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Activation of multiple hemopoietic growth factor genes in Abelson virus transformed myeloid cells Abraham, Samuel D. M. 1988

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ACTIVATION OF MULTIPLE HEMOPOIETIC GROWTH FACTOR GENES IN ABELSON VIRUS TRANSFORMED MYELOID CELLS. by SAMUEL D. M. ABRAHAM B.Sc , Simon Fraser U n i v e r s i t y , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Genetics Programme) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1988 ®Samuel D. M. Abraham, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 5~^ Z/uZ^i DE-6(3/81) i i ABSTRACT The s t r i n g e n t requirement f o r hemopoietic growth f a c t o r s (HGF) i n the in d u c t i o n of hemopoiesis in v i t r o has r a i s e d questions as to t h e i r p o s s i b l e r o l e ( s ) i n leukemogenesis. Several recent c l i n i c a l s t u d i e s have shown aberrant c e l l growth f a c t o r gene a c t i v a t i o n i n pa t i e n t derived leukemic c e l l s . Assessment of growth f a c t o r a c t i v i t y i s often based on i n v i t r o b i o a c t i v i t y assays of conditioned media or body f l u i d s . The s p e c i f i c i t y of t h i s type of endpoint i s , however, open to question due to the overlap i n b i o l o g i c a l a c t i v i t i e s of many HGFs. In assessing the r o l e of growth f a c t o r gene expression i n a murine myeloid leukemia model I have used a s e n s i t i v e RNA d e t e c t i o n procedure coupled w i t h a vector-probe system that enables the synthesis of uniformly l a b e l l e d r a d i o a c t i v e DNA probes to detect unambiguously the expression of p a r t i c u l a r growth f a c t o r genes. The Abelson murine leukemia v i r u s (A-MuLV) derived myeloid transformants used i n t h i s study had pr e v i o u s l y been shown to produce a m u l t i - l i n e a g e colony s t i m u l a t i n g a c t i v i t y (CSA). While these A-MuLV transformants were shown to produce GM-CSF, i t seemed l i k e l y that the m u l t i -l i n e a g e CSA was due to another f a c t o r . In a d d i t i o n to confirming the expression of GM-CSF mRNA, I was able to show that the c e l l s of a l l four A-MuLV transformed l i n e s tested a l s o expressed i n t e r l e u k i n - 3 mRNA. This f i n d i n g was s t r o n g l y corroborated by b i o - a c t i v i t y data obtained using the CM from the A-MuLV myeloid transformants. A d d i t i o n a l p r e l i m i n a r y a n a l y s i s by b i o a c t i v i t y assays have a l s o shown the p o s s i b l e presence of i n t e r l e u k i n - 6 (IL-6) and a r e c e n t l y described pre-B c e l l f a c t o r suggesting perhaps a common mechanism und e r l y i n g the a c t i v a t i o n of these various growth f a c t o r genes. i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i v LIST OF FIGURES v LIST OF ABBREVIATIONS v i i ACKNOWLEDGEMENTS i x CHAPTER I : INTRODUCTION 1) Overview of hemopoiesis 1 2) Hemopoiesis and growth f a c t o r s 2 A) Category I ( M u l t i - l i n e a g e hemopoietins) 5 B) Category I I (Lineage r e s t r i c t e d hemopoietins) 8 3) Leukemogenesis 10 4) Model systems f o r leukemic transformation 14 5) Thesis o b j e c t i v e s 18 References 19 CHAPTER I I : MATERIALS AND METHODS 1) DNA i s o l a t i o n and p u r i f i c a t i o n 29 2) DNA a n a l y s i s 30 3) RNA i s o l a t i o n / p u r i f i c a t i o n 32 4) RNA a n a l y s i s 34 5) Preparation of probes for S l - a n a l y s i s 35 6) C e l l c u l t u r e 46 7) Growth f a c t o r bioassays 47 References 49 CHAPTER I I I : RESULTS 1) I n i t i a l assesment of uniformly l a b e l l e d probes i n d e t e c t i n g growth f a c t o r gene expression 51 2) Baseline studies of IL-3 and GM-CSF gene expression i n murine c e l l s . 56 3) Growth f a c t o r a c t i v a t i o n i n Abelson-MuLV myeloid transformants 61 References 76 CHAPTER IV: DISCUSSION AND CONCLUSIONS 77 References 81 i v LIST OP TABLES page CHAPTER 1 Table 1. CHAPTER 2 Table 1. Murine hemopoietic colony s t i m u l a t i n g f a c t o r s I n d i c a t o r c e l l s f o r the d e t e c t i o n of growth f a c t o r a c t i v i t y 48 CHAPTER 3 Table 1. Table 2. Table 3. M u l t i l i n e a g e CSF produced by A-MuLV transformed c e l l s 63 B i o a c t i v i t y of conditioned media from l o g phase c u l t u r e s of A-MuLV transformed c e l l s 66 IL-6 and pre-B c e l l growth f a c t o r a c t i v i t y produced by Abelson transformed c e l l s 75 V LIST OP FIGURES CHAPTER 2 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Schematic of pTZ-vector system: c l o n i n g l o g i c with respect to anti-sense DNA probe synthesis Diagram representing 5' and 3' IL-3 fragments sub-cloned i n t o pTZ-vectors and the probes generated Diagram representing GM-CSF cDNA fragments sub-cloned i n t o the pTZ-vector system and the probes generated Schematic representation of uniformly l a b e l l e d s i n g l e stranded probes generated v i a the pTZ-vector system Diagram of c-myc fragment end-labelled f o r use as a S l - a n a l y s i s probe page 37 41 42 44 45 CHAPTER 3 Figure 1. Detection of mIL-3 by S l - a n a l y s i s using a uniformly l a b e l l e d double stranded DNA probe 52 Figure 2. Detection of mIL-3 by S l - a n a l y s i s using a uniformly l a b e l l e d s i n g l e stranded DNA probe 54 Figure 3. Northern b l o t a n a l y s i s showing d e t e c t i o n of 3-actin but not mIL-3 mRNA 55 Figure 4. S l - a n a l y s i s using a double stranded GM-CSF cDNA probe to detect GM-CSF gene a c t i v a t i o n i n murine c e l l s 57 Figure 5a. S l - a n a l y s i s using a s i n g l e stranded 5' IL-3 probe to detect IL-3 gene a c t i v a t i o n i n murine c e l l s 59/60 Figure 5b. S l - a n a l y s i s using a s i n g l e stranded 3' IL-3 probe to detect IL-3 gene a c t i v a t i o n i n murine c e l l s 59/60 Figure 6. S l - a n a l y s i s using a s i n g l e stranded GM-CSF cDNA probe to demonstrate GM-CSF gene a c t i v a t i o n i n some A-MuLV transformants 65 Figure 7. S l - a n a l y s i s using a s i n g l e stranded 3' IL-3 probe to demonstrate IL-3 gene a c t i v a t i o n ,in some A-MuLV transformants 68 Figure 8a. S l - a n a l y s i s assessing IL-3 expression of A-MuLV transformants when i n maximal growth phase and when maximal c e l l d e nsity i s reached 70/71 VI page Figure 8b. S l - a n a l y s i s assessing GM-CSF expression of A-MuLV transformants when i n maximal growth phase and when maximal c e l l density i s reached Figure 8c. S l - a n a l y s i s of c-myc l e v e l s of A-MuLV transformants when i n maximal growth phase and when maximal c e l l d e n s i t y i s reached Figure 9a. Southern b l o t a n a l y s i s of A-MuLV transformant DNA f o r GM-CSF gene rearrangements Figure 9b. Southern b l o t a n a l y s i s of A-MuLV transformant DNA f o r IL-3 gene rearrangements 70/71 70/71 73 73 LIST OF ABBREVIATIONS A-MuLV Abelson murine leukemia v i r u s bp base p a i r ( s ) BSA bovine serum albumin cm centimeter CM conditioned medium CSA colony s t i m u l a t i n g a c t i v i t y CSF colony s t i m u l a t i n g f a c t o r ( s ) DMEM Dulbecco's modified Eagles medium DNA deoxyribonucleic a c i d dATP deoxyriboadenosine 5'-triphosphate dCTP deo x y r i b o c y t i d i n e 5'-triphosphate dGTP deoxyribothymidine 5'-triphosphate dTTP deoxyribothymidine 5'-triphosphate dNTP deoxyribonucleotide 5'-triphosphate ddH 20 double d i s t i l l e d water DTT d i t h i o t h r e i t o l EDTA ethyle n e d i a m i n e t e t r a - a c e t i c a c i d EtBr ethidium bromide EtOH ethanol FCS f e t a l c a l f serum Fr-MuLV Friend murine leukemia v i r u s hr hour(s) IL i n t e r l e u k i n IPTG i s o p r o p y l t h i o g a l a c t o s i d a s e Kb k i l o b a s e p a i r ( s ) kd k i l o d a l t o n ( s ) 1 l i t r e y i m i c r o l i t r e ml m i l i l i t r e min minute(s) mol wt molecular weight NaAc sodium acetate NH 4Ac ammonium acetate nt n u c l e o t i d e PBS phosphate buffered s a l i n e PEG polyethylene g l y c o l PIPES piperazihe-N,N'-bis[2-ethane-sulfonic a c i d ] PMA phorbol m y r i s t i c acetate PWM pokeweed mitogen RNA r i b o n u c l e i c a c i d rpm r e v o l u t i o n s per minute SDS sodium dodecyl sulphate X-gal 5-dibromo-A-chloro 3-indolygalactosidase ACKNOWLEDGEMENTS I would l i k e to thank K e i t h Humphries f o r h i s advice and support throughout the years, and to Drs Connie J . Eaves and Fumio Takei f o r t h e i r c o n t r i b u t i o n s to t h i s study both l i t e r a l l y and f i g u r a t i v e l y . I a l s o wish to thank a l l other students and s t a f f at the Terry Fox Laboratory who have helped along the way. I a l s o wish to acknowledge those f r i e n d s ; Luutje D i j s , Sue Hayley and the Von Widmer's whose continued support has been i n v a l u a b l e . F i n a l l y , I e s p e c i a l l y wish to thank Sara f o r simply p u t t i n g up with me a l l these months. 1 C H A P T E R I INTRODUCTION 1) OVERVIEW OF HEMOPOIESIS Hemopoiesis i s a dynamic process i n which c i r c u l a t i n g mature blood c e l l s of m u l i t i p l e types are supplied throughout l i f e i n l a r g e numbers. These mature c e l l s have l i m i t e d or no p r o l i f e r a t i v e c a p a c i t y and most have a r e l a t i v e l y short l i f e span. Under normal circumstances, t h e i r numbers are q u i t e i n v a r i a n t and yet production of s e l e c t i v e c e l l populations can be markedly augmented i n response to s t r e s s e s such as bleeding or i n f e c t i o n . I t i s now c l e a r that the production of mature blood c e l l s depends on the 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 of a s e l f - m a i n t a i n i n g population of p r i m i t i v e p l u r i p o t e n t stem c e l l s . A major challenge now i s to unravel the r e g u l a t o r y mechanisms at play i n t h i s complex system. In a d u l t s , the major non-lymphoid hemopoietic organ i s the red bone marrow of the vertebrae, r i b s , sternum, p e l v i s , scapulae, s k u l l and proximal p o r t i o n s of the long bones. The hemopoietic (red) bone marrow at these s i t e s i s comprised mainly of mature hemopoietic c e l l s , t h e i r immediate morphologically recognizable precursors, and p h e n o t y p i c a l l y d i v e r s e stromal elements. C o n s t i t u t i n g a smaller and l e s s recognizable component are the hemopoietic stem c e l l s and t h e i r immediate progeny. These c e l l s are thought to be the c r i t i c a l targets for both r e g u l a t i o n and c e l l u l a r transformation events g i v i n g r i s e to the development of leukemia. While i t remains unclear as to the precise mechanisms governing hemopoiesis, r e s u l t s from both i n v i t r o and i n v i v o s t u d i e s suggest a 2 complex i n t e r p l a y between growth f a c t o r s , the e x t r a - c e l l u l a r matrix, the stromal environment and hemopoietic c e l l s themselves. Though i t remains the main o b j e c t i v e of t h i s t h e s i s to i n v e s t i g a t e the r o l e of growth f a c t o r s i n malignant hemopoiesis, the ul t i m a t e goal remains the be t t e r understanding of the i n t e r a c t i o n s between these growth f a c t o r s and p r i m i t i v e hemopoietic c e l l s and of t h e i r consequences to both normal and leukemic s t a t e s . 2) HEMOPOIESIS AND GROWTH FACTORS In the mouse, hemopoietic stem c e l l s were f i r s t assayed by t h e i r a b i l i t y to generate m u l t i - l i n e a g e colonies of c e l l s i n the spleens of syngeneic i r r a d i a t e d r e c i p i e n t s . This in v i v o stem c e l l assay system developed by T i l l and McCulloch (1) permitted both a q u a n t i t a t i o n of the stem c e l l pool and a q u a l i t a t i v e assessment of t h e i r p r o l i f e r a t i v e , d i f f e r e n t i a t i o n and self - r e n e w a l p r o p e r t i e s ( 2 ) . Further s t u d i e s using c e l l s marked by chromosome a l t e r a t i o n s (3) and more r e c e n t l y by r e t r o v i r u s e s (4) have provided a d d i t i o n a l support f o r the existence of p l u r i p o t e n t hemopoietic stem c e l l s with repopulation p o t e n t i a l . The changes that occur as p l u r i p o t e n t stem c e l l s become r e s t r i c t e d to and then d i f f e r e n t i a t e down a s i n g l e hemopoietic lineage are slowly being deciphered. I t has become apparent i n the past few years that a great complexity of c e l l u l a r i n t e r a c t i o n s r e q u i r i n g c e l l - c e l l contact and secreted f a c t o r s are involved i n r e g u l a t i n g the process of c e l l p r o l i f e r a t i o n that accompanies hemopoiesis. These c h a r a c t e r i z a t i o n s have been g r e a t l y aided by the advent of i n v i t r o colony assays (5,6). In p a r t i c u l a r , these assay systems have helped uncover those c e l l types intermediate to the stem c e l l and the e f f e c t o r c e l l s ( i e committed p r o g e n i t o r s ) . These assays i n v o l v e the im m o b i l i z a t i o n of hemopoietic c e l l s i n se m i - s o l i d medium co n t a i n i n g 3 appropriate n u t r i e n t s , serum and growth f a c t o r s . The l o c a l i z e d p r o g e nitors can then be scored f o r the colony s i z e and type of colony that grows. D i f f e r e n t c u l t u r e c o n d i t i o n s have now been developed that allow the d e t e c t i o n of committed progenitors f o r a l l hematopoietic pathways ( 7 ) . An important consequence stemming from the development of i n v i t r o assay systems has been the r e c o g n i t i o n that the various hematopoietic precursor c e l l s are unable to su r v i v e or p r o l i f e r a t e unless provided w i t h c e r t a i n s p e c i f i c growth f a c t o r s . The subsequent molecular c l o n i n g and c h a r a c t e r i z a t i o n of many such regulatory f a c t o r s , r e f e r r e d to as the colony s t i m u l a t i n g f a c t o r s (CSF's), has g r e a t l y f a c i l i t a t e d many st u d i e s i n t o the nature of normal and n e o p l a s t i c hematopoietic r e g u l a t i o n . The colony s t i m u l a t i n g f a c t o r s : "Colony s t i m u l a t i n g f a c t o r s (CSF)" were o p e r a t i o n a l l y defined from i n v i t r o observations of t h e i r a b i l i t y to st i m u l a t e progenitor c e l l s of d i f f e r e n t hemopoietic lineages to form c o l o n i e s of recognizable maturing c e l l s . In the past few years s e v e r a l hemopoietic growth f a c t o r s have been cloned, and recombinant forms of the pro t e i n s produced and p u r i f i e d . Table 1 l i s t s some of these f a c t o r s and hemopoietic c e l l types they can a f f e c t i n v i t r o . 4 TABLE 1. Murine Hemopoietic Growth Factors Colony Mol. S t i m u l a t i n g Wt. Factor (kd) A l t e r n a t i v e Names Major target c e l l s i n Chromosomal Location Multi-CSF GM-CSF 28 23 IL-3,BPA,HCGF PSF.MCGF CFU-S CFU-MIX BFU-E CFU-GM/G/M CFU-EO CFU-Meg Mast c e l l s CFU-GM CFU-MIX* BFU-E* CFU-Meg* CFU-EO* 11 11 G-CSF 25 M-CSF 70 ERYTHROPOIETIN 39 IL-4 IL-5 IL-6 20 26 DF ( d i f f e r e n t i a t i o n f a c t o r ) CSF-1 BSF-1 EDF ( 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 ) BSF-2 HGF,INF02 26 kDa p r o t e i n CFU-G CFU-MIX** BFU-E** CFU-M BFU-E CFU-E Pre-B c e l l s B - c e l l s T - c e l l s Mast c e l l s Eo-CSF B - c e l l s Pre-B c e l l s T - c e l l s CFU-MIX Pre-B c e l l s *: high cone's req'd; **: i n i t i a t e s but does not s u s t a i n p r o l i f e r a t i o n Attempts have been made to c l a s s i f y the CSFs under two broad c a t e g o r i e s : i ) those that act upon p r i m i t i v e hemopoietic c e l l s of m u l t i p l e l i n e a g e s and/or on p l u r i p o t e n t stem c e l l s and; i i ) those that act upon a s i n g l e l i n e a g e of progenitor c e l l s . As w i l l become apparent such a c l a s s i f i c a t i o n while convenient i s now l i k e l y too s i m p l i s t i c . 