a p p e a r s t h a t t he mechan ism by w h i c h v a n a d a t e c a r r i e s ou t t h e s e e f f e c t s i s e i t h e r by i n c r e a s i n g the amount o f m e m b r a n e - a s s o c i a t e d p h o s p h o t y r o s i n e o r by a c t i v a t i n g the i n s u l i n r e c e p t o r d i r e c t l y by e s t e r i f i c a t i o n ( 1 7 7 ) . I t has been w e l l documented t h a t v a n a d a t e i s a p o t e n t i n h i b i t o r o f p h o s p h a t a s e s t h a t s p e c i f i c a l l y remove p h o s p h a t e g r o u p s f rom t y r o s i n e r e s i d u e s ( 1 7 1 ) . T h i s may e x p l a i n o u r f i n d i n g t h a t v a n a d a t e (10 uM f i n a l ) was a b l e to i n c r e a s e the amount o f base s t a b l e p r o t e i n p h o s p h o r y l a t i o n s i g n i f i c a n t l y above c o n t r o l l e v e l s ( F i g u r e 9 ) , as w e l l a s s t i m u l a t e t h y m i d i n e u p t a k e i n t o the same c e l l s . T h e r e i s s t r o n g e v i d e n c e t o a r g u e t h a t the g r o w t h s t i m u l u s a f f o r d e d by v a n a d a t e u l t i m a t e l y r e s u l t s f r om t h e s e p h o s p h o r y l a t i o n c h a n g e s . R e c e n t l y , (188 ) v a n a d a t e has been shown t o i n d u c e t r a n s f o r m a t i o n o f NRK-1 c e l l s ( r a t k i d n e y c e l l s ) , w h i l e c o n c o m i t t a n t l y i n c r e a s i n g the l e v e l o f p h o s p h o t y r o s i n e i n NRK-1 c e l l p r o t e i n f o r t y f o l d . We t h u s t e n t a t i v e l y c o n c l u d e t h a t the v a n a d a t e - s t i m u l a t e d 3 H - t h y m i d i n e i n c o r p o r a t i o n we o b s e r v e w i t h PhZ s p l e e n c e l l s i s c a u s e d by t he d e m o n s t r a t e d i n c r e a s e i n p h o s p h o t y r o s i n e c o n t e n t o f PhZ membrane p r o t e i n s . D i m e t h y l s u l f o x i d e (DMSO) has a l s o been c l e a r l y i m p l i c a t e d i n e f f e c t i n g membrane p h o s p h o r y l a t i o n c h a n g e s . E a r p (173) d e m o n s t r a t e d t h a t DMSO was a b l e t o i n c r e a s e p h o s p h o t y r o s i n e c o n t e n t w i t h i n MEL c e l l s , and s p e c u l a t e d t h a t t h i s phenomenon m igh t be a p u t a t i v e mechanism by w h i c h DMSO i n d u c e d t h e d i f f e r e n t i a t i o n o f t h e s e c e l l s . We now f u r t h e r s u b s t a n t i a t e t h i s f i n d i n g by s h o w i n g t h a t DMSO s i g n i f i c a n t l y a l t e r s the p h o s p h o r y l a t i o n s t a t e o f membranes i s o l a t e d f r om E p - r e s p o n s i v e e r y t h r o i d c e l l s . S p e c i f i c a l l y , DMSO s t i m u l a t e s t he p h o s p h o r y l a t i o n o f a 35 Kd m e m b r a n e - a s s o c i a t e d p r o t e i n found on Ep r e s p o n s i v e PhZ s p l e e n c e l l s , bu t no t on JG-1 o r on n o r m a l s p l e e n c e l l s . Subsequen t c h a r a c t e r i z a t i o n showed t h i s p r o t e i n to be p h o s p h o r y l a t e d by a M g + 2 - d e p e n d e n t , p o s s i b l y t y r o s i n e - s p e c i f i c , p r o t e i n k i n a s e . I t i s t hus 57 c o n c e i v a b l e t h a t DMSO s t i m u l a t e s i n v i t r o e r y t h r o p o i e s i s t h r o u g h p h o s p h o r y l a t i o n changes i n d u c e d on PhZ s p l e e n c e l l membranes, and p e r h a p s , by i n c r e a s i n g the p h o s p h o r y l a t i o n o f a s p e c i f i c 35 Kd m e m b r a n e - a s s o c i a t e d p r o t e i n . From o t h e r s y s t e m s , i t i s becoming c l e a r t h a t a g r o u p o f membrane-a s s o c i a t e d p r o t e i n s , c o l l e c t i v e l y termed g u a n o s i n e t r i p h o s p h a t e ( G T P ) - b i n d i n g p r o t e i n s , G p r o t e i n s , may be r e s p o n s i b l e f o r g r o w t h s i g n a l t r a n s f e r f r om g r o w t h f a c t o r r e c e p t o r to c y t o s o l ( 1 6 2 ) . Of i n t e r e s t i s the r e c e n t f i n d i