5 (A) CATEGORY I ( M u l t i - l i n e a g e hemopoietins) I n t e r l e u k i n - 3 ; A prototype f o r the f i r s t category i s I n t e r l e u k i n - 3 ( I L - 3 ) . IL-3 (or multi-CSF), o r i g i n a l l y i s o l a t e d from a mouse myelomonocytic c e l l l i n e (WEHI-3B) ( 8 ) , i s a f a c t o r capable of s t i m u l a t i n g p l u r i p o t e n t progenitors as w e l l as committed precursors of the e r y t h r o i d , megakaryocytic, n e u t r o p h i l i c , macrophage, mast c e l l and lymphocytic li n e a g e s (9,10). Thus f a r the only normal c e l l s found to synthesize IL-3 are a c t i v a t e d T-lymphocytes (10). IL-3 has been shown to be a monomeric g l y c o p r o t e i n of about 28 KD, approximately 40% of which i s carbohydrate (11). The gene f o r IL-3 e x i s t s i n s i n g l e copy form on mouse chromosome 11 and i s comprised of 5 exons (12,13). The complete amino a c i d sequence of 166 residues includes a s i g n a l peptide and four p o t e n t i a l N - g l y c o s y l a t i o n s i t e s . In v i t r o t e s t i n g of deglycosylated IL-3 suggested that the carbohydrate moieties are not required f o r b i o l o g i c a l a c t i v i t y . The IL-3 monomer a l s o contains 4 c y s t e i n e residues l i k e l y to be involved i n d i s u l f i d e bridges as b i o l o g i c a l a c t i v i t y i s l o s t upon treatment with mercaptoethanol. Granulocyte macrophage colony s t i m u l a t i n g f a c t o r (GM-CSF); Another candidate f o r the f i r s t category of CSFs i s GM-CSF which, l i k e IL-3, s t i m u l a t e s the p r o l i f e r a t i o n of the same granulocyte-macrophage clones. To a l e s s e r extent i t a l s o stimulates p l u r i p o t e n t stem c e l l s (14,15). High concentrations of GM-CSF have a l s o been shown to s t i m u l a t e e o s i n o p h i l , megakaryocyte and e r y t h r o i d colony formation (16,17). In a d d i t i o n to a c t i v a t e d T-lymphocytes, GM-CSF appears al s o to be synthesized by macrophages, f i b r o b l a s t s and e n d o t h e l i a l c e l l s (7). Murine GM-CSF was f i r s t p u r i f i e d from endotoxin stimulated mouse lung conditioned media as a monomeric g l y c o p r o t e i n of 23 KD (18). Murine GM-CSF i s a s i n g l e copy gene comprised of 4 exons (18) and has been l o c a l i z e d on chromosome 11 (19). Sequencing of GM-CSF cDNA revealed that the mature polypeptide i s comprised 6 of 124 amino acids (19,20). There are 4 cysteine residues i n t h i s polypeptide. These are thought to be l i n k e d by d i s u l f i d e bridges as treatment with mercaptoethanol destroys a l l b i o l o g i c a l a c t i v i t y . B i o l o g i c a l a c t i v i t y of GM-CSF has als o been shown to be independent of g l y c o s y l a t i o n as b a c t e r i a l l y synthesized recombinant GM-CSF d i s p l a y s the same potency as na t i v e GM-CSF (21). I n t r i g u i n g s i m i l a r i t i e s between IL-3 and GM-CSF: While the amino a c i d and n u c l e o t i d e sequences of murine IL-3 and GM-CSF bear no s t a t i s t i c a l l y s i g n i f i c a n t homologies, s i m i l a r i t i e s a r i s i n g from t h e i r overlapping b i o l o g i c a l a c t i v i t i e s and chromosomal l o c a t i o n have generated much sp e c u l a t i o n concerning the r e g u l a t i o n of these two CSFs. Recently i t has been shown that murine GM-CSF and IL-3 l i e w i t h i n 230 Kb of each other on chromosome 11 (22). Sequence a n a l y s i s of t h e i r r e s p e c t i v e 5' noncoding regions has revealed a common decanucleotide (5' GPuGPuTTPyCAPy 3') (18). This sequence and the observed proximity of these CSFs might be involved i n b r i n g i n g about t h e i r co-ordinate t r a n s c r i p t i o n that i s r e a d i l y observed f o l l o w i n g l e c t i n s t i m u l a t i o n of T-lymphocytes (23). However, co-ordinate expression of GM-CSF and IL-3 i s not always observed (24). S t i m u l a t i o n of a T - c e l l l i n e with IL-2 y i e l d e d only GM-CSF and not IL-3 a c t i v i t y , i n c o n t r a s t , the co-ordinate production of GM-CSF and IL-3 was seen when these c e l l s were treated with Concanavalin A. Subsequent a n a l y s i s however revealed that the mRNA for both CSFs was present while only GM-CSF was apparently t r a n s l a t e d . At another l e v e l of gene r e g u l a t i o n both IL-3 and GM-CSF encode an AU-rich sequence i n t h e i r 3' untr a n s l a t e d regions, which has been shown to r e s u l t i n mRNA i n s t a b i l i t y (25). For both murine GM-CSF and IL-3 the existence of a longer rare v a r i a n t form of mRNA r e s u l t i n g from a l t e r n a t i v e promoters located upstream of the 7 main body of both genes has been described (18,26). The hydrophobic nature observed i n the 5' regions of these v a r i a n t s has led to the s p e c u l a t i o n that membrane bound forms of these two potent hemopoietic growth f a c t o r s may e x i s t on c e r t a i n c e l l types. B - c e l l f a c t o r s : Also included i n Category I are c e r t a i n " B - c e l l f a c t o r s " ; I n t e r l e u k i n - 4 , I n t e r l e u k i n - 5 and I n t e r l e u k i n - 6 which are i n c r e a s i n g l y being shown to e f f e c t a wide range of hemopoietic c e l l s (27). I n t e r l e u k i n - 4 ( I L - 4 ) : Also known as B - c e l l s t i m u l a t i n g f a c t o r - 1 , IL-4, a 20 kO g l y c o p r o t e i n produced by some a c t i v a t e d T - c e l l s , was f i r s t c h a r a c t e r i z e d f o r i t s a b i l i t y to st i m u l a t e r e s t i n g B - c e l l s (28,29). C u r r e n t l y , IL-4 i s a l s o known to stimulate growth of normal T - c e l l s (30), supports the p r o l i f e r a t i o n T - c e l l and mast c e l l l i n e s (31) as w e l l as a c t i n g on s e v e r a l other hemopoietic c e l l types; macrophages, granulocytes, . e r y t h r o i d precursors (32). I n t e r l e u k i n - 5 ( I L - 5 ) : I n i t i a l l y c h a r a c t e r i z e d as a B - c e l l growth and d i f f e r e n t i a t i o n f a c t o r (33), IL-5 i s now al s o known to have a very potent e f f e c t upon the growth and d i f f e r e n t i a t i o n of e o s i n o p h i l precursors (34). A product of T - c e l l s and T-lymphoma c e l l s , murine IL-5 has been shown to be able to s t i m u l a t e human 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 and a c t i v a t i o n (35). I n t e r l e u k i n - 6 ( I L - 6 ) : Though i n i t i a l l y i d e n t i f i e d as B - c e l l s t i m u l a t i n g f a c t o r - 2 (BSF-2), IL-6, a f a c t o r capable of inducing the f i n a l maturation of B - c e l l s i n t o antibody-forming c e l l s (36,37) was, once cloned, soon determined to be i d e n t i c a l to a number of a c t i v i t i e s described by d i f f e r e n t groups. These were known as 02 i n t e r f e r o n (38), 26 kDa p r o t e i n (39) and hybridoma growth f a c t o r (40) by v i r t u e of assays used to i d e n t i f y them. I t has now been reported that many d i f f e r e n t c e l l s i n c l u d i n g mononuclear blood c e l l s , f i b r o b l a s t s , e n d o t h e l i a l c e l l s , T - c e l l s , c a r d i a c myxoma c e l l s and a bladder carcinoma c e l l l i n e produce IL-6 (41). C e l l s responsive to IL-6 are now known expanded to include T - c e l l s (42), 8 hepatocytes (43), f i b r o b l a s t s (44) and even p l u r i p o t e n t hemopoietic stem c e l l s (45). (B) CATEGORY I I (Lineage r e s t r i c t e d hemopoietins) Prototypes f o r the category of CSF's that appear to be r e s t i c t e d to i n f l u e n c i n g the p r o l i f e r a t i o n , d i f f e r e n t i a t i o n and s u r v i v a l of only more mature hemopoietic c e l l s are: macrophage colony s t i m u l a t i n g f a c t o r (M-CSF), granulocyte colony s t i m u l a t i n g f a c t o r (G-CSF) and e r y t h r o p o i e t i n (Ep). In the murine and human systems a l l 3 of these CSFs e x i s t as s i n g l e copy genes (46-48) and show a remarkable degree of i n t e r s p e c i e s c r o s s - r e a c t i v i t y (49). Macrophage colony s t i m u l a t i n g f a c t o r (M-CSF): M-CSF was f i r s t p u r i f i e d to homogeneity from L - c e l l conditioned media. A homo-dimeric g l y c o p r o t e i n with a molecular weight of 70 KD (50), i t functions i n colony assay systems i n v i t r o to st i m u l a t e p r i m a r i l y the formation of macrophage c o l o n i e s and to a l e s s e r extent g r a n u l o c y t i c c o l o n i e s (51). Granulocyte colony s t i m u l a t i n g f a c t o r (G-CSF): O r i g i n a l l y c h a r a c t e r i z e d as a f a c t o r capable of inducing the d i f f e r e n t i a t i o n of murine myelomonocytic WEHI-3BD+ c e l l s (52), G-CSF was ev e n t u a l l y p u r i f i e d from endotoxin stimulated lung conditioned media (53). A 25 KD monomeric g l y c o p r o t e i n , G-CSF stimul a t e s the formation of a few c h a r a c t e r i s t i c a l l y s m a ll mature granulocyte colonies (54). E r y t h r o p o i e t i n (Ep): E r y t h r o p o i e t i n was f i r s t p u r i f i e d to homogeneity from the ur i n e of p a t i e n t s with a p l a s t i c anemia (55). Produced i n the kidney, t h i s 39 kd monomeric g l y c o p r o t e i n (56) stimul a t e s i n v i t r o the p r o l i f e r a t i o n and terminal maturation of CFU-E and some BFU-E (57,58). Other f a c t o r s a f f e c t i n g hemopoiesis: In the mouse, a l l t i s s u e s appear to contain some e x t r a c t a b l e hemopoietic growth f a c t o r s (16). This probably r e f l e c t s the i n v i t r o observations of CSF synthesis by s p e c i f i c c e l l types ( i . e . f i b r o b l a s t s , e n d o t h e l i a l c e l l s , lymphocytes, monocytes and 9 macrophages) which i n viv o are common to many organs. In s p i t e of what i s known regarding the i n v i t r o c e l l u l a r sources of the CSFs l i s t e d e a r l i e r , l i t t l e i s understood of the relevant i n viv o sources and even l e s s i s known about the mechanisms that e l i c i t and regulate CSF production. Moreover i t i s now c l e a r that the various CSFs whose r o l e s i n s t i m u l a t i n g hemopoiesis are w e l l e s t a b l i s h e d , are being found to i n t e r a c t with a broader range of c e l l types than p r e v i o u s l y thought. Thus previous attempts to c a t e g o r i z e these f a c t o r s are beginning to seem over s i m p l i s t i c . For example, under c e r t a i n c o n d i t i o n s G-CSF has been shown to i n i t i a t e the p r o l i f e r a t i o n of macrophage, e o s i n o p h i l , e r y t h r o i d and even mixed colony forming c e l l s (54), thus a c t i n g upon c e l l s at e i t h e r end of the d i f f e r e n t i a t i o n pathway. Other f a c t o r s such as IL-4, IL-5 and IL-6 were i n i t i a l l y c h a r a c t e r i z e d f o r t h e i r r o l e i n B-lymphocyte growth and maturation and have only r e c e n t l y been shown to have a much wider spectrum of b i o l o g i c a l a c t i v i t i e s as p r e v i o u s l y mentioned. Recently another b i o l o g i c a l a c t i v i t y , a s y n e r g i s t i c or co-operative a c t i v i t y , has been a t t r i b u t e d to a number of known CSFs as w e l l as a few yet uncharacterized CSFs. One such a c t i v i t y i n i t i a l l y r e f e r r e d to as hemopoietin 1 was f i r s t i s o l a t e d from a human bladder carcinoma l i n e (59). I t helps to induce the formation of giant macrophage c o l o n i e s by c e l l s i n 5 - f l u o r o u r a c i l treated mouse marrow when used i n conjunction with M-CSF (60,61). Recently hemopoietin-1 has been shown to be i n t e r l e u k i n 1 (62). Another study suggests that IL-6 can act i n synergy with IL-3 to boost the formation of b l a s t c e l l c o lonies by bone marrow c e l l s (45). An approximately 60 kd f a c t o r p a r t i a l y p u r i f i e d from a murine marrow adherent l i n e (Tc-1) was r e c e n t l y shown to enhance the e f f e c t s of IL-3, GM-CSF and M-CSF i n v i t r o (63). This f a c t o r was a l s o shown to possess pre-B c e l l inducing and B - c e l l d i f f e r e n t i a t i o n a c t i v i t i e s . S y n e r g i s t i c a c t i v i t i e s are 10 a l s o claimed of IL-3 (64); i n a d d i t i o n to i t s m u l t i - l i n e a g e a c t i v i t i e s , IL-3 i s seen as capable of enhancing the e f f e c t s of M-CSF and e r y t h r o p o i e t i n . There remain many other f a c t o r s , not discussed e a r l i e r , which a l s o i n f l u e n c e i n v i t r o hemopoiesis and hence are p o t e n t i a l l y relevant to i n v i v o hemopoiesis. For example, the products of a c t i v a t e d monocytes, tumor neucrosis f a c t o r - a (TNFa) and IL-1 have both been shown to help enhance CSF production by f i b r o b l a s t s and e n d o t h e l i a l c e l l s (65-69). Other f a c t o r s such as interferon-gamma (IFN-y) can act s y n e r g i s t i c a l l y with TNF-a i n inducing monocytes to synthesize M-CSF (70). Another f a c t o r , transforming growth f a c t o r type 3 (TGF0) was r e c e n t l y shown to induce chemotaxis and IL-1 production i n monocytes (71). More r e c e n t l y s e v e r a l groups have reported on the p r e l i m i n a r y c h a r a c t e r i z a t i o n of yet another c l a s s of f a c t o r s that s t i m u l a t e pre-B c e l l s and are produced by stromal c e l l s (72-74). I t i s therefore p o s s i b l e that there are many more growth f a c t o r s a f f e c t i n g the d i f f e r e n t lineages and stages of growth and d i f f e r e n t i a t i o n of hemopoiesis that remain as yet undiscovered. 