n g t h a t a m e m b r a n e - a s s o c i a t e d 35 Kd p r o t e i n ( a s a s s e s s e d u n d e r r e d u c i n g SDS/PAGE c o n d i t i o n s ) i s t he 3 s u b u n i t o f a s p e c i f i c G T P - b i n d i n g p r o t e i n . T h i s 35 Kd s u b u n i t has been shown to be t y r o s i n e - p h o s p h o r y l a t e d upon c e l l u l a r a c t i v a t i o n by e p i d e r m a l g r o w t h f a c t o r ( 1 7 8 ) . The p h o s p h o r y l a t i o n o f t h i s G p r o t e i n s u b u n i t p o t e n t i a l l y a l l o w s f o r the p r o p a g a t i o n o f the g r o w t h s i g n a l to t h e c e l l . I t has been p o s t u l a t e d t h a t the 3 s u b u n i t i s v e r y t i g h t l y a s s o c i a t e d w i t h a s m a l l p e p t i d e (Y s u b u n i n t ) , and t h a t t h i s a s s o c i a t i o n i s p o s s i b l y s t a b l e u n d e r SDS/PAGE n o n r e d u c i n g c o n d i t i o n s ( 1 6 3 ) . To d e t e r m i n e t he r e l a t i o n s h i p be tween the 35 Kd p r o t e i n p h o s p h o r y l a t e d by DMSO i n o u r PhZ s p l e e n c e l l s y s t e m and the 35 Kd 3 s u b u n i t o f the G p r o t e i n c o m p l e x , we examined o u r D M S O - s t i m u l a t e d p h o s p h o p r o t e i n u n d e r n o n r e d u c i n g c o n d i t i o n s . As d e s c r i b e d i n the R e s u l t s s e c t i o n ( F i g u r e 1 1 ) , t h i s p r o t e i n d i d m i g r a t e s l i g h t l y more s l o w l y s u g g e s t i n g t h a t t h i s p h o s p h o -p r o t e i n may i n d e e d r e p r e s e n t the G p r o t e i n s u b u n i t . Howeve r , f u r t h e r e x p e r -i m e n t a t i o n , n o t a b l y t he f a i l u r e to remove t h i s p r o t e i n f rom the membrane w i t h EDTA, f a i l e d t o s u b s t a n t i a t e t h i s c l a i m ( t hough t h i s p r o c e d u r e had p r e v i o u s l y been d e m o n s t r a t e d o n l y on human p l a c e n t a l membranes) . T h e r e f o r e , the q u e s t -i o n o f w h e t h e r t he D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n i s the 3 s u b u n i t o f t h e G T P - b i n d i n g comp lex r e m a i n s u n a n s w e r e d . F u r t h e r c h a r a c t e r i z a t i o n i s 58 n e c e s s a r y b e f o r e any d e f i n i t i v e c l a i m s can be made. The D M S O - s t i m u l a t e d 35 Kd p r o t e i n d e m o n s t r a t e d h e r e m igh t a l s o be e i t h e r l i p o c o r t i n o r c a l p a c t i n , two 35 Kd m e m b r a n e - a s s o c i a t e d t y r o s i n e - p h o s p h o r y l a t e d p r o t e i n s ( 1 8 9 ) . R e c e n t l y , t h e r e have been r e p o r t s i n the l i t e r a t u r e t h a t a m a j o r s u b s t r a t e o f the oncogene and g r o w t h - f a c t o r r e c e p t o r t y r o s i n e k i n a s e s i s a f a m i l y o f p r o t e i n s w i t h an a p p a r e n t m o l e c u l a r w e i g h t e s t i m a t e d a t 3 4 - 3 9 Kd on SDS/PAGE ( 1 9 0 , 1 9 1 ) . T h i s p r o t e i n f a m i l y shows r e m a r k a b l e c o n s e r v a t i o n among a v i a n and mammal ian s p e c i e s (192) and makes up a c o n s i d e r a b l e amount o f t o t a l f i b r o b l a s t p r o t e i n ( i . e . 0 . 1 - 0 . 4 % ) ( 1 9 3 ) . Two d i s t i n c t p r o t e i n s p e c i e s , s h a r i n g u n e x p e c t e d homology to each o t h e r , c o n s t i t u t e t h i s r e c e p t o r k i n a s e s u b s t r a t e s u p e r g r o u p . C a l p a c t i n s ( I and I I ) b i n d p h o s p h o l i p i d v e s i c l e s i n t he p r e s e n c e o f C a + 2 ( 1 9 4 ) , and l i p o c o r t i n s ( I and I I ) i n h i b i t t he a c t i o n o f p h o s p h o l i p a s e A 2 ( 1 9 5 ) . I t i s w o r t h y o f n o t e t h a t c a l p a c t i n ( p36 ) has been l o c a l i z e d by immunochemica l and c e l l f r a c t i o n a t i o n s t u d i e s t o t he c o r t i c a l s k e l e t o n , w h i c h u n d e r l i e s the p l a s m a membrane ( 1 9 3 ) . I t i s v e r y c o n c e i v a b l e t h a t t he D M S O - s t i m u l a t e d 35 Kd p h o s p h o p r o t e i n d e m o n s t r a t e d h e r e may be e i t h e r o f t h e s e two s p e c i e s . I m m u n o p r e c i p i t a t i o n o f ou r 35 Kd p r o t e i n w i t h a n t i s e r a a g a i n s t a G p r o t e i n 0 s u b u n i t o r a g a i n s t a n t i s e r a a g a i n s t c a l p a c t i n o r l i p o c o r t i n m igh t h e l p r e s o l v e t h e s e q u e s t i o n s . I t i s a l s o p o s s i b l e , though l e s s l i k e l y , t h a t DMSO s t i m u l a t e s e r y t h r o p o i e s i s by d e c r e a s i n g the p h o s p h o r y l a t i o n o f a s u b s e t o f membrane p r o t e i n s s i n c e i t was f ound t h a t some p r o t e i n s were a c t u a l l y l e s s p h o s p h o r y l a t e d ( a f t e r base t r e a t m e n t ) i n the p r e s e n c e o f DMSO when compared to a c o n t r o l samp le ( F i g u r e 9 ) . A t h i r d a l t e r n a t i v e i s t h a t the p h o s p h o r y l a t i o n changes s t i m u l a t e d by DMSO may r e f l e c t a r t i f a c t s o f the s y s t e m , and the p r o t e i n p h o s p h o r y l a t i o n c h a n g e s s e e n may have n o t h i n g to do w i t h r e d c e l l d i f f e r e n t i a t i o n . W i t h 59 r e g a r d to t h i s , i t has been d e m o n s t r a t e d by K r y s t a l t h a t t he o p t i m a l c o n c e n t r a t i o n o f DMSO r e q u i r e d to y i e l d the g r e a t e s t s t i m u l a t o r y e f f e c t i n b o t h ^ H - t h y m i d i n e and ^ F e i n c o r p o r a t i o n a s s a y s i s a p p r o x i m a t e l y 1.25%. T h i s c o n c e n t r a t i o n c a u s e d no s i g n i f i c a n t p h o s p h o r y l a t i o n e v e n t s ( d a t a no t s h o w n ) . From o u r s t u d i e s , a 20% DMSO c o n c e n t r a t i o n was n e c e s s a r y b e f o r e s i g n i f i c a n t p h o s p h o r y l a t i o n changes c o u l d be r e p o r t e d . However , a n o t h e r g r o u p has d e m o n s t r a t e d t h a t DMSO i n d u c e d the p h o s p h o r y l a t i o n o f t he EGF r e c e p t o r ( i n l i v e r c e l l s ) o n l y a t f i n a l c o n c e n t r a t i o n s i n e x c e s s o f 15% ( 1 7 5 ) , and a r g u e t h a t s u c h e x c e s s i v e c o n c e n t r a t i o n s a r e n e c e s s a r y to make an o t h e r w i s e u n d e t e c t a b l e l e v e l o f p h o s h o r y l a t i o n e a s i l y o b s e r v a b l e w i t h i n t he s h o r t t ime f r ame o f t h e i r i n v i t r o p h o s p h o r y l a t i o n a s s a y . T h u s , we c a n p o s t u l a t e t h a t i n o u r s y s t e m h i g h DMSO c o n c e n t r a t i o n s a r e r e q u i r e d m e r e l y to e x p e d i t e t h e membrane p h o s p h o r y l a t i o n changes t h a t n o r m a l l y accompany t e r m i n a l e r y t h r o i d d i f f e r e n t i a t i o n . F i n a l l y , t he s p e c i f i c i t y o f the DMSO p h o s p h o r y l a t i o n e f f e c t f o r l a t e e r y t h r o i d c e l l s ( a s opposed to l y m p h o i d and e a r l y e r y t h r o i d c e l l s ) h e l p s t o v a l i d a t e ou r c l a i m t h a t DMSO s t i m u l a t e s e r y t h r o b l a s t p r o l i f e r a t i o n and d i f f e r e n t i a t i o n t h r o u g h a p r o t e i n p h o s p h o r y l a t i o n mechan i sm. The r e s u l t s o b t a i n e d f rom the ^ H - t h y m i d i n e i n c o r p o r a t i o n s t u d i e s w i t h FVA c e l l s a t d i f f e r e n t s t a g e s o f m a t u r i t y s u g g e s t t h a t Ep and the E p -m i m i c k i n g a g e n t s a c t on d i f f e r e n t s u b p o p u l a t i o n s o f d e v e l o p i n g e r y t h r o b l a s t s . Ep s t i m u l a t e d b o t h e a r l y and m a t u r i n g e r y t h r o b l a s t s ( o n l y 15% h e m o g l o b i n p o s i t i v e ) w h i l e t he c o l l e c t i o n o f E p - m i m i c k i n g a g e n t s were o p e r a t i v e o n l y on t h e more ma tu re