3) LEUKEMOGENISIS Myeloid leukemias are believed to a r i s e from the n e o p l a s t i c transformation and c l o n a l expansion of p r i m i t i v e hemopoietic c e l l s . Key features of the disease are the abnormal production of large numbers of c e l l s that remain ph e n o t y p i c a l l y immature. The presence of these c e l l s e v e n t u a l l y d i s r u p t s normal hemopoiesis g i v i n g r i s e to anemia, thrombocytopenia and neutropenia. Evidence f o r the c l o n a l nature of neoplasia i s exemplified by a n a l y s i s of chronic myelogenous leukemia (CML) i n man. In CML, the P h i l a d e l p h i a chromosome (Ph*) which i s present i n over 90% of cases (75) has served as a 11 cytogenetic marker i n c l o n a l i t y s t u d i e s . These s t u d i e s , corroborated by glucose-6-phosphate dehydrogenase (G6PD) a n a l y s i s (76,77), have revealed the presence of the Ph^ - chromosome or the monoclonal expression of one G6PD i s o -enzyme i n granulocytes, monocytes, macrophages, er y t h r o c y t e s , megakaryocytes, e o s i n o p h i l s , basophils and t h e i r committed progenitors (78-83), implying that CML i s a c l o n a l d i s o r d e r o r i g i n a t i n g i n the p l u r i p o t e n t hemopoietic stem c e l l compartment. Analogous f i n d i n g s i n v o l v i n g cytogenetic or other techniques have documented the c l o n a l o r i g i n of many other human leukemias. S t i l l at i s s u e , however, are the s p e c i f i c growth a l t e r a t i o n s and mechanisms that underly the leukemic transformation. Considerable evidence now points to the involvement of proto-oncogenes i n hemopoietic c e l l transformation, as has been observed i n other malignancies. S p e c i f i c chromosomal t r a n s l o c a t i o n s associated with c e r t a i n hematological malignancies have pinpointed s e v e r a l such genes. For example, i n CML, a r e c i p r o c a l exchange between chromosomes 9 and 22 r e s u l t i n g i n the Ph* chromosome i s now known to r e l o c a t e a p o r t i o n of the proto-oncogene c-abl to a newly recognized region (breakpoint c l u s t e r region or bcr) w i t h i n a genetic locus now known a phl-1 (84-87). The r e s u l t a n t c-abl f u s i o n p r o t e i n ( p 2 1 0 c - a b l ) , l i k e i t s transforming r e t r o v i r a l counterpart ( p l 6 0 v _ a ^ ^ ) , e x h i b i t s a more robust t y r o s i n e kinase a c t i v i t y than the normal c-abl product (88). Another example of a genetic l e s i o n r e s u l t i n g from a t r a n s l o c a t i o n event i s seen i n B u r k i t t lymphoma. Here the proto-oncogene c-myc i s c o n s i s t e n t l y involved i n a t r a n s l o c a t i o n to various t r a n s c r i p t i o n a l l y a c t i v e immunoglobulin l o c i (89). The r e s u l t a n t c o n s i t i t u t i v e expression of the t r a n s l o c a t e d c-myc gene i s thought to be c r i t i c a l to the malignant phenotype seen i n B u r k i t t lymphomas (90). The involvement of the ras family of oncogenes i n hemopoietic tumors was f i r s t detected i n the NIH-3T3 c e l l t r a n s f e c t i o n assay (91). In 12 subsequent s t u d i e s of acute myelogenous leukemia (AML) point mutations i n N-ras and K-ras, i n d i c a t i v e of an a c t i v a t e d s t a t e (92), were seen i n approximately 25% of the p a t i e n t s studied (93). Also of i n t e r e s t was the f i n d i n g of such a c t i v a t e d ras-oncogenes i n pa t i e n t s with a myelodysplastic syndrome ( i . e . a pre-leukemic c o n d i t i o n ) , suggesting a r o l e f o r these oncogenes i n the onset of malignancy (94-96). Growth f a c t o r s and leukemogenisis: An important stage i n tumor development might be the release of a c e l l from i t s dependence on exogenous growth f a c t o r s e i t h e r as an i n i t i a t i n g event or as part of tumor progression. Indeed, i t i s presently thought that many i f not a l l oncogenes encode f o r p r o t e i n s that are c o n t r o l l i n g elements along the normal mitogenic pathway i n a c e l l . P r o t e ins such as growth f a c t o r s , growth f a c t o r receptors and p r o t e i n s involved i n i n t r a c e l l u l a r s i g n a l transduction c o n s t i t u t e elements i n t h i s pathway. The a b l , myc and ras oncogenes i m p l i c a t e d i n hematologic malignancies may be considered i n t h i s context as being i n v o l v e d i n i n t r a c e l l u l a r s i g n a l transduction. Some e a r l i e r i n s i g h t s l i n k i n g growth f a c t o r s to a transformed phenotype were derived from s t u d i e s e s t a b l i s h i n g a r e l a t i o n s h i p between oncogenes and s p e c i f i c growth f a c t o r s . One proto-oncogene, c - s i s , has been shown to encode f o r the B-chain of p l a t e l e t derived growth f a c t o r (PDGF) (97) and i s only capable of transforming c e l l s that express the PDGF receptor (98). The c e l l u l a r homologues to the two oncogenes erb-B and fms have been shown to encode the epidermal growth f a c t o r receptor and the macrophage colony s t i m u l a t i n g f a c t o r receptor r e s p e c t i v e l y (99-101). Both oncogenes v-erb-B and v-fms are thought to mimic receptor occupancy, thereby c o n s t i t u t i v e l y s t i m u l a t i n g the c e l l s i n which they are expressed. I n s e r t i o n a l mutagenic a c t i v a t i o n of growth f a c t o r s and growth f a c t o r r e l a t e d genes involved i n hemopoiesis has provided a d d i t i o n a l information 13 l i n k i n g growth f a c t o r s to transformation. In the gibbon leukemic c e l l l i n e MLA 144, c o n s t i t u t i v e synthesis of IL-2 i s due to the i n s e r t i o n of a v i r a l long terminal repeat (LTR) i n the 3' non-coding region of the gene (102). S i m i l a r l y , the murine myelomonocytic c e l l l i n e , WEHI-3B, which i s thought to have a r i s e n from an IL-3 dependent neutrophil-macrophage progenitor c e l l , c o n s t i t u t i v e l y synthesizes IL-3, presumably as a r e s u l t of the i n s e r t i o n of an i n t r a c i s t e r n a l A p a r t i c l e (IAP) i n the 5' non-coding region of the IL-3 gene (103). A recent i n vivo study i n v o l v i n g the i n f e c t i o n of mice with the Friend murine leukemia v i r u s (a non-transforming r e t r o v i r u s ) demonstrated that 20% of the m y e l o b l a s t i c leukemias induced by t h i s v i r u s were as s o c i a t e d w i t h an i n t e g r a t i o n of the p r o v i r a l DNA next to the M-CSF receptor gene, c-fms (104). High expression of the c-fms gene was observed i n a l l tumors and was accompanied by the l o s s of the normal c-fms a l l e l e i n some. The i n t r o d u c t i o n of t r a n s c r i p t i o n a l l y a c t i v e CSF genes i n t o f a c t o r dependent c e l l l i n e s has provided d i r e c t evidence that autonomous growth can confer a leukemic phenotype. In one such study a r e t r o v i r a l expression vec t o r encoding a GM-CSF cDNA was introduced i n t o the f a c t o r dependant murine c e l l l i n e FDCP-1 (105). These transfected c e l l s were then shown to grow independently of exogenous CSF and u n l i k e t h e i r p a r e n t a l FDCP-1 c e l l s , gave r i s e to tumors i n syngeneic mice. A s i m i l a r study i n v o l v i n g a murine T - c e l l l i n e t r a n s f e c t e d with an IL-2 expression vector a l s o r e s u l t e d i n growth autonomy and tumorogenicity (106). S i m i l a r r e s u l t s have been obtained from s t u d i e s of spontaneaously a r i s i n g f a c t o r independent mutants from f a c t o r dependent c e l l l i n e s . In one such instance, the a b i l i t y of the mutant l i n e to grow autonomously because of i t s aquired c a p a c i t y f o r IL-3 synt h e s i s was concominant with an a b i l i t y to e s t a b l i s h tumors i n syngeneic mice (107). Recent s t u d i e s have provided data l i n k i n g aberant hemopoietic growth f a c t o r production to human leukemia. In one study, leukemic c e l l s i s o l a t e d 14 from three AML p a t i e n t s were shown to p r o l i f e r a t e autonomously i n v i t r o v i a an a u t o c r i n e mechanism i n v o l v i n g the c o n s t i t u t i v e synthesis of GM-CSF (108). One mechanism f o r t h i s deranged CSF synthesis may be s t r u c t u r a l rearrangement of the genes f o r the a f f e c t e d growth f a c t o r . This has r e c e n t l y been observed i n the leukemic c e l l s of some AML p a t i e n t s (109). Another study i n v o l v i n g AML pa t i e n t s demonstrated the production by leukemic b l a s t c e l l s of b i o l o g i c a l l y a c t i v e IL-1 that i n turn could promote the rele a s e i n v i t r o of GM-CSF from stromal c e l l s (110,66,67). In m u l t i p l e myeloma, a malignancy of plasma c e l l s , c o n s t i t u t i v e expression of IL-6 was observed i n 26 of 26 pa t i e n t s studied i n one report and autocrine induced p r o l i f e r a t i o n was documented i n v i t r o i n 46% of these (111). 4) MODEL SYSTEMS FOR LEUKEMIC TRANSFORMATION The t r a n s i t i o n of a normal c e l l i n t o a n e o p l a s t i c c e l l i s p r e s e n t l y thought to be a m u l t i - f a c t o r i a l process. Dir e c t experimental evidence f o r t h i s has been provided by gene t r a n s f e r i n t o primary embryo c e l l c u l t u r e s w i t h the notio n of two broad classes of oncogene products termed competence f a c t o r s and progression f a c t o r s (112,113). In v i t r o , the p r o l i f e r a t i o n of normal hemopoietic c e l l s demonstrates a s t r i c t requirement f o r exogenous growth f a c t o r s . In co n t r a s t , t h e i r f u l l y transformed counterparts may appear to obviate t h i s need. In attempting to decipher the molecular events that give r i s e to the n e o p l a s t i c phenotype many groups have developed models where the e f f e c t s of various oncogenes can be assayed by growth f a c t o r independence and tum o r i g e n i c i t y . In examining the e f f e c t s of v-myc and v-raf upon f r e s h murine bone marrow c e l l s , B l a s i et a l (114) demonstrated that only the simultaneous expression of both oncogenes r e s u l t e d i n continuous i n v i t r o growth 15 ( i m m o r t a l i z a t i o n ) of macrophages. B i o - a c t i v i t y a n a l y s i s of the conditioned media from these t r a n s f e c t e d c e l l s was negative f o r the synthesis of CSFs i n s p i t e of t h e i r autonomous growth. Studies of v-ra f transformed hemopoietic c e l l s have shown that while these c e l l s are capable of continuous i n v i t r o p r o l i f e r a t i o n they remain f a c t o r (IL-3) dependent (115) suggesting therefore that v-myc s u b s t i t u t e s f o r growth f a c t o r s i n the v-myc/v-raf immortalized macrophages. Another study examining the e f f e c t s of v-myc on f a c t o r dependent c e l l l i n e s concluded that v-myc expression i n c e r t a i n IL-2 or IL-3 dependent hemopoietic c e l l l i n e s were capable of abrogating t h e i r requirement f o r added growth f a c t o r s by a non-autocrine mechanism (116). As with the myc oncogene, the abelson oncogene i s a l s o thought to operate i n the transformation of c e l l s v i a a non-autocrine mechanism. In a model system using Friend murine leukemia v i r u s immortalized, f a c t o r (IL-3) dependent c e l l l i n e s , O l i f f et a l . (117) were able to demonstrate that the onset of f a c t o r independence conferred by s u p e r i n f e c t i n g these c e l l l i n e s w i t h the Abelson murine leukemia v i r u s (A-MuLV) was l i n k e d w i t h t h e i r a b i l i t y to e s t a b l i s h tumors i n syngeneic mice. The abelson oncogene i n t h i s study appeared to fu n c t i o n as a progression f a c t o r by a l l o w i n g the c e l l s to express a f u l l y malignant phenotype. As no CSF production was detected from these transformed clones i t was concluded that a non-autocrine mechanism of p r o l i f e r a t i o n was involved. S i m i l a r approaches to understanding the mechanism of transformation of myeloid c e l l s have involved i n f e c t i o n of another w e l l c h a r a c t e r i z e d f a c t o r dependent (GM-CSF and IL-3) c e l l l i n e with an A-MuLV complex (118). Again i t was concluded that the tumorigenic l i n e s derived were not p r o l i f e r a t i n g due to endogenous expression of e i t h e r IL-3 or GM-CSF. In another study, i t was demonstrated that while both A-MuLV and Harvey murine sarcoma v i r u s (Ha-MSV) are capable of s t i m u l a t i n g the growth and d i f f e r e n t i a t i o n of e r y t h r o i d precursor c e l l s , only those i n f e c t e d w i t h A-MuLV grew independently of e r y t h r o p o i e t i n (Epo) (119) with no concomitant 16 a b i l i t y to synthesize Epo. A model system using normal mast c e l l s as targe t s f o r A-MuLV transformation both i n v i t r o and i n v i v o f u r t h e r emphasized the abrogation of f a c t o r dependance by the A-MuLV derived transformants. Here too no synthesis of mast c e l l growth f a c t o r (IL-3) could be detected i n the malignant A-MuLV transformed c e l l s (120). In a l l the above v i r a l abelson (v-abl) induced myeloid leukemia models, one u n i f y i n g feature has been the concurrence of a non-autocrine mechanism as that which promotes f a c t o r independence i n the various malignant A-MuLV derived clones. The abnormal ty r o s i n e kinase a c t i v i t y a s s o ciated with an a c t i v a t e d abelson oncogene (121) i s thus thought to obviate the need f o r an e x t r a c e l l u l a r mitogenic s i g n a l transduced by growth f a c t o r - r e c e p t o r complexes i n the plasma membrane. Autocrine growth was reported of f a c t o r dependent v-myc or v-myb transformed chicken myeloid c e l l s that were i n f e c t e d with r e t r o v i r u s e s encoding oncogenes of the s r c family (122). These su p e r i n f e c t e d chicken myeloid c e l l s were shown to synthesize t h e i r own growth f a c t o r only i f p p 6 0 v - s r c was continuously being expressed. This p a r t i c u l a r observation suggests that c e r t a i n members of the s r c v i r a l oncogene f a m i l y mimick c e l l u l a r pathways involved i n growth f a c t o r s s y n t h e s i s . In the study of myeloid leukemia c e l l l i n e s derived from A-MuLV i n f e c t e d mice, Schrader et a l (123) demonstrated that these l i n e s grew autonomously by v i r t u e of an I L -3 mediated autocrine loop. A-MuLV myeloid leukemia model: A-MuLV i s an acut e l y transforming, r e p l i c a t i o n d e f e c t i v e r e t r o v i r u s that r e s u l t e d from a recombination event between the Moloney murine leukemia v i r u s (M-MuLv) and the c e l l u l a r proto-oncogene, c-abl (124). In a d d i t i o n to thymus independent B - c e l l lymphomas, the A-MuLV i s now a l s o known to give r i s e to a v a r i e t y of myeloid leukemias i n v i v o . In studying the s u s c e p t i b i l i t y of the various myeloid hemopoietic lineages to A-MuLV transformation, an i n v i t r o experiment by C.J. Eaves et 17 a l (125) looked at i n f e c t i o n of m u l t i - l i n e a g e hemopoietic c o l o n i e s of s i n g l e c e l l o r i g i n derived from Balb/c mice. I t was shown that transformed c e l l l i n e s could be derived at high frequency from these m u l t i - l i n e a g e c o l o n i e s only i f these A-MuLV exposed c e l l s were subsequently co-cultured upon i r r a d i a t e d 3T3 c e l l s f o r up to 3 months. The myeloid c e l l s generated i n t h i s way are continuously growing, tumorigenic and d i s p l a y c h a r a c t e r i s t i c s of mast c e l l s (histamine p o s i t i v e , express high a f f i n i t y receptors f o r IgE, metachromatic s t a i n i n g of granules with toluidene b l u e ) . Of p a r t i c u l a r i n t e r e s t i s that these l i n e s grow without the need of a feeder l a y e r or any exogenous f a c t o r s . The need f o r c o - c u l t i v a t i o n upon 3T3 feeders subsequent to A-MuLV i n f e c t i o n suggested that v-abl was required f o r i n i t i a t i o n of the transformation process but that secondary events were necessary f o r progression to a malignant phenotype. This notion i s supported by work of V h i t l o c k and U i t t e (126) who showed that i n v i t r o i n f e c t i o n of bone marrow c e l l s w i t h A-MuLV required c o - c u l t u r i n g f o r s e v e r a l weeks on feeders before the i n f e c t e d pre-B c e l l s evolved a f u l l y malignant phenotype. Others have demonstrated that while A-MuLV plays a r o l e i n the i n i t i a t i o n of transformation i t appears not to be necessary f o r the maintanance of t h i s s t a t e (127). In a l a t e r study using s i m i l a r l y derived i n v i t r o A-MuLV myeloid l i n e s i t was shown that the conditioned media (CM) from the c e l l s s t i m u l a t e d the formation of large e r y t h r o i d c o l o n i e s , erythroid-mixed c o l o n i e s and i n some cases l a r g e non-erythroid colonies (128). I t was subsequently demonstrated that these c e l l l i n e s do i n fact synthesize and r e l e a s e J ) i o - a c t i v e GM-CSF. However attempts to detect IL-3 (to account f or the p l u r i p o t e n t a c t i v i t y ) by Northern b l o t a n a l y s i s were negative. 