e r y t h r o i d compartment (>50% h e m o g l o b i n p o s i t i v e ) . T h e s e r e s u l t s echo t h o s e found when c o m p a r i n g Ep to EEF ( s e e I n t r o d u c t i o n ) — w h e r e EEF s t i m u l a t e d m a i n l y p o s t m i t o t i c e r y t h r o b l a s t s . I t i s t hus no t u n r e a s o n a b l e to t h i n k t h a t Ep and the E p - m i m i c k i n g a g e n t s may f u n c t i o n t h r o u g h d i f f e r e n t mechan isms ( i . e . E p - m i m i c k i n g a g e n t s t h r o u g h p r o t e i n p h o s p h o r y l a t i o n , and Ep by an u n c h a r a c t e r i z e d m e c h a n i s m ) . 60 T h i s p r o j e c t was u n d e r t a k e n on the a s s u m p t i o n t h a t E p , v a n a d a t e , and DMSO m igh t s h a r e a common mechanism o f a c t i o n ( i . e . , p r o t e i n p h o s p h o r y l a t i o n ) . However , f rom ou r i n v i t r o p h o s p h o r y l a t i o n r e s u l t s , i t does no t a p p e a r as though Ep f u n c t i o n s t h r o u g h t h i s mechan i sm. Y e t , p r o t e i n p h o s p h o r y l a t i o n c a n n o t be c o m p l e t e l y d i s c o u n t e d . I t i s g e n e r a l l y a c c e p t e d t h a t g r o w t h f a c t o r s i g n a l t r a n s d u c t i o n , a t l e a s t f o r some g r o w t h f a c t o r s , o c c u r s t h r o u g h t y r o s i n e - s p e c i f i c p h o s p h o r y l a t i o n , and t h a t r e c e p t o r s f o r t h e s e g r o w t h f a c t o r s ( i n s u l i n , E G F , p l a t e l e t - d e r i v e d g r o w t h f a c t o r ) a r e t y r o s i n e k i n a s e s . The k i n a s e a c t i v i t y ( a u t o p h o s p h o r y l a t i o n p r o p e r t y ) o f t h e s e r e c e p t o r s can e a s i l y be d e m o n s t r a t e d i n a p h o s p h o r y l a t i o n a s s a y l i k e t h a t emp loyed h e r e . I t i s p o s s i b l e t h a t t h e r e a r e s i m p l y no t enough Ep r e c e p t o r s f o r a s i g n i f i c a n t change i n p r o t e i n p h o s p h o r y l a t i o n t o be d e t e c t e d . I t was r e c e n t l y r e p o r t e d (127) t h a t t h e r e a r e a p p r o x i m a t e l y 660 Ep r e c e p t o r s on Ep r e s p o n s i v e c e l l s ( u s i n g a F r i e n d v i r u s i n f e c t e d c e l l p o p u l a t i o n ) . T h i s work was done u s i n g t r i t i a t e d Ep ( p e r i o d a t e o x i d a t i o n o f Ep f o l l o w e d by r e d u c t i o n w i t h t r i t i a t e d sod ium c y a n o b o r o h y d r i d e ) and may no t be c o m p l e t e l y a c c u r a t e ( b e c a u s e o f low Ep s p e c i f i c r a d i o a c t i v i t y ) . N o n e t h e l e s s , t he number o f r e c e p t o r s f ound by G o l d w a s s e r i s c o n s i s t e n t w i t h r e c e p t o r numbers o f o t h e r h e m o p o i e t i n s and i s one t e n t h t o one h u n d r e t h f ewe r t h a n the number o f r e c e p t o r s f o r t h o s e g r o w t h f a c t o r s l i s t e d a b o v e . A l t e r n a t i v e l y , t he Ep r e c e p t o r may no t a u t o p h o s p h o r y l a t e , and y e t s t i l l have t y r o s i n e k i n a s e a c t i v i t y . I t has been a r g u e d t h a t Ep may no t be a g r o w t h f a c t o r , bu t r a t h e r , a s u r v i v a l f a c t o r , p e r m i t t i n g t e r m i n a l l y d i f f e r e n t i a t i n g e r y t h r o i d elements to c o m p l e t e t h e i r p r o g r a m . A c c o r d i n g l y , the ho rmona l message t r a n s f e r mechanism may no t i n v o l v e t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n , bu t r a t h e r , an as y e t , u n c h a r a c t e r i z e d mechan ism. I t i s wo r t hy o f n o t e , h o w e v e r , t h a t a n o t h e r 61 h e m o p o i e t i c f a c t o r , I L - 3 , t h a t i s t hough t to a c t p r i m a r i l y a s a s u r v i v a l f a c t o r ( 1 9 6 ) , has been shown to s t i m u l a t e m e m b r a n e - a s s o c i a t e d p h o s p h o r y l a t i o n changes ( 1 9 7 ) . Ep i s e s s e n t i a l f o r t he t e r m i n a l d i f f e r e n t i a t i o n o f e r y t h r o i d p r e c u r s o r s . E a r l y e v e n t s a s s o c i a t e d w i t h Ep s t i m u l a t i o n o f d e v e l o p i n g e r y t h r o b l a s t s i n c l u d e r a p i d c a l c i u m f l u x e s and h e m o g l o b i n s y n t h e s i s ( 1 3 7 , 1 3 5 ) . However , membrane a l t e r a t i o n s p r e d i s p o s i n g e r y t h r o i d c e l l s t o t h e s e changes a r e c o m p l e t e l y unknown. The p u r p o s e o f t h i s s t u d y was t o d e t e r m i n e w h e t h e r p r o t e i n p h o s p h o r y l a t i o n p l a y s a r o l e i n t he mechan ism by w h i c h Ep a c t i v i t y i s e f f e c t e d . Two a g e n t s w h i c h were a b l e t o m i m i c Ep a c t i v i t y i n v i t r o d e m o n s t r a t e d the a b i l i t y to i n d u c e s i g n i f i c a n t p h o s p h o r y l a t i o n changes i n membranes f rom E p - r e s p o n s i v e c e l l s . M o r e o v e r , t he r e s u l t s o b t a i n e d w i t h t h e s e a g e n t s were c o n s i s t e n t w i t h t h o s e r e p o r t e d e l s e w h e r e ( i n o t h e r s y s t e m s ) , and t h e s e a g e n t s o p e r a t e d on a d i s t i n c t l y d i f f e r e n t e r y t h r o i d compar tment t han E p . We thus c o n c l u d e t h a t p l a s m a membrane p r o t e i n p h o s p h o r y l a t i o n e v e n t s may p l a y an i m p o r t a n t r o l e i n t h e l a t e s t a g e s o f e r y t h r o p o i e s i s i n v i v o , bu t t h a t the mechan ism o f a c t i o n o f Ep r e m a i n s u n r e s o l v e d . 6 2 REFERENCES: 1 . M e t c a l f D. "The H a e m o p o i e t i c C o l o n y S t i m u l a t i n g F a c t o r s " ( E l s e v i e r , Ams te rdam, 1 9 8 4 ) . 2 . Z u c k e r S , B e c k G , Y i P , L y s i k RM and J F D i S t e p h a n o . R a t h e m o p o i e t i c , c e l l p r e c u r s o r s f o r v e r y low d e n s i t y l i p o p r o t e i n (VLDL) g r o w t h i n h i b i t o r . Exp H e m a t o l 9 : 5 5 0 , 1 9 8 1 . 3 . A x e l r a d AA e t a l . I n : Hemog lob i ns i n Deve lopment and D i f f e r e n t i a t i o n , New Y o r k , A l a n R L i s s I n c . , p 4 5 , 1 9 8 1 . 4 . B roxmeye r H E . I n : " L o n g - T e r m Bone Marrow C u l t u r e " , A l a n R L i s s I n c . , p 3 6 3 , 1984 . 5 . K r y s t a l G , E a v e s AC & Eaves C J . D e t e r m i n a t i o n o f n o r m a l human serum e r y t h r o p o i e t i n l e v e l s , u s i n g mouse bone marrow. J Lab C l i n Med 9 7 : 1 5 8 , 1 9 8 1 . 6 . Zoumbos NC, G a s c o n P , D j e u J Y & Young N S . I n t e r f e r o n i s a m e d i a t o r o f h e m a t o p o i e t i c s u p p r e s s i o n i n a p l a s t i c anemia i n v i t r o and p o s s i b l y i n v i v o . P r o c N a t l Acad S c i USA 8 2 : 188 , 1 9 8 5 . 7 . W r i g h t E G , G a r l a n d JM & L o r d B I . S p e c i f i c i n h i b i t i o n o f h a e m o p o i e t i c s tem c e l l p r o l i f e r a t i o n : c h a r a c t e r i s t i c s o f the i n h i b i t o r p r o d u c i n g c e l l s . L e u k e m i a Res 4 : 5 3 7 , 1980 . 8 . B u r g e s s AW, C a m a k a r i s J & M e t c a l f D. P u r i f i c a t i o n and p r o p e r t i e s o f c o l o n y - s t i m u l a t i n g f a c t o r f rom mouse l u n g - c o n d i t i o n e d medium. J B i o l Chem 2 5 2 : 1998 , 1977 . 9 . M e t c a l f D, J o h n s o n GR & B u r g e s s AW. D i r e c t s t i m u l a t i o n by p u r i f i e d GM-CSF o f the p r o l i f e r a t i o n o f m u l t i p o t e n t i a l and e r y t h r o i d p r e c u r s o r c e l l s . B l o o d 5 5 : 1 3 8 , 1980 . 1 0 . Waheed A & Shadduck K. P u r i f i c a t i o n and p r o p e r t i e s o f L c e l l - d e r i v e d c o l o n y - s t i m u l a t i n g f a c t o r . J Lab C l i n Med 9 4 : 1 8 0 , 1979 . 