5) THESIS OBJECTIVES The o v e r a l l o b j e c t i v e of the work, described i n t h i s t h e s i s i s to understand b e t t e r the r o l e of growth f a c t o r s i n normal and leukemic hemopoiesis. In v i t r o growth f a c t o r expression has been documented i n a number of hemopoietic (lymphocytes, monocytes and macrophages) and non-hemopoietic c e l l s ( f i b r o b l a s t s and e n d o t h e l i a l c e l l s ) . While l i t t l e i s known about the mechanisms governing i n vivo hemopoiesis, even l e s s i s understood about the mechanisms that e l i c i t and regulate CSF production both i n v i t r o and i n v i v o . The e l u c i d a t i o n of c e l l and t i s s u e sources of the CSFs, as w e l l as an understanding of the modes of gene r e g u l a t i o n involved i s needed i n order to bette r c l a r i f y t h e i r r o l e ( s ) i n p h y s i o l o g i c and patho-p h y s i o l o g i c s t a t e s . In the majority of studies the documenting of growth f a c t o r gene expression both i n v i t r o and i n vivo has r e l i e d on r e s u l t s from of b i o -a c t i v i t y assays. These assay systems are l i m i t e d by the s e n s i t i v i t y and s p e c i f i c i t y of the responder c e l l s used. I t i s now c l e a r that many b i o -a c t i v i t y assays can not c l e a r l y d i s t i n g u i s h between d i f f e r e n t f a c t o r s due to t h e i r overlapping spectra of a c t i v i t i e s on the same c e l l s . Such ambiguity could however be minimized through the use of a very s p e c i f i c molecular h y b r i d i z a t i o n technique such as Sl-nuclease mapping. I t was the o b j e c t i v e of t h i s t h e s i s to use such a technique to evaluate the expression of murine GM-CSF and IL-3 genes i n a v a r i e t y of normal and malignant c e l l types. Of p a r t i c u l a r i n t e r e s t were the A-MuLV myeloid " m a s t - c e l l l i k e " transformants that are known to produce GM-CSF and p o s s i b l y another m u l t i -l i n e a g e CSF. 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Grunwald DJ, Dale B, Dudley J , Lamph W, Sugden B, Ozanne B, R i s s e r R: Loss of v i r a l gene expression and r e t e n t i o n of t u m o r i g e n i c i t y by Abelson lymphoma c e l l s . J V i r o l 43:92, 1982 128. Chung SW, Wong PMC, Shen-Ong G, R u s c e t t i S, I s h i z a k a T, Eaves CJ: Production of granulocyte macrophage colony s t i m u l a t i n g f a c t o r (GM-CSF) by Abelson v i r u s induced tumorigenic mast c e l l l i n e s . Blood 68:1074, 1986 29 C H A P T E R II MATERIALS AND METHODS 1) DNA ISOLATION AND PURIFICATION I s o l a t i o n of high molecular weight genomic DNA: To i s o l a t e and p u r i f y genomic DNA (1) c e l l s were washed 2 x i n phosphate b u f f e r s a l i n e (PBS). To approximately 4 x 10 7 c e l l s , 2 ml of TNE Buffer (10 mM T r i s pH 8.0, 150 mM NaCl, 10 mM EDTA) was added and gently mixed. To t h i s c e l l l y s a t e was added 20 y l of 20% SDS and 10 p i of proteinase K (stock 10 mg/ml; Sigma Chemical Company). This was then allowed to incubate f o r a minimum of 6 hrs at 37°C. Fol l o w i n g t h i s the l y s a t e was extracted 3 x with TNE e q u i l i b r a t e d phenol, 1 x with a 1:1 mixture of TNE e q u i l i b r a t e d phenol and chloroform and f i n a l l y 3 x with a 24:1 mixture of chloroform to isoamyl a l c o h o l . The aqueous phase recovered was then d i a l y z e d against a 100 x volume of TE b u f f e r (10 mM T r i s , 1 mM EDTA) with 3 changes over 36 hours. The DNA was then q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y (A260/A280 = 2) and stored i n s t e r i l e a l i q u o t s at 4°C. I s o l a t i o n of plasmid DNA: Plasmid mini preps were conducted as described by Holmes and Quigley (2). B r i e f l y , a 5 ml overnight b a c t e r i a l c u l t u r e (AgQO ~ 1-2) was p e l l e t e d and resuspended i n 0.35 ml STET b u f f e r (8X sucrose, 5% T r i t o n X-100, 50 mM EDTA, 50 mM T r i s pH 8.0). Lysozyme (25 y l at 10 mg/ml) was added to f u r t h e r weaken the b a c t e r i a l w a l l before a 30 second incubation i n b o i l i n g water brought about gentle l y s i s . The l y s a t e was spun i n a microfuge (15,000 g) at 4°C and the c l e a r supernatant c o n t a i n i n g the plasmid was removed. An equal volume of isopropanol was then 30 added and the DNA was allowed to p r e c i p i t a t e by incubation on dry i c e f o r 10 minutes. The r e s u l t i n g p e l l e t when spun and d r i e d was then resuspended i n 50 u l 1 x TE. T y p i c a l l y 5 u l contained s u f f i c i e n t plasmid DNA f o r r e s t r i c t i o n enzyme a n a l y s i s . Large s c a l e plasmid preps were done by a modified method of Birnboim and Dolby ( 3 ) . One l i t r e b a c t e r i a l c u l t u r e s were p e l l e t e d i n 250 ml p l a s t i c b o t t l e s w i t h sealed l i d s at 7000 rpm f o r 10 mins. The p e l l e t s were c o l l e c t i v e l y resuspended i n 20 mis of 25% sucrose, 0.05M T r i s ; pH 8.0. F r e s h l y made lysozyme (2 mis from 10 mg/ml stock) was then added. The s o l u t i o n was allowed to incubate on i c e f o r 5 mins before 1.5 ml EDTA (250 mM, pH 8.0) was added f o r a f u r t h e r 5 min incubation at room temperature. Then 15 ml of a T r i t o n - X s o l u t i o n ( 1 % Triton-X, 62.5 mM EDTA, 50 mM T r i s pH 8.0) was added dropwise over a 1 minute period with g e n t l e constant s w i r l i n g of the f l a s k . The r e s u l t i n g l y s a t e was then spun (19,000 rpm f o r 35 mins at 4°C) i n Oakridge c e n t r i f u g e tubes. CsCl was added to the c a r e f u l l y decanted c l e a r supernatant (0.9 gm/ml l y s a t e ) , followed by a 1/20 v o l of EtBr (10 mg/ml). The s o l u t i o n s were t r a n s f e r r e d to heat s e a l a b l e S o r v a l l c e n t r i f u g e tubes to be spun at 42,000 rpm f o r a minimum of 18 hrs at room temperature. The lower plasmid band was removed with a hypodermic needle. This s o l u t i o n was mixed with s e v e r a l changes of CsCl saturated isopropanol and d i a l y z e d against 100 x v o l of 1 x TE. The d i a l y z e d samples were q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y and stored at -20°C. 2) DNA ANALYSIS Southern b l o t a n a l y s i s : R e s t r i c t i o n enzymes used were obtained from BRL or Pharmacia and used as per s u p p l i e r s s p e c i f i c a t i o n s . Southern b l o t a n a l y s i s (Southern 1975; Maniatis et a l . , 1982) (4,5) was c a r r i e d out on DNA 31 samples which were digested with a v a r i e t y of r e s t r i c t i o n enzymes and subjected to agarose slab g e l e l e c t r o p h o r e s i s . The e l e c t r o p h o r e t i c b u f f e r used was TBE (89 mM T r i s , 89 mM borate, 2.5 mM EDTA pH 8.3). A f t e r an overnight e l e c t r o p h o r e s i s at 30V the g e l was stained i n an ethidium bromide s o l u t i o n (10 mg/ml i n ddf^O), photographed, and then treated w i t h 0.25 M HCL; 0.5 M NaOH/1.5 M NaCl and 1 M NH4Ac/0.02 M NaOH i n succession (Smith, GE and Summers, M.D., 1980) (6). The DNA was then t r a n s f e r r e d onto c e l l u l o s e n i t r a t e paper i n a u n i d i r e c t i o n a l manner (Smith and Summers, 1980; Southern, EM, 1975). The f i l t e r s were a i r d r i e d and then oven baked under vacuum at 80°C f o r 2 hours. Subsequent h y b r i d i z a t i o n with nick t r a n s l a t e d (-^P) probes was conducted i n polyethylene envelopes i n 3 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium c i t r a t e [pH 7.0]), 4 x Denhardt's (50 x: 5 g f i c o l l , 5 g p o l y v i n y l p y r r o l i d o n e , 5 g BSA pentax V f r a c t i o n to a f i n a l v o l of 500 mis) and 0.1% sodium dodecyl sulphate (5). Heat denatured probe was added to a conc e n t r a t i o n o f 10 6 cpm/ml/cm^ of n i t r o c e l l u l o s e with 250 ug/ml denatured salmon sperm DNA. H y b r i d i z a t i o n was allowed to proceed f o r approximately 20 hrs at 68°C i n a shaking water bath. Post h y b r i d i z a t i o n washing (repeated 4 x at 30 minutes per wash) of the f i l t e r s were done i n 100 mis of 0.1 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate at 55°C. A f i f t h wash was done i n the same b u f f e r at 65°C f o r 30 minutes. The f i l t e r was then sealed i n a p l a s t i c bag and placed on Kodak XAR-5 brand X-ray f i l m with an i n t e n s i f y i n g screen (Cronex, Dupont) f o r autoradiography at -70°C for 1-7 days depending on s i g n a l i n t e n s i t y . Nick t r a n s l a t i o n s of DNA probes: A l l DNA probes that were n i c k t r a n s l a t e d (7) were e i t h e r CsCl density gradient p u r i f i e d as described i n l a r g e s c a l e plasmid preps, or fragments e l e c t r o e l u t e d from agarose g e l s ( 8 ) . DNA probes l a b e l l e d by t h i s procedure were used i n both Southern and Northern b l o t a n a l y s i s . A n i c k t r a n s l a t i o n k i t , purchased from BRL was used 32 as per s u p p l i e r s s p e c i f i c a t i o n s . T y p i c a l l y , i n a 50 u l r e a c t i o n 50 uCi of a- 3 2PdCTP (800 Ci/m mole) was used i n n i c k t r a n s l a t i n g 1 ug of DNA. F o l l o w i n g a 1 hr incubation at 15°C the n i c k t r a n s l a t e d probe was p u r i f i e d by 2 passes through a Sephadex G-50 s p i n column. Probes had a s p e c i f i c a c t i v i t y of - 10 8 cpm/ug. Oligo l a b e l l e d DNA probes: T y p i c a l l y 20 ng of a g e l p u r i f i e d DNA fragment was added to 200 ng of hexanucleotides i n a f i n a l volume of 14 u l , b o i l e d f o r 3 min and allowed to cool on i c e . To t h i s was added 2 u l of 10 x HLB (500 mM pH 6.9; 100 mM MgCl 2; 60 mM-Mercaptoethanol), 2 u l of n u c l e o t i d e mix (dGTP, dTTP, dATP at 2.5 mM each), 2 y l of <x-32PdCTP (3000 Ci/m mole) and 2 u n i t s of Klenow polymerase. The r e a c t i o n was allowed to incubate at room temperature f o r 2 hours. The r e a c t i o n mix was then d i l u t e d to a f i n a l volume of 100 p i before l a b e l l e d DNA was p u r i f i e d over a Sephadex G-50 s p i n column. 3) RNA ISOLATION/PURIFICATION R i b o n u c l e i c a c i d p u r i f i c a t i o n followed one of two procedures depending on the number of c e l l s being dealt with. In the f i r s t procedure (5) 2.7 ml of 6M Guanidine h y d r o c l o r i d e was added and vortexed f o r 10 seconds followed by the a d d i t i o n of 0.3 ml of 2M KAc (pH 5.0) with v o r t e x i n g f o r 5-10' u n t i l the s o l u t i o n became c l e a r . Following t h i s 6 ml of a 1% sarcosyl/lOOmM T r i s pH 8.0 was added and thoroughly mixed. F i n a l l y CsCl s a l t was added at 1 gm/2.5 ml lysate-mix and d i s s o l v e d by gentle mixing. The r e s u l t a n t s o l u t i o n was then layered upon 2.0 ml of a 5.6 M CsCl-0.1 M EDTA s o l u t i o n i n a polyallomer tube. This gradient was then centrifuged i n an SW-41 r o t o r at 32,000 rpm (22-24°C) for approximately 48 hrs. The RNA which p e l l e t s to the bottom was c a r e f u l l y kept c l e a r of the DNA found at the l y s a t e - C s C l cushion 33 i n t e r f a c e . The p e l l e t was resuspended i n 1 ml of NETS bu f f e r (100 mM NaCl, 10 mM T r i s HC1, pH 7.6, 1 mM EDTA and 0.5% SDS). I f the c e l l s were obtained from t i s s u e c u l t u r e the RNA was u s u a l l y s u f f i c i e n t l y pure as to obviate the need f o r phenol e x t r a c t i o n s . In t h i s case the RNA was recovered by a d d i t i o n of a 1/20 v o l of 3 M NaAc (pH 5.0) and 2 v o l of absolute ethanol. This procedure allows f o r 5 x l 0 7 c e l l s to be processed per polyallomer tube. The a l t e r n a t i v e procedure (9) used allowed f or the processing of up to 2x10^ c e l l s per tube, thus f a c i l i t a t i n g e x t r a c t i o n s of l a r g e amounts of RNA. Washed c e l l s were resuspended i n a Urea l y s i s b u f f e r (10^ c e l l s / 40 mis) 7 M Urea, 2% s a r c o s y l , 350 mM NaCl, 10 mM T r i s pH 7.9, 1 mM EDTA. This was then thoroughly mixed and poured i n t o an autoclaved g l a s s douncer. The mixture was c a r e f u l l y and thoroughly dounced (15-30 strokes) at 4°C u n t i l i t s v i s c o s i t y was s u f f i c i e n t l y low as to allow drops to form upon being passed through a 5 ml p i p e t t e . Then 1 gm CsCl s a l t was added per 2.5 ml c e l l l y s a t e d i s s o l v e d and layered upon a 3.5 ml CsCl cushion (5.6 M CsC l , 0.1 M EDTA) i n a polyallomer tube. This was cen t r i f u g e d i n an SW-41 r o t o r at 29,000 rpm (22-24°C) f o r approximately 24 hours and RNA recovered as above. A l l RNAs obtained were q u a n t i f i e d s p e c t r o p h o t o m e t r i c a l l y at h-260 a n c* ^280 and stored as ethanol p r e c i p i t a t e s at -70°C. Further mRNA p u r i f i c a t i o n was sometimes performed using oligo-dT columns to e n r i c h f o r polyadenylated RNA (10). Oligo-dT c e l l u l o s e was weighed according to i t s binding capacity as determined by the s u p p l i e r (Pharmacia). The oligo-dT was f i r s t washed i n 0.1 N NaOH and allowed to s e t t l e . Removal of the aqueous phase was followed by s e v e r a l r i n s e s i n a low s a l t b u f f e r (10 mM T r i s pH 7.0) u n t i l a pH of 7 was reached. The o l i g o -dT c e l l u l o s e was then placed i n a s t e r i l e pasture p i p e t t e f i l l e d w i t h s t e r i l e g l a s s wool at one end. The column of oligo-dT c e l l u l o s e was then r i n s e d w e l l with high s a l t b u f f e r (0.6 M NaCl, 10 mM T r i s pH 7.5). F i n a l l y the column was ri n s e d with prewarmed (37°C) high salt-SDS b u f f e r (0.6 M 34 NaCl, 0.5% SDS, 10 mM T r i s pH 7.5) before the t o t a l genomic RNA sample was a p p l i e d to i t . The RNA sample was heated at 65°C f o r 5 minutes then copied to room temperature before being a p p l i e d to the column. The column was immediately r i n s e d with high s a l t b u f f e r and the e f f l u e n t c o l l e c t e d , reheated, cooled and re-run through the same column. Then the column was r i n s e d w i t h the low s a l t b u f f e r (10 mM T r i s pH 7.0) and the e f f l u e n t c o l l e c t e d i n 1 ml f r a c t i o n s . T y p i c a l l y a l l of the polyadenylated RNA was c o l l e c t e d i n the f i r s t 2-3 f r a c t i o n s . Polyadenylated RNA y i e l d s of 2-5% of the t o t a l RNA a p p l i e d were r o u t i n e l y obtained by t h i s method. 4) RNA ANALYSIS Northern b l o t a n a l y s i s : RNA samples were f r a c t i o n a t e d on 1% agarose-2.2M formaldehyde g e l s i n 1 x MOPS buffe r (50 mM morpholino-propanesulphonic a c i d [pH 7.0], 1 mM EDTA) (Leonard G. Davis et a l , 1986) (11). E l e c t r o p h o r e s i s f o r 45 mins at 100 v o l t s i n a m i n i - g e l apparatus, followed by s t a i n i n g i n 5 yg/ml EtBr i n s t e r i l e ddl^O allowed f o r v i s u a l i z a t i o n of w e l l resolved ribosomal bands and RNA-ladder markers (BRL s u p p l i e r ) by UV fluorescence. The g e l was then allowed to soak i n two changes of 2 x SSC f o r a t o t a l of 40 minutes and then set to t r a n s f e r onto nylon membrane f i l t e r s (Zetaprobe; Biorad) i n a 1 x SSC t r a n s f e r b u f f e r . F i l t e r s were baked under vacuum f o r 2 hrs at 80°C. P r e h y b r i d i z a t i o n was done at 65°C, i n 1.5 x SSPE (20 x SSPE: 3.6M NaCl; 200 mM NaH 2P0 4, pH 7.4; 20 mM EDTA, pH 7.4) 1% SDS, 0.5% skim milk p r o t e i n and 0.5 mg/ml denatured salmon sperm DNA f o r at l e a s t 4 hours and h y b r i d i z e d at 65°C i n the same s o l u t i o n w i t h 10^ cpm/ml 3 2 P l a b e l l e d probe for 18-24 hrs. Upon completion of h y b r i d i z a t i o n , membranes were b r i e f l y r insed i n 2 x SSC before being washed with vigorous a g i t a t i o n at room temperature i n the f o l l o w i n g s o l u t i o n s f o r 15 35 minutes/wash: i ) 2 x SSC/0.1% SDS; i i ) 0.5 x SSC/0.1% SDS; i i i ) 0.1 x SSC/0.1% SDS. A f i n a l wash was conducted at 60-65°C i n 1% SDS/0.1 x SSC f o r up to 1 hr (12). Following t h i s , the f i l t e r was set up f o r autoradiography at -70°C f o r 1-7 days depending upon s i g n a l i n t e n s i t y . S l - a n a l y s i s (13): H y b r i d i z a t i o n s f o r S l - a n a l y s i s were conducted at 52°C i n an 80% formamide b u f f e r (80% formamide, AO mM PIPES, 0.4 M NaCl, 1 mM EDTA). T y p i c a l l y , 1 to 50 ug of t o t a l genomic RNA were taken up i n t o DNA coated c a p i l l a r y tubes along with 30-100,000 cpm of s i n g l e stranded or double stranded DNA probes i n a t o t a l volume of 10 u l . The c a p i l l a r y tube was heat sealed at both ends and the contents denatured at 85°C f o r 15 minutes i n a water bath and incubated at the required temperature f o r 12-18 hrs. The contents of each c a p i l l a r y tube were then e x p e l l e d i n t o 300 p i of S i d i g e s t b u f f e r (280 mM NaCl, 30 mM NaAc pH 4.4, 4.5 mM Zn [ A c ] 2 , 20 ug/ml denatured c a l f thymus DNA, 200 units/ml SI nuclease [Sigma]) and incubated f o r 30 minutes at 37°C. The r e a c t i o n was terminated with 75 p i of 2.5 M NH4Ac/50 mM EDTA and Ethanol p r e c i p i t a t e d with 10 ug c a r r i e r E. c o l i tRNA i n i c e c o l d 70% ethanol, d r i e d , and resuspended i n 5 p i of l o a d i n g b u f f e r 0.1% xylene cyanol, 0.1% bromphenol blue 10 mM EDTA, 95% deionized formamide). Samples were heat denatured at 90°C f o r 5 min quick cooled on i c e and loaded on a 5% acrylamide/8 M Urea mini-sequencing g e l and run at 450V. Gels were d r i e d and set up f o r autoradiography at -70°C for 1-5 days with an i n t e n s i f y i n g screen. 5) PREPARATION OF PROBES FOR SI ANALYSIS D e s c r i p t i o n of vector: The pTZ 18/19R vectors are a new generation of recombinant plasmids that combine the features of many vector systems both phage and plasmid (14). Both pTZ 18R (2860 bp) and pTZ 19R (2863 bp) d i f f e r 36 i n the o r i e n t a t i o n of t h e i r puc 18 and puc 19 derived p o l y l i n k e r regions (Figure 1). This m u l t i p l e c l o n i n g region contains over 10 unique r e s t r i c t i o n s i t e s making many c l o n i n g s t r a t e g i e s p o s s i b l e . Adjacent to the p o l y l i n k e r i s a T7 RNA polymerase promoter w i t h i n a l a c Z' gene. The T7 promoter f a c i l i t a t e s the synthesis of s i n g l e stranded RNA both i n v i t r o and i n v i v o , beginning at nucleotide 253 and proceding rightward through the m u l t i p l e c l o n i n g s i t e . Thus, the choice of e i t h e r vector or r e s t r i c t i o n endonuclease w i l l determine i f sense or anti-sense message w i l l be produced. I n s e r t i o n of any DNA of even a few hundred nuc l e o t i d e s w i l l d i s r u p t the l a c Z' gene thus a l l o w i n g f o r the s e l e c t i o n of recombinant plasmid transformed b a c t e r i a (white c o l o n i e s ) over the pare n t a l plasmid transformants (blue c o l o n i e s ) when plated on LB-plates c o n t a i n i n g X-gal, IPTG and a m p i c i l l i n (40, 150 and 50 ug/ml, r e s p e c t i v e l y ) . The plasmids a l s o have a high copy-number plasmid r e p l i c a t i o n o r i g i n , thus f a c i l i t a t i n g higher y i e l d s i n plasmid preps without any need f o r a m p l i f i c a t i o n procedures. In a d d i t i o n , the plasmids a l s o have the i n t e r g e n i c (IG) region of filamentous phage f l . 37 5' B H 3 ' s A schematic representation of the pTZ 18/19 R v e c t o r -c l o n i n g system. The main features of t h i s vector system shown above are: the filamentous phage o r i g i n of r e p l i c a t i o n ( f l ) , the pBR322 o r i g i n (ORI), the a m p i c i l l i n r e s i s t a n c e gene (AMP r) and the l a c Z' gene. Within a s t r e t c h of approximately 100 bp are encoded the m u l t i p l e c l o n i n g region, the T7 RNA polymerase promoter and the H-13 reverse primer sequence. Also shown i s the c l o n i n g strategy used ( i . e . 5'-3' o r i e n t a t i o n of sub-fragment with respect to vector) when i t i s required that a n t i -sense probes be synthesized v i a the M-13 reverse primer sequence. 38 As t h i s filamentous phage r e p l i c a t i o n o r i g i n i s v i r t u a l l y i d e n t i c a l to that of most other filamentous phages, a c e l l that harbours the pTZ plasmid vector can be used to produce s i n g l e stranded plasmid DNA by i n f e c t i o n w i t h a f u n c t i o n a l "helper" M-13 phage. Under proper s e l e c t i v e c o n d i t i o n s , the high plasmid copy number r e s u l t s i n the production of much more plasmid DNA than helper phage DNA. Another feature of the pTZ vectors i s the M-13 reverse primer sequence. With the use of commercially a v a i l a b l e complementary M-13 reverse primer oligomers, complementary strand s y n t h e s i s primed from the M-13 reverse primer s i t e can be synthesized i n v i t r o u s i n g DNA polymerase (Klenow fragment). This technology can be used both i n dideoxy sequencing of i n s e r t DNA or as a means of making high s p e c i f i c a c t i v i t y sense or antisense DNA probes. Use of pTZ vector system to generate s i n g l e stranded sense strand DNA  templates (15): Single stranded DNA templates f o r probe s y n t h e s i s were prepared by i n t r o d u c t i o n of pTZ vector containing the i n s e r t of i n t e r e s t i n t o an F + E. c o l i s t r a i n (NM522). A 5 ml b a c t e r i a l c u l t u r e was grown i n 2 x YT medium c o n t a i n i n g 50 Mg/ml a m p i c i l l i n overnight and a 1 ml a l i q u o t used to i n n o c u l a t e 50 ml of 2 x YT (with 100 ug/ml a m p i c i l l i n ) and incubated a f u r t h e r 30 minutes. Cultures were then i n f e c t e d with 100 y l of M-13K07 (a phage helper stock, with a m u l t i p l i c i t y of i n f e c t i o n of 20 pfu/ml). A f t e r 30 min incubation at 37°C kanamycin was added to 70 yg/ml, and a f t e r a f u r t h e r 14-18 hr incubation b a c t e r i a were p e l l e t e d at 17,000 rpm f o r 15 min at 4°C and phage-like-single-stranded DNA p a r t i c l e s p r e c i p i t a t e d from the cl e a r e d supernatant by a d d i t i o n of 1/4 v o l p r e c i p i t a t i o n b u f f e r (20% p o l y e t h y l e n e g l y c o l [PEG 8,000]/3.5 M NH 4Ac). This s o l u t i o n was g e n t l y i n v e r t e d a few times and stored on i c e for 30 minutes. Tubes were ce n t r i f u g e d at 17,000 rpm for 15 mins at 4°C and the p r e c i p i t a t e resuspended i n 0.2 ml of 1 x TE b u f f e r . Phage p a r t i c l e s were extracted against TNE e q u i l i b r a t e d phenol and chloroform (1:1 r a t i o ) with vigorous a g i t a t i o n 5-6 39 times u n t i l no m a t e r i a l was seen at the i n t e r f a c e . A f t e r chloroform e x t r a c t i o n s the s i n g l e stranded DNA was p r e c i p i t a t e d by a d d i t i o n of 0.1 ml 7.5 M NH^Ac and 0.6 ml 100% ethanol followed by an incubation on dry i c e f o r 15 minutes. The p r e c i p i t a t e was spun down and resuspended i n 200 p i of s t e r i l e ddh^O, and stored at -20°C. DNA y i e l d s were i n the range of 500-700 ug as determined by ^260' Clones constructed i n pTZ vectors: 5' IL-3. A 1.6 kb BamHI/Hindlll fragment encompassing -1.5 kb of 5' untranscribed sequence was cloned i n t o the BamHI/Hindlll s i t e of pTZ 19R by a standard l i g a t i o n procedure (Figure 2). This c l o n i n g o r i e n t a t i o n allows f o r i s o l a t i o n of s i n g l e stranded plasmid template of only the sense strand (with respect to the cloned fragment). When used i n an M-13 primer i n i t i a t e d s y n t h e s i s r e a c t i o n the r a d i o l a b e l e d anti-sense strand obtained can be cleaved at the BamHI s i t e , to y i e l d a smaller s i n g l e stranded fragment when denatured and run out on a sequencing g e l . In a Sl-nuclease r e a c t i o n the protected fragment i s expected to correspond to only the 104 bp encoded by exon 1. 3' I L - 3 . A 568 bp B g l l l r e s t r i c t i o n enzyme fragment encompassing most of exon 5 of the mIL-3 genomic s t r u c t u r e (455 bp) and 113 bp of 3' untranscribed sequence was cloned i n t o the BamHI s i t e of pTZ 18R w i t h the concomitant l o s s of the BamHI s i t e (Figure 2). In t h i s case s i n g l e stranded probes generated are 670 bp i n length (some plasmid sequence i s included) and an SI a n a l y s i s i s expected to give a 455 bp fragment corresponding to a l l of the 3' t r a n s c r i b e d sequence encoded by t h i s clone. GM-CSF. A BamHI/TaqI fragment (200 bp) i s o l a t e d from a nearly f u l l l ength cDNA clone of murine GM-CSF, a g i f t from Dr N. Gough of the Ludwig I n s t i t u t e , (16) was cloned i n t o the BamHI/AccI s i t e s of pTZ 19R (Figure 3). In s y n t h e s i z i n g s i n g l e stranded probes a B g l l digest generates a probe fragment of 431 bp, 231 of which i s vector sequence. Hence the protected species i n an SI a n a l y s i s i s expected to be 200 n u c l e o t i d e s (nt) long. 40 A BamHI/EcoRI fragment (720 bp) encompassing a nearly f u l l length GM-CSF cDNA was cloned i n t o the BamHI/EcoRI s i t e s i n pTZ 18R (Figure 3). Double stranded probes were generated i n a B g l l / H h a l digest which y i e l d a fragment of 386 bp; 146 bp vector sequence and 240 bp GM-CSF cDNA sequence. 41 Bam HI Hind Bgl II Bgl II 1 2 -D-LH 3 4 •Q 1.6 Kb • 104 bp J-| 568 bp ] 455 bp FIGURE 2. A schematic representation of the murine IL-3 gene and the regions sub-cloned i n t o pTZ vectors. The BamH I/Hind I I I 5' genomic fragment (-1.6 Kb) was subcloned i n t o pTZ 19R and i s expected to y i e l d a 104 bp fragment when used as a probe i n an SI a n a l y s i s . The Bgl I I fragment at the 3' end of the gene was subcloned i n t o pTZ 18R and i s expected to give a protected species of 455 bp i n a SI a n a l y s i s . 42 Bam HI Taq I Hha I 1 1 \ 200 bo 1 431 bp 386 bp FIGURE 3. A schematic representation of a nearly f u l l length (720 bp) murine GM-CSF cDNA that was cloned i n t o pTZ 18R and used to generate a double stranded 386 bp probe (shown at bottom of diagram). A BamH I/Taq I 5' cDNA sub-cloned i n t o pTZ 19R was used i n generating s i n g l e stranded probes of 431 bp. In a d d i t i o n to the 146 bp of vector sequence common to both probes (wavy l i n e ) t h i s 431 bp probe a l s o encodes an a d d i t i o n a l 85 bp of vector sequence o r i g i n a t i n g from the M-13 reverse primer sequence. The predicted fragment s i z e s of e i t h e r GM-CSF probes i s shown i n the open boxes which correspond to the t r a n s c r i b e d regions of the gene; vector sequences represented by the wavy l i n e terminate at a Bgl I s i t e w i t h i n the ve c t o r . 43 Probe s y n t h e s i s : A digramatic representation of the probe s y n t h e s i s procedure using the pTZ vector system i s provided i n Figure 4. To synthesize r a d i o - l a b e l l e d probes 1 yg of s i n g l e stranded template was f i r s t combined with 0.2 yg M-13 reverse primer, heated at 90°C f o r 5 minutes, and then annealing was allowed to take place at room temperature over 30 minutes. F o l l o w i n g t h i s , 1 u n i t of DNA polymerase and 8x10"^ mM dATP, dGTP, dTTP and 5 x l 0 ~ 4 mM dCTP, with 62.5 y moles of a 3 2P-dCTP (New England Nuclear, 800 Ci/m mole) a l l i n 70 mM T r i s (pH 7.5), 120 mM MgCl 2 500 mM NaCl were added to the M-13 annealed template mix and incubated at 37°C f o r 1 hour. The r e a c t i o n was then ethanol p r e c i p i t a t e d , resuspended i n ddH 20 and cut w i t h the appropriate r e s t r i c t i o n enzyme(s) f o r eventual i s o l a t i o n of the cloned sequence as e i t h e r s i n g l e stranded or double stranded DNA. Probes were resolved on an 8 M Urea/5% acrylamide g e l and i s o l a t e d a f t e r v i s u a l i z a t i o n on an autoradiogram by standard e l e c t r o e l u t i o n . The s p e c i f i c a c t i v i t y of the probes synthesized i n t h i s system was g e n e r a l l y about 1.0 x 10* cpm/yg. A murine c-myc genomic fragment ( H i n d l l l / S s t I ) i s o l a t e d from the clone, pM104, (18) was 5'-end l a b e l l e d at the H i n d l l l s i t e and used i n S l - a n a l y s i s of c-myc RNA l e v e l s (Figure 5). 44 D) » W M D ^ FIGURE 4. A diagramatic representation of probe synthesis using the pTZ vector system. A) Single stranded DNA template generated v i a a M-13 helper phage. B) This was annealed to DNA oligomers (primers; depicted by open boxes) complementary to the M-13 reverse primer sequence i n the vector (blackened boxes). DNA polymerase along with dATP, dTTP, dGTP, dCTP and a 3 2P-dCTP was added and incubated at 37°C f o r 1 f o r complementary strand synthesis to occur (dashed l i n e ) . C) The now double stranded DNA i s cut once downstream of the s i t e of DNA synthesis i n i t i a t i o n . D) The r e s u l t a n t m a t e r i a l i s heat denatured r e l e a s i n g the s i n g l e stranded probe m a t e r i a l of i n t r e s t (wavy dashed l i n e ) which can be run on a denaturing g e l and p u r i f i e d . 45 SSt I Hind Y -I I 1.7 Kb I I 701 bp FIGURE 5. A schematic representation of the murine c-myc gene and the 3' fragment (Sst I/Hind I I I ) used i n a Sl-nuclease a n a l y s i s . The 1.7 Kb fragment was end l a b e l l e d at the Hind I I I s i t e to y i e l d a 701 bp fragment (corresponding to most of exon 3) i n a SI a n a l y s i s . 46 6) CELL CULTURE E s t a b l i s h e d c e l l l i n e s : A-MuLV transformed mast c e l l lines. The procedure used to generate the transformed l i n e s studied and t h e i r c h a r a c t e r i z a t i o n has been described i n d e t a i l p r e v i o u s l y (22). B r i e f l y , c e l l s from i n d i v i d u a l m u l t i l i n e a g e hemopoietic c o l o n i e s of Balb/c or (C57B1/6 x C3H/HeJ) F x o r i g i n had been exposed to A-MuLV. A f t e r 2-3 months c u l t i v a t i o n on i r r a d i a t e d NIH 3T3 c e l l s , or M2-10 B4 c e l l s (a cloned marrow stromal c e l l l i n e i s o l a t e d i n t h i s l a b o r a t o r y , 8 ) , feeder-independent mast c e l l l i n e s were e s t a b l i s h e d and i n some cases cloned. A l l l i n e s were maintained i n RPMI 1640 supplemented with 20% f e t a l c a l f serum (FCS) as continuous suspension c u l t u r e s . Routinely they were s p l i t 1:5 each week which r e s u l t e d i n a lowering of the c e l l concentration to ~ 5 x l 0 4 c e l l s / m l . Conditioned media (CM) were c o l l e c t e d from spent (7 day old) c u l t u r e s , or 5 or 7 days a f t e r seeding c e l l s at 10^ c e l l s / m l i n fr e s h medium, where i n d i c a t e d . A l l CM were heated to 56°C f o r 1 hour and then stored at -20°C p r i o r to t e s t i n g f o r b i o a c t i v i t y . M210B4 ce l l s . M210B4 c e l l s were propagated by weekly s u b c u l t u r i n g at 5 x 10 A c e l l s per 25 cm 2 f l a s k i n RPMI 1640 plus 15% FCS. B6SUtA c e l l s . B6SUtA c e l l s (20) are a Friend v i r u s i n f e c t e d c u l t u r e of C57B/6 bone marrow c e l l s . They were propagated i n RPMI 1640 with 20% FCS and 5% PWM stim u l a t e d mouse spleen c e l l s (PWM-SCCM) (21). EL-4 cell s . The murine lymphoma c e l l l i n e EL-4 was c a r r i e d i n DMEM supplemented with 5% FCS. To s t i m u l a t e f a c t o r production by the EL-4 l i n e (23), these c e l l s were c u l t u r e d at 1 x 10^/ml with 10 ng/ml PMA. WEHI-3B cell s . The mouse myelomonocytic c e l l l i n e WEHI-3B (24) was c a r r i e d i n Alpha medium supplemented with 15% FCS. Primary c e l l c u l t u r e s : Spleen c e l l s . Spleen c e l l suspensions were prepared from BALB-C male mice i n 2 mis Alpha medium supplemented with 2% FCS. For pokeweed mitogen s t i m u l a t i o n , 5 x 10^ c e l l s / m l f r e s h l y prepared 47 mouse spleen c e l l s were c u l t u r e d i n Alpha medium supplemented with 10% FCS, 1 0 - 6 M 2-mercaptoethanol and 0.33% PWM (Gibco). The c u l t u r e s were incubated i n 75 cm 2 t i s s u e c u l t u r e f l a s k s f o r 48 hours at 37°C, 5% C0 2 i n a i r . 