1 1 . Das SK , S t a n l e y ER, G u i l b e r t L J & Forman LW. Human c o l o n y - s t i m u l a t i n g f a c t o r ( C S F - 1 ) r a d i o i m m u n o a s s a y : r e s o l u t i o n o f t h r e e s u b c l a s s e s o f human c o l o n y - s t i m u l a t i n g f a c t o r . B l o o d 5 8 : 6 3 0 , 1 9 8 1 . 1 2 . I h l e J N , K e l l e r J , H e n d e r s o n L , K l e i n F & P a l a s z y n s k i E . P r o c e d u r e s f o r t he p u r i f i c a t i o n o f i n t e r l e u k i n 3 to h o m o g e n e i t y . J Immunol 1 2 9 : 2 4 3 1 , 1 9 8 2 . 1 3 . C l a r k - L e w i s I, Ken t SB & S c h r a d e r JW. P u r i f i c a t i o n to a p p a r e n t h o m o g e n e i t y o f a f a c t o r s t i m u l a t i n g the g r o w t h o f m u l t i p l e l i n e a g e s o f h e m o p o i e t i c c e l l s . J B i o l Chem 2 5 9 : 7488 , 1984 . 1 4 . I s c o v e NN, R o i t s c h CA, W i l l i a m s N & G u i l b e r t L J . M o l e c u l e s s t i m u l a t i n g e a r l y r e d c e l l , g r a n u l o c y t e , mac rophage , and m e g a k a r y o c y t e p r e c u r s o r s i n c u l t u r e : s i m i l a r i t y i n s i z e , h y d r o p h o b i c i t y , and c h a r g e . J C e l l P h y s i o l S u p p l 1: 6 5 , 1982 . 63 1 5 . B a r t e l m e z S H , S a c c a R & S t a n l e y ER. L i n e a g e s p e c i f i c r e c e p t o r s u s e d to i d e n t i f y a g r o w t h f a c t o r f o r d e v e l o p m e n t a l l y e a r l y h e m o p o i e t i c c e l l s : a s s a y o f h e m o p o i e t i n - 2 . J C e l l P h y s i o l 1 2 2 : 3 6 2 , 1 9 8 5 . 1 6 . I h l e J N , K e l l e r J , O r o s z l a n S , Hende rson L E , C o p e l a n d TD , F i t c h F , P r y s t o w s k y MB, G o l d w a s s e r E , S c h r a d e r J V , P a l a s z y n s k i E , Dy M & L e b e l B . B i o l o g i c p r o p e r t i e s o f homoseneous i n t e r l e u k i n - 3 . I. D e m o n s t r a t i o n o f WEHI-3 g r o w t h f a c t o r a c t i v i t y , mast c e l l g r o w t h f a c t o r a c t i v i t y , p c e l l -s t i m u l a t i n g f a c t o r a c t i v i t y , c o l o n y - s t i m u l a t i n g f a c t o r a c t i v i t y , and h i s t a m i n e - p r o d u c i n g c e l l - s t i m u l a t i n g f a c t o r a c t i v i t y . J Immunol 1 3 1 : 2 8 2 , 1 9 8 3 . 1 7 . 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I n : C e l l u l a r and M o l e c u l a r B i o l o g y o f Lymphok ines ( e d s S o r g C & S c h i m p l A ) , Acadamec, O r l a n d o , F L ) , p 1 9 5 , 1 9 8 5 . 3 1 . Co f fman RL & C a r t y J . J Immunol ( i n p r e s s ) 3 2 . Lee F , Y o k o t a T , O t s u k a T , Meye rson P , V i l l a r e t D, C o f f m a n R, Mosmann T , R e n n i c k D, Roehm N , S m i t h C e t a l . I s o l a t i o n and c h a r a c t e r i z a t i o n o f a mouse i n t e r l e u k i n cDNA c l o n e t h a t e x p r e s s e s B - c e l l s t i m u l a t o r y f a c t o r 1 a c t i v i t i e s and T - c e l l - a n d m a s t - c e l l - s t i m u l a t i n g a c t i v i t i e s . P r o c N a t l Acad S c i USA 8 3 : 2 0 6 1 , 1986 . 3 3 . R e n n i c k D. P r e s e n t e d a t the 2 1 s t C o n g r e s s o f the I n t e r n a t i o n a l S o c i e t y H a e m a t o l , S y d n e y , A u s t r a l i a , May 1 1 - 1 6 , 1986 . 3 4 . N i c o l a NA, M e t c a l f D, J o h n s o n GR & B u r g e s s HV. 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S i e f f C A , Emerson S g , Donahue R E , Na than DG, Wang E A , Wong GC & C l a r k S C . Human r e c o m b i n a n t g r a n u l o c y t e - m a c r o p h a g e c o l o n y - s t i m u l a t i n g f a c t o r : A m u l t i l i n e a g e h e m a t o p o i e t i n . S c i e n c e 2 3 0 : 1 1 7 1 , 1 9 8 5 . 3 8 . G a s s o n J C , Kaufman S E , W e i s b a r t RH , Tomonaga M & G o l d e DW. H i g h - a f f i n i t y b i n d i n g o f g r a n u l o c y t e - m a c r o p h a g e c o l o n y - s t i m u l a t i n g f a c t o r to n o r m a l and l e u k e m i c human m y e l o i d c e l l s . P r o c N a t l Acad S c i USA 8 3 : 6 6 9 , 1 9 8 6 . 