7) GROWTH FACTOR BIOASSAYS B i o a c t i v i t y assays were conducted by Dr. C. Eaves, Dr. G. K r y s t a l , Dr. F. Lemoine and Dr. P. Lansdorp i n the Terry Fox Lab. CM were tested f o r t h e i r a b i l i t y to support colony formation by progenitors from f r e s h l y suspended normal mouse marrow, or marrow from mice pretreated w i t h 150 mg/kg 5 - f l u o r o u r a c i l , or from 7-9 day o l d b l a s t c o l o n i e s generated i n primary assays of marrow from mice pretreated with 150 mg/kg 5 - f l u o r o u r a c i l . C e l l s were p l a t e d i n standard m e t h y l c e l l u l o s e assays c o n t a i n i n g 30% FCS, 1% deionize d BSA, 10 - 4M 2 mercaptoethanol, 3 units/ml of e r y t h r o p o i e t i n , CM or a c o n t r o l source of growth f a c t o r , and a-medium (25). A l l c o l o n i e s c o n t a i n i n g >20 c e l l s were scored a f t e r 2-3 weeks inc u b a t i o n and were c l a s s i f i e d i n s i t u as e r y t h r o i d (with or without other l i n e a g e s ) or pure granulocyte-macrophage. In some cases, the number of m u l t i l i n e a g e c o l o n i e s that achieved macroscopic s i z e (>10,000 c e l l s ) i s shown s e p a r a t e l y . CM were a l s o tested i n short-term -^-thymidine i n c o r p o r a t i o n assays f o r t h e i r a b i l i t y to s t i m u l a t e the p r o l i f e r a t i o n of the c e l l l i n e s 32D clone 23 (20), B13.29 (26) and H9 (27) that are responsive to s p e c i f i c growth f a c t o r s . Table 1 l i s t s the c e l l l i n e s used and the growth f a c t o r s to which each i s known to respond. 48 Table 1. I n d i c a t o r C e l l s f o r Detection of Growth Factor A c t i v i t y I n d i c a t o r C e l l s D e r i v a t i o n Stimulatory Factors Non-Stimulatory Factors 32D Clone 23 Long-term myeloid marrow c u l t u r e IL-3 GM-CSF, IL-4 B13.29 Hybridoma IL-6 (=HGF, If n 0 2 ) , IL-4 (weak) GM-CSF, I L - 1 , IL-2, IL-3, H9 Long-term pre-B "pre-B c e l l f a c t o r " c e l l marrow c u l t u r e I L - 1 , IL-2, IL-3, IL-6, GM-CSF, G-CSF, M-CSF, I f n Y 49 REFERENCES 1. B l i n N, and S t a f f o r d D.W. I s o l a t i o n of high molecular weight DNA. Nucl A c i d Res 3:2303, 1976. 2. Holmes DS, and Quigley M. A ra p i d b o i l i n g method f o r preparation of b a c t e r i a l plasmids. Anal Biochem 114:193, 1981. 3. Birnboim HC, and Dolby J . A ra p i d a l k a l i n e e x t r a c t i o n procedure f o r screening recombinant plasmid DNA. Nucl Acid Res 7:1513, 1979. 4. Southern EM. Detection of s p e c i f i c sequences among DNA fragments separated by g e l e l e c t r o p h o r e s i s . J Mol B i o l 98:503, 1975. 5. Maniatis T, F r i t s c h EF, and Sambrook J . Molecular c l o n i n g . I n : "A Laboratory Manual", Cold Spring Harbor Laboratory P u b l i c a t i o n s , New York 1982. 6. Smith GE, and Summers MD. The b i d i r e c t i o n a l t r a n s f e r of DNA and RNA to n i t r o c e l l u l o s e or diazobenzlyoxymethyl-paper. Anal Biochem 109:123, 1980. 7. Rigby PWJ, Dickmann M, Rhodes C, and Berg P. L a b e l l i n g d e o x y r i b o n u c l e i c a c i d to high s p e c i f i c a c t i v i t y i n v i t r o by n i c k t r a n s l a t i o n with DNA polymerase. J Mol B i o l 133:237, 1977. 8. McDonald MW, Simon MN, and Studier FW. A n a l y s i s of r e s t r i c t i o n fragments of T7 DNA and determination of molecular weights by e l e c t r o p h o r e s i s i n n e u t r a l and a l k a l i n e g e l s . J Mol B i o l 110:119, 1977. 9. G l i s i n V, Crkvenjakov R, and Byus C. R i b o n u c l e i c a c i d i s o l a t i o n by cesium c h l o r i d e c e n t r i f u g a t i o n . Biochemistry 13:2633, 1974. 10. Aviv H, and Leder P. P u r i f i c a t i o n of b i o l o g i c a l l y a c t i v e g l o b i n mRNA by chromatography on oligothymidine a c i d c e l l u l o s e . Proc N a t l Acad S c i USA 69:1408, 1972. 11. Davis LG, Dibner MD, and Battey JF. Basic Methods i n Molecular B i o l o g y , E l s e v i e r Science P u b l i s h i n g Co., Inc., New York, pp 143, 1986. 12. Reed KC. N u c l e i c a c i d h y b r i d i z a t i o n with DNA bound to Zeta-probe membrane. Bio Rad B u l l e t i n 1234, p 6-9, 1987. 13. Favaloro J , Treisman R, and Kamen R. T r a n s c r i p t i o n maps of polyoma v i r u s s p e c i f i c RNA: a n a l y s i s by two-dimensional nuclease SI g e l mapping, meth Enzymol 65:718, 1980. 14. Mead DA, Skorupa ES, and Kemper B. Sin g l e stranded DNA SP6 promoter plasmids f o r engineering mutant RNAs and p r o t i e n s : synthesis of a " s t r e t c h e d " preproparathyroid hormone. NAR 13:1103, 1985. 15. USB Research Center, 1985. G e n e s c r i b e - Z t m ( D e s c r i p t i o n and P r o t o c o l s ) , United States Biochemical Corporation. 50 16. Gough NM, Metcalf D, Gough J , G r a i l D, Dunn A: St r u c t u r e and expression of the mRNA fo r murine granulocyte-macrophage colony s t i m u l a t i n g f a c t o r . EMBO J 4:645, 1985 17. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, and Kir s c h n e r MW. Number and evo l u t i o n a r y conservation of a- and |3-Tubulin and cytoplasmic 3 - and y- a c t i n genes using s p e c i f i c cloned cDNA probes. C e l l 20:95, 1980. 18. Grace LC, Shen 0, Keath EJ, P i c c o l i SP, and Cole MD. Novel myc oncogene RNA from a b o r t i v e immunoglobulin gene recombination i n mouse plasmacytomas. C e l l 31:443, 1982. 19. Eaves C, Coulombel L, and Eaves A. An a l y s i s of hematopoiesis i n long term marrow c u l t u r e s . In: "Haemopoietic Stem C e l l s " , (Sv-Aa Killmann, EP Cronkite and CN Muller-Berat eds), Muskard, Copenhagen, p 287, 1983. 20. Greenberger JS, Eckner RJ, Sakakeeny M, Marks P, Reid D, Nable G, Hapel A, I h l e JN, and Humphries KC. I n t e r l e u k i n - 3 dependant hematopoietic progenitor c e l l l i n e s . Federation Proceedings 42:2762, 1983. 21. Iscove NN, R o i t s c h CA, Williams N, and G u i l b e r t L J . Molecules s t i m u l a t i n g e a r l y red c e l l , granulocyte, macrophage, and megakaryocyte precursors 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 , hydrophobicity, and charge. J C e l l P h y s i o l (Suppl) 1:65, 1982. 22. Wong PMC, Humphries RK, Chen TR, Eaves CJ. Evidence f o r a m u l t i - s t e p pathogenesis i n the generation of tumorigenic c e l l l i n e s from hemopoietic c o l o n i e s exposed to Abelson v i r u s i n v i t r o . Exp Hematol 15:280, 1987. 23. Goier PA. Studies i n antibody response of mice to tumor i n o c u l a t e s . Br J Cancer 4:372, 1950. 24. Metcalf D, Moore MA, and Warner NL. Colony formation i n v i t r o by myelomonocytic leukemia c e l l s . J N a t l Cancer Inst 43:938, 1969. 25. Eaves CJ, K r y s t a l G, Eaves AC. E r y t h r o p o i e t i c c e l l s . In: Baum SJ (ed) B i b l i o t h e c a Hematologica—Current Methodology i n Experimental Hematology, Karger, Basel p 811, 1984. 26. Lansdorp PM, Aarden LA, Ca l a f a t J , Zeiljemaker WP. A growth Factor dependent B - c e l l hybridoma. Curr Topics M i c r o b i o l Immunol 132:105, 1986. 27. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l derived pre-B c e l l growth f a c t o r a c t i v e On normal and immortalized pre-B c e l l s . Exp Hematol ( i n p r e s s ) . 51 C H A P T E R III RESULTS 1) INITIAL ASSESSMENT OF UNIFORMLY LABELLED PROBES IN DETECTING GROWTH  FACTOR GENE EXPRESSION. I n i t i a l experiments using pTZ-vector derived probes were done to v e r i f y the u t i l i t y of the probe system developed. A number of known sources of growth f a c t o r s as w e l l as s e v e r a l c e l l and t i s s u e RNAs known not to synthesize growth f a c t o r s or that have yet to be ch a r a c t e r i z e d as sources of growth f a c t o r s were surveyed. Use of uniformly l a b e l l e d double stranded probes: In assessi n g the s e n s i t i v i t y of s i n g l e and double stranded probes generated by the pTZ-vector system i n d e t e c t i n g s p e c i f i c mRNAs r e a d i l y , Sl-nuclease a n a l y s i s was performed on two known sources of murine IL-3 by using the pTZ 18R construct encoding the 3' IL-3 sequence as the probe sources (Chapter 2, f i g . 2). In the f i r s t experiment t o t a l c e l l u l a r RNA from the murine myelomonocytic c e l l l i n e WEHI-3B and pokeweed mitogen stimulated splenocytes (PWM-spleen) was h y b r i d i z e d to double stranded, pTZ generated 3' IL-3 anti-sense sequence (Figure 1). The WEHI-3B c e l l l i n e i s known to c o n s t i t u t i v e l y t r a n s c r i b e the IL-3 gene ( 1 ) . Varying amounts of t o t a l c e l l u l a r WEHI-3B RNA (l-20ug, Lanes 1-4) probed with a constant amount of 3' IL-3 probe (50,000 cpm) revealed the expected protected species of 455 bp with a threshold of s e n s i t i v i t y of approximately 5 ug RNA for a 3 day autoradiograph exposure. 52 FIGURE 1. Detection of IL-3 mRNA i n t o t a l c e l l u l a r RNA by Sl-nuclease a n a l y s i s . Lane 1, 20ug of VEHI-3B RNA; Lane 2, 15ug of WEHI-3B RNA; Lane 3, 5ug of WEHI-3B RNA; Lane A, lug of WEHI-3B RNA; Lane 5, 5ug of PVM-spleen RNA; Lane 6, 20ug of PWM-spleen RNA; Lane 7, 20ug of l i v e r RNA; Lane 8, 20ug of E . c o l i tRNA. M denotes the 4>x Hae I I I molecular weight marker. Input probe i s seen as a 627 nuc l e o t i d e (nt) fragment and the protected species as a A55 nt fragment. A l l samples were probed against 50,000 cpm of the 3'IL-3 (Bgl I I fragment) double stranded DNA probe. Exposure time was 3 days. 53 Strong IL-3 p o s i t i v e s i g n a l s were seen f o r the PWM-stimulated spleen samples, (5 ug, lane 5; 20 ug, lane 6), while no s i g n a l s were d i s c e r n a b l e i n the negative c o n t r o l s of tRNA and l i v e r RNA (20 ug/lane, lanes 8 and 7 r e s p e c t i v e l y ) . From autoradiograph band i n t e n s i t i e s mIL-3 mRNA concentrations are estimated to be 4-5 times higher i n PWM-stimulated spleen c e l l s compared to WEHI-3B c e l l s , (eg. compare lanes 3 and 5). Use of uniformly l a b e l l e d s i n g l e stranded probes: SI mapping r e s u l t s w i t h a si n g l e - s t r a n d e d DNA probe f o r the 3' region of IL-3 mRNA are presented i n Figure 2 f o r t o t a l c e l l u l a r PWM-spleen RNA over the range of 15 to 0.1 yg. In t h i s instance the protected species of 455 bp i s r e a d i l y seen with as l i t t l e as 0.1 ug RNA a f t e r only 24 hrs of autoradiography. S i n g l e stranded probes appeared to y i e l d improved s e n s i t i v i t y and l e s s background noise. Comparing the de t e c t i o n of IL-3 mRNA by Northern B l o t a n a l y s i s : Using Northern b l o t a n a l y s i s to detect IL-3 message i n WEHI-3B from both t o t a l c e l l u l a r RNA and chromatographically enriched polyadenylated RNA samples (Figure 3) f a i l e d to rev e a l a detectable s i g n a l . The same b l o t s were reprobed w i t h a chicken 0-actin cDNA fragment (2) and gave c l e a r l y d i s c e r n a b l e s i g n a l s w i t h i n 12 hrs. Repeated attempts to detect IL-3 mRNA from t h i s and other sources of IL-3 (PWM-stimulated spleen, PMA-stimulated EL-4IL.2 c e l l s ) have not yet revealed an appropriate s i g n a l . S i m i l a r l y , these b l o t s a l s o r e a d i l y r e v e a l both p-actin and c-myc mRNA species when probed a c c o r d i n g l y . These same RNA samples when analyzed v i a SI mapping using the pTZ-probes gave r e a d i l y detectable IL-3 p o s i t i v e s i g n a l s . From these observations i t was concluded that the pTZ-vector-probe system when used i n Sl-nuclease a n a l y s i s would be u s e f u l i n the d e t e c t i o n of rare mRNA species and might be superior to Northern a n a l y s i s f o r the de t e c t i o n of IL-3 mRNA. 54 1 2 3 4 5 6 i i i i i i FIGURE 2. Detection of IL-3 mRNA i n t o t a l c e l l u l a r RNA by Sl-nuclease a n a l y s i s . Lane 1, 20ug E . c o l i tRNA; Lane 2, 15ug Pwm-spleen RNA; Lane 3, lOug PVM-spleen RNA; Lane 4, lug PWM-spleen RNA; Lane 5, 0.5ug PWM-spleen RNA; Lane 6, O.lug PWM-spleen RNA. M denotes the <|>x Hae I I I molecular weight marker. Input probe i s seen as a 670 nt fragment and the protected species as a 455 nt fragment. A l l samples were probed against 50,000 cpm of 3'IL-3 (Bgl I I fragment) s i n g l e stranded DNA probe. Exposure time was 24 h r s . 55 FIGURE 3. Northern Blot a n a l y s i s for murine IL-3 and (3-actin. Lane 1, 25ug WEHI-3B t o t a l c e l l u l a r RNA; Lane 2, 25ug M210B4 t o t a l c e l l u l a r RNA; Lane 3, 5ug WEHI-3B poly A + RNA. Panel (A), probed w i t h an o l i g o l a b e l l e d 3' IL-3 (Bgl I I ) fragment. Panel (B), probed with an o l i g o l a b e l l e d 2Kb chicken 0-actin cDNA fragment. Exposure time was 9 hrs. 2) BASELINE STUDIES OF IL-3 AND GM-CSF GENE EXPRESSION IN MURINE CELLS GM-CSF expression: As an i n i t i a l test of SI a n a l y s i s f o r d e t e c t i n g murine GM-CSF expression a number of murine c e l l l i n e s and f r e s h mouse t i s s u e was analyzed (Figure 4). These included PWM stimulated splenocytes; a multipotent f a c t o r dependent c e l l l i n e , B6SUtA; the promyelocytic WEHI-3B c e l l l i n e ; the mouse T lymphoma l i n e EL4-IL-2 before and a f t e r s t i m u l a t i o n by PMA and a v a r i e t y of f i b r o b l a s t l i n e s . The l a t t e r i n c l u d e a marrow stromal l i n e , M210B4 as w e l l as m u l t i p l y passed f i b r o b l a s t c u l t u r e s from 15 day S l / S l d mouse embryos and t h e i r +/+ l i t t e r m a t e s . Samples were assayed with a pTZ generated GM-CSF cDNA probe encompassing 240 n u c l e o t i d e s (nt) of the 5' t r a n s c r i b e d sequence described e a r l i e r (Chapter 2, Figure 3). A strong band at 240 nt was r e a d i l y detected f o r the PWM-spleen RNA sample (Figure 4, lane 6) and a weak, though d i s c e r n a b l e p o s i t i v e s i g n a l detected i n the +/+ f i b r o b l a s t s . A l l other c e l l sources were negative. 57 M 1 2 3 4 5 6 7 8 M P i i i i i i i i i I i FIGURE 4. Detection of GM-CSF mRNA i n t o t a l c e l l u l a r RNA by Sl-nuclease a n a l y s i s . Lane 1, 25yg M210B4 RNA: Lane 2, 25yg +/+ bone marrow f i b r o b l a s t RNA; Lane 3, 25yg S l / S l ^ bone marrow f i b r o b l a s t RNA; Lane 4, 25ug PMA stimulated EL4IL-2 RNA; Lane 5, 25ug unstimulated EL4IL-2RNA; Lane 6, 2.4yg PWM-spleen RNA; Lane 7, 25ug B6SUtA RNA; Lane 8, 25yg WEHI-3B RNA. Each sample was probed a g a i s t 50,000cpm of a 386 bp GM-CSF cDNA double stranded fragment generated by a Hha I (cuts 40 bp 3' of the Taq I s i t e ) / B g l I (cuts 146 bp int o the pTZ vector sequence) d i g e s t of the vector construct (Chapter 2, Figure 3). P denotes the input probe fragment of 386 nt, while M denotes the <J>x Hae I I I molecular weight marker. P o s i t i v e s i g n a l s are seen by a 240 nt protected species. Exposure time was 16 hrs. 58 IL-3 expression: Probes generated from e i t h e r the 5' or 3' regions of the murine IL-3 genomic sequence (Chapter 2, Figure 2 ) , were used f o r Sl-nuclease a n a l y s i s i n surveying a number of c e l l u l a r sources f o r IL-3 expression. In Figure 5a, r e s u l t s are shown f o r an assay on normal mouse and PWM-stimulated spleen i n a d d i t i o n to the f i b r o b l a s t l i n e M210B4 and myeloid c e l l l i n e s B6SUtA and WEHI-3B. A 600 bp s i n g l e stranded 5' IL-3 probe was used (Chapter 2, Figure 2). Only the PWM-spleen and WEHI-3B samples expressed the gene, as seen by the presence of a 104 nt protected fragment. Another SI a n a l y s i s using a probe generated from 3' IL-3 clone (Chapter 2, Figure 2) i s presented i n Figure 5b. Again IL-3 mRNA was r e a d i l y detected i n PWM-spleen c e l l s but even with prolonged autoradiography no IL-3 mRNA was observed i n a number of f i b r o b l a s t l i n e s and unstimulated spleen c e l l s . In a l l analyses ( i e 5' or 3' Sl-nuclease mapping) only a s i n g l e protected probe species was observed i n d i c a t i n g no heterogeneity i n t r a n s c r i p t i n i t i a t i o n or polyadenylation. 59 FIGURE 5. Detection of IL-3 mRNA i n t o t a l c e l l u l a r RNA by Sl-nuclease a n a l y s i s . (A) Lane 1, 2.5ug PWM-spleen RNA; Lane 2, 25ug WEHI-3B RNA; Lane 3, 25ug B6SUtA RNA; Lane 4, 25ug kidney RNA; Lane 5, 25pg l i v e r RNA; Lane 6, 25pg b r a i n RNA; Lane 7, 25ug M210B4 RNA. A l l samples were probed against 50,000cpm of a 5' IL-3 s i n g l e stranded probe (Chapter 2, Figure 2). (B) Lane 1, 25pg M210B4 RNA; Lane 2, 25yg 1° c u l t u r e of adult mouse marrow, stroma 1, RNA; Lane 3, 25ug S l / S l d marrow f r i b r o b l a s t RNA; Lane 4, 25ug +/+ marrow f i b r o b l a s t RNA; Lane 5, 2.5 ug PWM-spleen RNA; Lane 6, 25pg normal spleen RNA; Lane 7, 25pg E . c o l i tRNA. A l l samples were probed against 50,000 cpm of 3'IL-3 (Bgl I I fragment) s i n g l e stranded DNA probe. P denotes the input probe of 670 n t , while M denotes the <J>x Hae I I I molecular weight marker. Exposure time was ~24 hrs. 60 3) GROWTH FACTOR ACTIVATION IN ABELSON-MuLV MYELOID TRANSFORMANTS In a previous study of growth f a c t o r s produced by the A-MuLV myeloid transformants used here ( 3 ) , i t was demonstrated that GM-CSF production was c o n s i s t e n t l y a c t i v a t e d i n these c e l l l i n e s . However, i n a d d i t i o n to GM-CSF a c t i v i t y , media conditioned by these c e l l s was shown to be capable of s t i m u l a t i n g s i n g l e and m u l t i l i n e a g e c o l o n i e s i n assays of mouse marrow c e l l s . The m u l t i l i n e a g e colony s t i m u l a t i n g a c t i v i t y present was d i f f i c u l t to e x p l a i n on the basis of GM-CSF alone, since murine GM-CSF has only minimal e r y t h r o i d and m u l t i l i n e a g e colony s t i m u l a t i n g a c t i v i t y ( 4 ) . Thus i t became a key o b j e c t i v e of t h i s study to determine the nature of t h i s m u l t i l i n e a g e colony s t i m u l a t i n g a c t i v i t y . GM-CSF and IL-3 bioassays: I n i t i a l a t t e n t i o n was focused on two w e l l e s t a b l i s h e d A-MuLV transformed mast c e l l l i n e s that had been maintained i n suspension c u l t u r e i n the absence of feeders or growth f a c t o r s f o r over a year. Both were found to be producing a m u l t i l i n e a g e CSF. When i s o l a t e d clones were obtained from these l i n e s and t h e i r i n d i v i d u a l CM tested, v a r i a t i o n i n CSF l e v e l s were demonstrable. Two clones from each of the two l i n e s were then studied i n f u r t h e r d e t a i l . Three of these (22-2B clones 4 and 6, and 19B-5A clone 5) were c o n s i s t e n t l y p o s i t i v e when t h e i r CM was tested i n m e t h y l c e l l u l o s e assays, whereas the fourth (19B-5A clone 7) has r a r e l y shown detectable a c t i v i t y i n such assays. To e s t a b l i s h whether the m u l t i l i n e a g e CSF produced by these c e l l s was a t t r i b u t a b l e to a f a c t o r d i f f e r e n t from GM-CSF, a n e u t r a l i z i n g anti-GM-CSF antiserum [ ( 5 ) , k i n d l y provided by D. Mochizuki, Immunex, S e a t t l e , Wash.J was added to m e t h y l c e l l u l o s e assays of the CM from these l i n e s . As shown i n Table 1, a concentration of t h i s antiserum that completely n e u t r a l i z e d 2 ng/ml of rGM-CSF [ ( 6 ) , k i n d l y provided by J . DeLamarter of Biogen, Boston, Mass.] had no e f f e c t on the m u l t i l i n e a g e CSF produced by A-MuLV transformed 62 mast c e l l s . These r e s u l t s suggested that production of another f a c t o r , i n a d d i t i o n to GM-CSF, had been a c t i v a t e d . Although previous experiments had f a i l e d to detect IL-3 mRNA by Northern a n a l y s i s ( 3 ) , my f i n d i n g s w i t h known IL-3 producing c e l l s suggested that t h i s might have been missed due to the i n s e n s i t i v i t y of t h i s method. Since many CM were able to s t i m u l a t e 32D clone 23 c e l l s , a c e l l l i n e that, thus f a r , has been found to respond only to IL-3 (see Table 2 below), a search for IL-3 message by S l - a n a l y s i s was i n i t i a t e d . 63 Table 1. M u l t i l i n e a g e CSF Produced by A-MuLV Transformed C e l l s A d d i t i o n No. of Colonies Produced^ Macroscopic Granulocyte/ M u l t i l i n e a g e E r y t h r o i d Macrophage None 0 0.5 0 1% PWM-SCCM2 3.0 18.5 78.0 " + 0.3% anti-GM-CSF 0.5 10.0 29.5 2 ng/ml rmGM-CSF3 0 0 55.0 " + 0.3% anti-GM-CSF 0 0 0 20% 22-2BC16 CM 1.0 4.0 91.5 " + 0.3% anti-GM-CSF 1.5 7.5 99.0 20% 19B-5AC15 CM 0 2.0 66.0 " + 0.3% anti-GM-CSF 0 1.5 53.5 Assays contained 3 x 10* B6C3Fj marrow c e l l s / 1 . 1 ml c u l t u r e . PWM-SCCM = media conditioned under serum-free c o n d i t i o n s [9] by 2 x 10*> mouse spleen c e l l s stimulated by 1% PWM f o r 5 days. rmGM-CSF = pure recombinant murine GM-CSF expressed i n E. C o l i and obtained from Biogen, Geneva, Switzerland. 64 SI Mapping of GM-CSF and IL-3 mRNA: RNA from the same four clones of A-MuLV transformed mast c e l l s ( t e s t e d i n Table 2) was analyzed. Since previous s t u d i e s had shown the expression of GM-CSF mRNA i n other Abelson MuLV l i n e s (3) we chose to f i r s t evaluate these l i n e s f o r the presence of GM-CSF mRNA, using a s i n g l e stranded uniformly l a b e l e d DNA probe encompassing 200 bp of murine GM-CSF cDNA sequence (Figure 6). Although expression was r e a d i l y d etectable, considerable clone to clone v a r i a t i o n was observed (Figure 8b). For example GM-CSF mRNA l e v e l s were c o n s i s t e n t l y higher i n l i n e 22-2B clone 4 (Figure 6, Lane 9) while not detec t a b l e i n l i n e 19B-5A clone 7 (Figure 6, Lane 8). From the r e l a t i v e i n t e n s i t i e s of the autoradiograph bands, accumulated GM-CSF mRNA i n the highest expressing l i n e was estimated at approximately 4% of that harvested from 1 day o l d c u l t u r e s of PWM-stimulated mouse spleen c e l l s ( F i g . 6, lane 3). A weak, p o s i t i v e s i g n a l was again observed i n the RNA derived from unstimulated +/+ f i b r o b l a s t s (Figure 6,Lane 4). No expression i s detectable i n the IL-3 factor-dependent myeloid l i n e B6SUtA (7) ( F i g . 6, lane 6) or WEHI-3B c e l l s . These samples were shown to contain i n t a c t RNA by Northern b l o t a n a l y s i s f o r 3 - a c t i n and/or c-myc mRNA. 65 P M 1 2 3 4 5 6 7 8 9 M i i i i i i i i i i i i FIGURE 6. Detection of GM-CSF mRNA i n t o t a l c e l l u l a r RNA by Sl-nuclease a n a l y s i s . Lane 1, normal spleen (10 ug); lane 2, E . c o l i tRNA (25 Ug); lane 3, PWM-stimulated spleen (10 ug); lanes 4 and 5, mouse bone marrow f i b r o b l a s t s (50 ug); lane 6 B6SUtA myeloid c e l l l i n e (22] (50 ug); lane 7, WEHI-3B (50 ug); lane 8, 19B-5A clone 7 (50 Ug); lane 9.22-2B clone 4 (50 ug). P denotes the input s i n g l e stranded probe of 431 nt (Chapter2, Figure3) which y i e l d s a p o s i t i v e s i g n a l of 200 nt. M denotes the <f>X Hae I I I molecular weight marker. Exposure time was -20 hrs. 66 Table 2. B i o a c t i v i t y of Conditioned Media From Log Phase Cultures of A-MuLV Transformed C e l l s . Colony Formation S t i m u l a t i o n of by Bone Marrow C e l l s 2 32D Clone 23 C e l l s 3 Macroscopic Granulocyte/ A d d i t i o n M u l t i l i n e a g e E r y t h r o i d Macrophage cpm None 0 0.5 0 118 + 18 1% PWM-SCCM 6 23 64 2500 ± 50 mGM-CSF ND ND ND 116 + 28 10% 22-2B C14 1 0.5 72 1854 ± 76 10% 22-2B C16 1 0.5 65.5 2026 ± 82 10% 19B-5A C15 0 0 0.5 198 ± 6 10% 19B-5A C17 0 2.5 9.0 1222 + 166 CM from A-MuLV transformed l i n e s was harvested 5 days a f t e r d i l u t i o n to 10 c e l l s / m l at the same time that c e l l s were harvested f o r RNA (see Figure 3). Assays contained 3 x 10 4 B6C3Fj marrow c e l l s / 1.1 ml c u l t u r e Assayed by 3H-thymidine i n c o r p o r a t i o n . 67 SI mapping with a probe from the 3' end of the IL-3 gene revealed d e t e c t a b l e l e v e l s of IL-3 mRNA i n a l l four of the l i n e s ( F i g . 7). Some clone to clone v a r i a t i o n was again apparent with 22-2B clone 4 showing the highest l e v e l of IL-3 mRNA. While l e v e l s were low they were rep r o d u c i b l e and d i f f e r e n c e s i n IL-3 mRNA l e v e l s i n the 4 l i n e s were seen c o n s i s t e n t l y when RNA's were i s o l a t e d on d i f f e r e n t occasions over a period of a month (see F i g . 7a and 7b). Accumulated IL-3 mRNA l e v e l s were comparable to those i n an EL-4 s u b l i n e stimulated with PMA ( F i g . 7a, lane 2), were approximately 20% those measured f o r WEHI-3B c e l l s ( F i g . 7a, lane 4 ) , and were IX of those measured f o r PWM-stimulated splenocytes ( F i g . 7a, lane 3). IL-3 mRNA was al s o d e t e c t a b l e using a probe for the 5' region of the IL-3 t r a n s c r i p t (data not shown). Protected probe lengths were as predicted f o r c o r r e c t l y i n i t i a t e d and polyadenylated IL-3 mRNA. 68 FIGURE 7. Detection of murine IL-3 mRNA i n t o t a l c e l l u l a r RNA by S l - a n a l y s i s using a s i n g l e stranded probe f o r the 3' end of the IL-3 gene, a) Lane 1, E . c o l i tRNA (25 ug); lane 2, PMA-stimulated EL-4 (25 ug); lane 3, PWM-stimulated spleen RNA (5 Ug); lane 4, VEHI-3B (5 ug); lane 5, B6SUtA (25 ug); lane 6, 22-2B clone 6 (25 ug); lane 7, 22-2B clone 4 (25 ug); lane 8, 19B-5A clone 7 (25 ug); lane 9, 19B-5A clone 5 (25 ug). b) A n a l y s i s of RNA i s o l a t e d at a second time (3 weeks l a t e r ) . Lane 1, 22-2B clone 4 (25 ug); lane 2, 19B-5A clone 7 (25 ug); lane 3, 19B-5A clone 5 (25 ug); lane 4, B6SUtA (25 ug); lane 5, PWM-stimulated spleen (5 ug). P denotes the input probe and M the $X Hae I I I marker. Exposure time was ~24 hrs. 69 V a r i a t i o n i n GM-CSF and IL-3 gene expression: To examine the k i n e t i c s of growth f a c t o r mRNA accumulation, c e l l s were d i l u t e d to 10 4 c e l l s / m l and then RNA i s o l a t e d a f t e r 5 days while the c e l l s were s t i l l i n a l o g a r i t h m i c growth phase, and again a f t e r 7 days when the c e l l s had reached t h e i r maximal c e l l d e n s i t y . For a l l 3 p o s i t i v e l i n e s of the 4 tes t e d , l e v e l s of GM-CSF mRNA appeared to be s i m i l a r at both times examined ( F i g . 8b). GM-CSF mRNA remained undetectable i n l i n e 19B-5A clone 7 ( F i g . 8b, lanes 7 and 8). In c o n t r a s t , IL-3 t r a n s c r i p t s showed a higher accumulated l e v e l i n l o g phase as opposed to plateau phase c e l l s i n each of the same 4 l i n e s ( F i g . 8a). C-myc mRNA l e v e l s , l i k e IL-3 mRNA l e v e l s , were sharply decreased i n plateau phase c e l l s ( F i g . 8c). CM from the l o g phase c u l t u r e s harvested f o r RNA stu d i e s were saved and tested by Dr. Connie Eaves and Dr. Gerald K r y s t a l f o r b i o a c t i v i t y on normal marrow progenitors and on 32D c e l l s r e s p e c t i v e l y . The r e s u l t s , shown i n Table 2, confirm the production of detectable colony s t i m u l a t i n g a c t i v i t y (CSA), i n c l u d i n g IL-3, by a l l 4 l i n e s i n t h i s experiment. Higher l e v e l s of CSA are observed i n CM from l i n e s 22-2B clone 4 and 6, while the lowest l e v e l i s seen i n the CM from l i n e 19B-5A clone 5. These r e s u l t s can be seen to approximate the mRNA l e v e l s i n the d i f f e r e n t A-MuLV clones (Figure 8a) as judged by t h e i r r e l a t i v e s i g n a l i n t e n s i t i e s (455 nt band). 70 FIGURE 8. Growth f a c t o r gene expression i n transformants at d i f f e r e n t times i n c u l t u r e . T o t a l c e l l u l a r RNA was i s o l a t e d from c u l t u r e s i n l o g phase (day 5) or at s a t u r a t i o n (day 7) and assayed by SI a n a l y s i s as f o r IL-3 (panel a ) , GM-CSF (panel b) or c-myc (panel c) mRNA. a. IL-3 mRNA l e v e l s . Lanes 1,3,5 and 7 are day 5 RNA i s o l a t e s (20 ug/lane) of 19B-5A, Clone 5, 19B-5A Clone 7, 22-2B Clone 4, and 22-2B Clone 6, r e s p e c t i v e l y . Lanes 2,4,6 and 8 are day 7 RNA samples (20 ug/lane) i n the same order; Lane 9, 2.5 yg of PWM-stimulated spleen RNA; Lane 10, 5 yg WEHI-3B RNA; Lane 11, 25 yg of normal unstimulated spleen RNA. P denotes input probe; M denotes <J>X Hae I I I marker lanes. The probe was as described i n F i g . 2. b. GM-CSF mRNA l e v e l s . Lanes 1,3,5 and 7 are day 5 RNA i s o l a t e s (20 yg/lane) of 19B-5A, Clone 5, 19B-5A Clone 7, 22-2B Clone 4, and 22-2B Clone 6, r e s p e c t i v e l y . Lanes 2,4,6 and 8 are day 7 RNA samples (20 yg/lane) i n the same order. Lane 9, 2.5 yg of PWM-stimulated spleen RNA. Lane 10, 25 yg of normal unstimulated spleen RNA. M denotes <j>X Hae I I I molecular weight markers. The probe was as described i n F i g . 6. c. c-myc RNA l e v e l s . Lanes 1,3,5 and 7 are day 5 RNA i s o l a t e s (20 yg/lane) of 19B-5A Clone 5, 19B-5A Clone 7, 22-2B Clone 4 and 22-2B Clone 6, r e s p e c t i v e l y ; Lanes 2,4,6, and 8 are day 7 RNA samples (20 yg/lane) i n the same order; Lane 9, 20 yg B6SUtA RNA; Lane 10, 20 yg WEHI-3B RNA; Lane 11, 25 yg E . c o l i tRNA. P denotes the input probe; M denotes <j>X Hae I I I molecular weight marker lanes. A 3 2P-end l a b e l l e d probe as described i n Chapter 2, Figure 5 was used. 71 7 8 M 9 10 11 7 8 M 9 10 7 8 9 10 M 11 *_1700 nt. —701 nt. 72 GM-CSF and IL-3 genomic s t r u c t u r e : Southern b l o t a n a l y s i s of the A-MuLV tranformants and c o n t r o l spleen DNA from the same s t r a i n revealed no gross rearrangement i n e i t h e r GM-CSF or IL-3 genes associated with t h e i r increased mRNA l e v e l s ( F i g . 9a and 9b). R e s t r i c t i o n enzymes BamHI and Hind I I I were used i n a n a l y z i n g the GM-CSF genomic s t r u c t u r e as these enzymes f l a n k the gene g i v i n g r e s t r i c t i o n fragments of 9.0 and 5.6 Kb r e s p e c t i v e l y when probed with a 720 bp GM-CSF cDNA fragment. Using an EcoRI digest the IL-3 gene s t r u c t u r e was probed with a Bgl I I fragment (encodes most of exon 5 of the mIL-3 gene) to give a fragment s i z e of 8.7 Kb as the enzyme cuts once 5.3 Kb 5' of exon 1 and at another s i t e 1.3 Kb 3' of exon 5. Using the same probe the region 3' of the IL-3 gene was f u r t h e r i n v e s t i g a t e d using a Hind I I I di g e s t which cuts w i t h exon 1 as w a l l as 4.8 Kb 3'of exon 5 to give an expected fragment s i z e of 6.5 Kb. As these A-MuLV l i n e s were derived from Balb/c mice, normal spleen DNA was ext r a c t e d from male Balb/c mice to serve as a c o n t r o l . 73 A kb 1 2 3 4 5 6 i i i i i i 9.0 5.6 B kb 8.7 6.5 1 2 3 4 5 6 i i i i i i FIGURE 9. Southern b l o t a n a l y s i s of A-MuLV transformants 22-2B clone 4 and clone 6 and Balb/c spleen DNA for GM-CSF (panel A) and IL-3 (panel B) sequences. 