3 9 . S o u z a LM, Boone T C , G a b r i l o v e J , L a i PH , Zsebo KM, Murdock DC, C h a z i n VR, B r u s z e w s k i J , Lu H, Chen KK e t a l . Recomb inan t human g r a n u l o c y t e c o l o n y -s t i m u l a t i n g f a c t o r : e f f e c t s on n o r m a l and l e u k e m i c m y e l o i d c e l l s . S c i e n c e 2 3 2 : 6 1 , 1986 . 4 0 . S t a n l e y ER & G u i l b e r t L J . Methods f o r the p u r i f i c a t i o n , a s s a y , c h a r a c t e r i z a t i o n and t a r g e t c e l l b i n d i n g o f a c o l o n y s t i m u l a t i n g f a c t o r ( C S F - 1 ) . J Immunol Methods 4 2 : 2 5 3 , 1 9 8 1 . 6 5 4 1 . Robb R J , K u t n y RM, P a n i c o M, M o r r i s HR & Chowdhry V . Amino a c i d s e q u e n c e and p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f human i n t e r l e u k i n - 2 . P r o c N a t l A c a d S c i 8 1 : 6 4 8 6 , 1984 . 4 2 . M i n g a r i MC, G e r o s a F , C a r r a G , A c c o l l a R S , M o v e t t a A , Z u b l e r R H , Waldmann TA & M o r e t t a L . Human i n t e r l e u k i n - 2 p romotes p r o l i f e r a t i o n o f a c t i v a t e d B c e l l s v i a s u r f a c e r e c e p t o r s s i m i l a r to t h o s e o f a c t i v a t e d T c e l l s . N a t u r e : 3 1 2 : 6 4 1 , 1984 . 4 3 . W e l t e K, P l a t z e r E , Lu L , G a b r i l o r e J L , L e v i E , M e r t e l s m a n n R & Moore M. P u r i f i c a t i o n and b i o c h e m i c a l c h a r a c t e r i z a t i o n o f human p l u r i p o t e n t h e m o p o i e t i c c o l o n y - s t i m u l a t i n g f a c t o r . P r o c Acad S c i USA 8 2 : 1526 , 1 9 8 5 . 4 4 . N i c o l a NA, B e g l e y CG & M e t c a l f D. I d e n t i f i c a t i o n o f t he human a n a l o g u e o f a r e g u l a t o r t h a t i n d u c e s d i f f e r e n t i a t i o n i n m u r i n e l e u k a e m i c c e l l s . N a t u r e 3 1 4 : 6 2 5 , 1985 . 4 5 . M i y a k i T , Kung CK & G o l d w a s s e r E . P u r i f i c a t i o n o f human e r y t h r o p o i e t i n . J B i o l Chem 2 5 2 : 5 5 5 8 , 1977 . 4 6 . G r a b e r SE & K r a n t z S B . E r y t h r o p o i e t i n and the c o n t r o l o f r e d c e l l p r o d u c t i o n . Ann Rev Med 2 9 : 5 1 , 1978 . 4 7 . E a v e s C J e t a l . I n : " H e m o g l o b i n s i n Deve lopment and D i f f e r e n t i a t i o n " ( eds S t a m a t o y a n n o p o u l o s G & N i e n h u i s AW) A l a n R L i s s I n c , New Y o r k , p 6 3 , 1 9 8 1 . 4 8 . D e s s y p r i s EN & K r a n t z S B . E f f e c t o f pu re e r y t h r o p o i e t i n on D N A - s y n t h e s i s by human marrow day 15 e r y t h r o i d b u r s t f o r m i n g u n i t s i n s h o r t - t e r m l i q u i d c u l t u r e . B r J Haema to l 5 9 : 2 9 5 , 1984. 4 9 . A n n a b l e L , C o t e s PM & M u s s e t t MV. The s e c o n d i n t e r n a t i o n a l r e f e r e n c e p r e p a r a t i o n o f e r y t h r o p o i e t i n , human, u r i n a r y , f o r b i o a s s a y . B u l l Wld H l t h Org 4 7 : 9 9 , 1972 . 5 0 . W e s t b r o o k C A , G a s s o n J C , G e r b e r S E , S e l s t e d ME & G o l d e DW. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f human T - l y m p h o c y t e - d e r i v e d e r y t h r o i d - p o t e n t i a t i n g a c t i v i t y . J B i o l Chem 2 5 9 : 9 9 9 2 , 1984 . 5 1 . D o c h e r t y A J P , Lyons A , S m i t h B J , W r i g h t EM, S t e p h e n s PE & H a r r i s T J R . Sequence o f human t i s s u e i n h i b i t o r o f m e t a l l o p r o t e i n a s e s and i t s i d e n t i t y to e r y t h r o i d - p o t e n t i a t i n g a c t i v i t y . N a t u r e 3 1 8 : 6 6 , 1 9 8 5 . 5 2 . Gauwerky C E , L u s i s A J & G o l d e DW. Human l e u k e m i a c e l l l i n e K562 r e s p o n d s t o e r y t h r o i d - p o t e n t i a t i n g a c t i v i t y . B l o o d 5 9 : 3 0 0 , 1 9 8 2 . 5 3 . J u b i n s k y PT & S t a n l e y ER. P u r i f i c a t i o n o f h e m o p o i e t i n 1: A m u l t i l i n e a g e h e m o p o i e t i c g r o w t h f a c t o r . P r o c N a t l Acad S c i USA 8 2 : 2 7 6 4 , 1 9 8 5 . 5 4 . R e n n i c k D. P r e s e n t e d a t the 2 1 s t C o n g r e s s o f the I n t e r n a t i o n a l S o c i e t y o f H e m a t o l o g y , S y d n e y , A u s t r a l i a , May 1 1 - 1 6 , 1986 . 5 5 . Abe E , T a n a k a H, I s h i m i Y , M i y a u r a C , H a y a s h i T , Nagasawa H, Tomida M, Yamaguch i Y , Hozumi M & Suda T . D i f f e r e n t i a t i o n - i n d u c i n g f a c t o r p u r i f i e d f rom c o n d i t i o n e d medium o f m i t o g e n - t r e a t e d s p l e e n c e l l c u l t u r e s s t i m u l a t e s bone r e s o r p t i o n . P r o c N a t l Acad S c i USA 8 3 : 5 9 5 8 , 1 9 8 6 . 6 6 5 6 . H i r a n o T e t a l . P u r i f i c a t i o n to homogene i t y and c h a r a c t e r i z a t i o n o f human B - c e l l d i f f e r e n t i a t i o n f a c t o r (BCDF o r B S F p - 2 ) . P r o c N a t l A c a d S c i USA 8 2 : 5 4 9 0 , 1 9 8 5 . 5 7 . S a n d e r s o n C J , O ' G a r r a A , War ren DJ & K l a u s GG. E o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r a l s o has B - c e l l g r o w t h f a c t o r a c t i v i t y : p r o p o s e d name i n t e r l e u k i n - 4 . P r o c N a t l Acad S c i USA 8 3 : 4 3 7 , 1 9 8 6 . 5 8 . Werber MM, Goldman J , Radnay J , K l e i n S & R o z e n s z a j n L H . I d e n t i f i c a t i o n and p u r i f i c a t i o n o f human T - l y m p h o c y t e c o l o n y - e n h a n c i n g f a c t o r , T L C E F : i n c r e a s e d p r o d u c t i o n by p h o r b o l m y r i s t a t e a c e t a t e . Immunology 5 6 : 2 8 5 , 1 9 8 5 . 5 9 . L o m e d i c o P T , G u b l e r V , H e l l m a n n C , D u k o v i c h M, Pan Y C , C o l l i e l K , Semionow R, Chua AO & M i z e l S B . C l o n i n g and e x p r e s s i o n o f m u r i n e i n t e r l e u k i n - 2 cDNA i n E s c h e r i c h i a c o l i . N a t u r e 3 1 2 : 4 5 8 , 1984 . 6 0 . Y o k o t a T , A v a i N , Lee F , R e n n i c k D, Mosmann T & A v a i K. Use o f cDNA e x p r e s s i o n v e c t o r f o r i s o l a t i o n o f mouse i n t e r l e u k i n 2 cDNA c l o n e s : E x p r e s s i o n o f T - c e l l g r o w t h - f a c t o r a c t i v i t y a f t e r t r a n s f e c t i o n o f monkey c e l l s . P r o c N a t l Acad S c i USA 8 2 : 6 8 , 1 9 8 5 . 6 1 . Fung MC, Hopes A J , Ymer S , Cohen DR, J o h n s o n RM, C a m p b e l l HD & Young I G . M o l e c u l a r c l o n i n g o f cDNA f o r m u r i n e i n t e r l e u k i n - 3 . N a t u r e 3 0 7 : 2 3 3 , 1984 . 6 2 . Lee F , Y o k o t a T , O t s u k a T , Meye rson P , V i l l a r e t D, Co f fman R, Mosmann T , R e n n i c k D, Roehm N, S m i t h C e t a l . I s o l a t i o n and c h a r a c t e r i z a t i o n o f a mouse i n t e r l e u k i n cDNA c l o n e t h a t e x p r e s s e s B - c e l l s t i m u l a t o r y f a c t o r 1 a c t i v i t i e s and T - c e l l - a n d m a s t - c e l l - s t i m u l a t i n g a c t i v i t i e s . P r o c N a t l A c a d S c i USA 8 3 : 2 0 6 1 , 1986 . 6 3 . Gough NM, Gough J , M e t c a l f D, K e l s o A , G r a i l D, N i c o l a NA, G u r g e s s AW & Dunn AR. M o l e c u l a r c l o n i n g o f cDNA e n c o d i n g a m u r i n e h a e m a t o p o i e t i c g r o w t h r e g u l a t o r , g r a n u l o c y t e - m a c r o p h a g e c o l o n y s t i m u l a t i n g f a c t o r . N a t u r e 3 0 9 : 7 6 3 , 1984 . 6 4 . McDona ld J D , L i n F K , G o l d w a s s e r E . C l o n i n g , s e q u e n c i n g , and e v o l u t i o n a r y a n a l y s i s o f the mouse e r y t h r o p o i e t i n g e n e . Mo l C e l l B i o l 6 : 8 4 2 , 1 9 8 6 . 6 5 . Wong GG e t a l . Human GM-CSF m o l e c u l a r c l o n i n g o f the c o m p l e m e n t a r y DNA and p u r i f i c a t i o n o f the n a t u r a l and r e c o m b i n a n t p r o t e i n s . S c i e n c e 2 2 8 : 8 1 0 , 1 9 8 5 . 6 6 . 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