22-2B clone 4, lanes 1 and 4; 22-2B clone 6, lanes 2 and 5; Balb/c, lanes 3 and 6. A l l lanes c o n t a i n 5 ug t o t a l c e l l u l a r DNA. A. GM-CSF: DNA samples were digested with BamH I (lanes 1-3) or Hind I I I (lanes 4-6) as these r e s t r i c t i o n s i t e s flank, the GM-CSF gene to give expected fragment s i z e s of 9.0 and 5.6 Kb, r e s p e c t i v e l y . The probe used was an o l i g o - l a b e l l e d 720 bp GM-CSF cDNA fragment spanning most of the t r a n s l a t e d sequence and some 3' u n t r a n s l a t e d sequence. B. IL-3: DNA samples i n lanes 1-3 were digested with EcoR I which f l a n k s the IL-3 gene, 5.3 Kb 5' of exon 1 and 1.3 Kb 3' of exon 5 to give an expected fragment s i z e of 8.7 Kb using an o l i g o -l a b e l l e d Bgl I I fragment spanning most of exon 5 of the IL-3 gene. In lanes 4-6 the DNA samples were digested w i t h Hind I I I which cuts w i t h i n exon 1 and a l s o 4.8 Kb 3' of exon 5 to g i v e an expected fragment of 6.5 Kb using the same Bgl I I genomic IL-3 fragment. 74 Non-IL-3 and GM-CSF growth f a c t o r a c t i v i t i e s : At the time t h i s study was undertaken the only murine hemopoietic molecular probes a v a i l a b l e were those of GM-CSF and IL-3. Consequently, to explore the p o s s i b i l i t y of a d d i t i o n a l growth f a c t o r a c t i v a t i o n i n these A-MuLV transformed c e l l s , i t was necessary to use growth f a c t o r bioassays (Chapter 2, Table 1). Hybridoma growth f a c t o r , now known as IL-6 or I n t e r f e r o n 02 ( 8 ) , was assayed on the mouse hybridoma l i n e B13.29 ( 9 ) . This l i n e i s not known to be responsive to any other hemopoietic growth f a c t o r , although i t has r e c e n t l y been discovered that i t can be weakly stimulated by murine IL-4 at concentrations >500 units/ml (Dr. Peter Lansdorp, Terry Fox Laboratory). B13.29 c e l l s t i m u l a t o r y a c t i v i t y was c l e a r l y detectable i n CM from a l l 4 l i n e s , although again v a r i a t i o n between l i n e s was seen (Table 3). As f u r t h e r shown i n Table 3, CM from a l l 4 l i n e s were a l s o able to s t i m u l a t e H9 c e l l s , a spontaneously immortalized cloned pre-B c e l l l i n e i s o l a t e d i n the Terry Fox Laboratory. This l i n e can be maintained as a suspension c u l t u r e at high c e l l concentrations but becomes feeder-dependent (e.g. on M2-10B4 c e l l s or a f a c t o r produced by M2-10B4 c e l l s ) when d i l u t e d below 3000 c e l l s / m l . H9 c e l l s do not respond to any other known hemopoietic growth f a c t o r . P r e l i m i n a r y biochemical c h a r a c t e r i z a t i o n of the H9 s t i m u l a t i n g f a c t o r produced by M2-10B4 c e l l s i n d i c a t e s that i t may be a new pre-B c e l l growth f a c t o r (10). CM from 22-2B clone 4 c e l l s was f u r t h e r tested f o r IL-4 b i o a c t i v i t y but found to be negative (F. Lee, DNAX, personal communication). 75 Table 3. IL-6 and Pre-B C e l l Growth Factor A c t i v i t y Produced by Abelson Transformed C e l l s Conditioned medium IL-6 A c t i v i t y 1 Pre-B C e l l 2 (units/ml) Stimulatory A c t i v i t y (cpm) c o n t r o l media <3 533 + 51 22-2B Clone 4 2000 1339 + 141 22-2B Clone 6 5000 849 ± 52 19B-5A Clone 5 30 1388 ± 88 19B-5A Clone 7 30 1410 ± 201 1 Assayed on B13.29 c e l l s . One un i t of a c t i v i t y i s defined as that g i v i n g 50% maximal s t i m u l a t i o n . 1 2 Assayed by s t i m u l a t i o n of 3H-thymidine i n c o r p o r a t i o n i n t o H9 pre-B c e l l s when CM present at 10% f i n a l concentration. 76 REFERENCES 1. Ymer S, Tucker WQJ, Sanderson CJ, Hapel AJ, Campbell HD & Young IG. C o n s t i t u t i v e synthesis of i n t e r l e u k i n - 3 by leukemia c e l l l i n e WEHI-3B i s due to r e t r o v i r a l i n s e r t i o n near the gene. Nature 371:255, 1985. 2. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, K i r s c h n e r MW. Number and e v o l u t i o n a r y conservation of a-and 3 - t u b u l i n and cytoplasmic 3-and y- a c t i n genes using s p e c i f i c cloned cDNA probes. C e l l 20:95, 1980. 3. Chung SW, Wong PMC, Shen-Ong G, R u s c e t t i S, I s h i z a k a T, Eaves CJ. Production of granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r by Abelson virus-induced tumorigenic mast c e l l l i n e s . Blood 68:1074, 1986. 4. Metcalf D, Burgess AW, Johnson GR, N i c o l a NA, Nice EC, DeLamarter J , Thatcher DR, Mermod J J . In v i t r o a c t i o n s on hemopoietic c e l l s of recombinant murine GM-CSF p u r i f i e d a f t e r production i n E s c h e r i c h i a c o l i : Comparison with p u r i f i e d n a t i v e GM-CSF. J C e l l P h y s i o l 128:421, 1986. 5. Muchizuki DY, Eisenman JR, Conlon PJ, Park LS, Urd a l , DL. Development and c h a r a c t e r i z a t i o n of antiserum to murine granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r . J Immunol 136:3706, 1986. 6. DeLamarter JF, Mermod J J , Liang CM, E l i a s o n JF, Thatcher DR. Recombinant murine GM-CSF from E. c o l i has b i o l o g i c a l a c t i v i t y and i s n e u t r a l i z e d by a s p e c i f i c antiserum. EMBO J 10:2575, 1985. 7. Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ. Demonstration of permanent f a c t o r dependent m u l t i p o t e n t i a l ( e r y t h r o i d / n e u t r o p h i l / b a s o p h i l ) hematopoietic progenitor c e l l l i n e s . Proc N a t l Acad S c i USA 80:2931, 1983. 8. Brakenhoff J P J , De Groot ER, Evers RF, Pannekoek H, Aarden LA. Molecular c l o n i n g and expression of hybridoma growth f a c t o r i n E s c h e r i c h c i a c o l i . J Immunol 139:4116, 1987. 9. Lansdorp PM, Aarden LA, C a l a f a t J , Zeiljemaker WP. A growth-factor dependent B - c e l l hybridoma. Curr Topics M i c r o b i o l Immunol 132:105, 1986. 10. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . Exp Hematol ( i n p r e s s ) . 77 C H A P T E R IV DISCUSSION & CONCLUSIONS The p l e t h o r a of growth f a c t o r s known to a f f e c t hemopoiesis o f t e n show overlap i n t h e i r i n v i t r o b i o l o g i c a l a c t i v i t i e s making i t d i f f i c u l t to use the assay to uniquely i d e n t i f y a p a r t i c u l a r growth f a c t o r . This i s f u r t h e r compounded by the fa c t that many responder c e l l l i n e s used i n bio-assays can be acted upon by more than one growth f a c t o r . In a d d i t i o n low l e v e l s of growth f a c t o r gene expression may go undetected i n a conventional bioassay. For these reasons I have used a s e n s i t i v e molecular h y b r i d i z a t i o n technique f o r the d e t e c t i o n of mRNA, Sl-mapping. This technique made use of high s p e c i f i c a c t i v i t y (>4xl0 8 cpm/yg) DNA probes generated v i a the pTZ vector system. This vector system enabled the r e l a t i v e l y easy generation of uniformly l a b e l l e d double or s i n g l e stranded probes through a s i n g l e stranded sense-strand intermediate. P r e l i m i n a r y assesment of various murine c e l l types has confirmed the s p e c i f i c i t y (eg IL-3 p o s i t i v e r e s u l t only f o r PWM-spleen and WEHI-3B c e l l s ; Chapter I I I , Figure 5a) and s e n s i t i v i t y (the d e t e c t i o n of IL-3 mRNA i n as l i t t l e as 0.1 ug of t o t a l c e l l u l a r RNA PWM-spleen c e l l s : Chapter I I I , Figure 2; and the d e t e c t i o n of GM-CSF mRNA i n a h i t h e r t o uncharacterized f e t a l f i b r o b l a s t c u l t u r e : Chapter I T I , Figure 4, Lane 2 and Figure 6, Lane 3) of t h i s technique. While not an exhaustive study, repeated attempts to detect growth f a c t o r gene expression (mIL-3) by the Northern b l o t technique were c o n s i s t e n t l y negative even though s i m i l a r or s u b s t a n t i a l l y higher concentrations of RNA were used. C e r t a i n r e l a t i v e l y abundant mRNA species such as c-myc or 3-actin ( -105 c o p i e s / c e l l ) were e a s i l y detected using the same b l o t s . 78 The major goal of t h i s p r o j e c t was to examine the p o s s i b l e a s s o c i a t i o n of growth f a c t o r gene a c t i v a t i o n i n the A-MuLV induced myeloid transformants. Previous s t u d i e s i n d i c a t e d that s i m i l a r l y d erived l i n e s produced GM-CSF as w e l l as a m u l t i l i n e a g e colony s t i m u l a t i n g a c t i v i t y , although no IL-3 mRNA could be demonstrated i n these c e l l s using Northern a n a l y s i s ( 1 ) . Using a s e n s i t i v e Sl-nuclease mapping technique I have now been able to show that IL-3 i s i n fact c o n s t i t u t i v e l y produced by a l l four of the transformants studied to date i n t h i s way. In a d d i t i o n , i t was demonstrated that GM-CSF and IL-3 are not the only hemopoietic growth f a c t o r genes whose expression i s a c t i v a t e d i n these transformed c e l l s . Other f a c t o r s i n c l u d e IL-6 (HGF) which has a l s o r e c e n t l y been shown to act as a weak. CSF on granulocyte-macrophage progenitors and to synergize w i t h IL-3 i n s t i m u l a t i n g very p r i m i t i v e multipotent progenitors ( 2 ) , and a newly described and d i s t i n c t pre-B c e l l s t i m u l a t o r y f a c t o r ( 3 ) . Thus, expression of at l e a s t A separate growth f a c t o r genes appears to be a common occurrence during the e v o l u t i o n of malignant mast c e l l l i n e s from A-MuLV i n f e c t e d precursors. Southern b l o t a n a l y s i s revealed no gross gene rearrangements e i t h e r 5' or 3' of e i t h e r the IL-3 or GM-CSF genomic sequences that might account f o r the t r a n s c r i p t i o n a l up-regulation of these genes. This r e s u l t i n i t s e l f does not however preclude the p o s s i b i l i t y of point mutations w i t h i n a gene or f o r that matter gross chromosomal a l t e r a t i o n l y i n g beyond the scope of the region v i s u a l i z e d by the probe. However, i t seems more l i k e l y that the pa n - a c t i v a t i o n phenomenon observed r e f l e c t s the operation of a more general mechanism, perhaps i n v o l v i n g t r a n s - a c t i v a t i n g f a c t o r s . This i s a p a r t i c u l a r l y a t t r a c t i v e p o s s i b i l i t y i n the case of the GM-CSF and IL-3 genes s i n c e these are very c l o s e l y l i n k e d on chromosome 11 and share c e r t a i n consensus sequences i n both t h e i r 5' and 3' noncoding regions (4). Recent evidence of t h e i r co-ordinate expression i n l e c t i n , but not IL-2 s t i m u l a t e d 79 T lymphocytes i s a l s o consistent with sharing of at l e a s t one amongst s e v e r a l c o n t r o l mechanisms (5). On the other hand, the murine IL-6 gene i s u n l i k e l y to be l i n k e d to the GM-CSF or IL-3 genes si n c e i n man i t i s lo c a t e d on a d i f f e r e n t chromosome (6). The gene encoding the pre-B c e l l growth f a c t o r to which H9 c e l l s respond has not yet been i s o l a t e d . I t i s a l s o p o s s i b l e that some or even a l l of the e f f e c t s on growth f a c t o r production w i l l prove to be secondary to other A-MuLV induced p e r t u r b a t i o n s of the metabolic a c t i v i t i e s of mast c e l l s . In the past, observations that A-MuLV derived mast c e l l transformants could circumvent a previous requirement f o r exogenous IL-3 i n the absence of det e c t a b l e growth f a c t o r gene expression (7,8) suggested a non-autocrine mechanism. In an analogous study, O l i f f et a l (9) demonstrated that an e x i s t i n g Fr-MuLV immortalized c e l l l i n e became both independent of exogenous IL-3 and tumorigenic when i n f e c t e d with A-MuLV, again without evidence of det e c t a b l e a c t i v a t i o n of growth f a c t o r gene expression. In c o n t r a s t , Schrader et a l (10), noted prolonged IL-3 s t i m u l a t i o n of RX-6 c e l l s , another IL-3 dependent mast c e l l l i n e , r e s u l t e d i n the e v o l u t i o n of a s u b l i n e that could grow at high c e l l concentrations i n the absence of exogenous IL-3. These c e l l s were s t i l l responsive to IL-3, but had acquired the a b i l i t y to produce IL-3 suggesting a c t i v a t i o n of an autocrine mechanism i n t h i s s u b l i n e . More r e c e n t l y , frequent production of IL-4 and o c c a s i o n a l l y IL-3 a l s o were reported i n some spontaneous A-MuLV transformed mast c e l l l i n e s generated under d i f f e r e n t conditions (11). This apparent spectrum of growth f a c t o r gene behavior i n various transformed mast c e l l s may i n part r e f l e c t the d i f f e r e n t s e n s i t i v i t i e s of the assays used to detect e i t h e r b i o a c t i v i t y or messenger RNA production although there are a l s o s i g n i f i c a n t v a r i a t i o n s i n the l e v e l s of message and gene product detected i n d i f f e r e n t s u b l i n e s even using the same techniques as shown here. A l t e r n a t i v e l y true heterogeneity i n gene a c t i v a t i o n may r e f l e c t m u l t i p l e transformation 80 mechanisms and c e l l t a r g e t s . Nevertheless, the frequency of growth f a c t o r a c t i v a t i o n i n A-MuLV transformed myeloid c e l l s that we have now documented suggests that t h i s i s a more common event than p r e v i o u s l y a n t i c i p a t e d . Whether or how many of the f a c t o r s a c t i v a t e d play a r o l e i n the malignant progression of A-MuLV transformed mast c e l l s i s not known. The p o s s i b i l i t y that an autocrine growth mechanism i s involved r e s u l t i n g from the production of one or more of these f a c t o r s has not yet been i n v e s t i g a t e d . One approach to answering t h i s question would be to use an t i b o d i e s d i r e c t e d against these f a c t o r s i n hope of i n h i b i t i n g the p r o l i f e r a t i o n of these A-MuLV transformants i n v i t r o . The f i n d i n g s presented here have i m p l i c a t i o n s f o r the deranged autosynthesis of CSFs r e c e n t l y observed i n some human leukemias. Of p a r t i c u l a r i n t e r e s t i s the example i n the A-MuLV transformants of the combined expression of the IL-3 and IL-6 genes. Together the products of these two genes can have an important s y n e r g i s t i c e f f e c t on very p r i m i t i v e multipotent c e l l s (2) and may thus be able to s t i m u l a t e c e l l s that otherwise would remain quiescent. C o n s t i t u t i v e production of one or more growth f a c t o r genes i n primary human leukemic c e l l s (12,13,14) has sparked an increased i n t e r e s t i n t h i s area. The p o s s i b i l i t y that the v-a b l oncogene may be a cause of the pa n - a c t i v a t i o n of c e r t a i n hemopoietic growth f a c t o r s i s t h e r e f o r e i n t r i g u i n g . More extensive i n v e s t i g a t i o n s of the mouse model used i n t h i s study could help to e l u c i d a t e the mechanisms inv o l v e d . 81 REFERENCES 1. Chung SW, Wong PMC, Shen-Ong G, R u s c e t t i S, I s h i z a k a T, Eaves CJ. Production of granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r by Abelson virus-induced tumorigenic mast c e l l l i n e s . Blood 68:1074, 1986. 2. Ikebuchi K, Wong GG, Clark SC, I h l e JN, H i r a i Y, Ogawa M. I n t e r l e u k i n -6 enhancement of interleukin-3-dependent p r o l i f e r a t i o n of m u l t i p o t e n t i a l hemopoietic progenitors. Pro N a t l Acad S c i USA 84:9035, 1987. 3. Lemoine FM, Humphries RK, Abraham SDM, K r y s t a l G, Eaves CJ. P a r t i a l c h a r a c t e r i z a t i o n of a novel stromal c e l l - d e r i v e d pre-B c e l l growth f a c t o r a c t i v e on normal and immortalized pre-B c e l l s . Exp Hematol ( i n p r e s s ) . 4. Barlow DP, Bucan M, Lehrach H, Hogan BLM, Gough N. Close genetic and p h y s i c a l l i n k a g e between the murine haemopoietic growth f a c t o r genes GM-CSF and multi-CSF ( I L - 3 ) . EMBO J 6:617, 1987. 5. Kelso A, Metcalf D, Gough NM. Independent r e g u l a t i o n of granulocyte-macrophage colony s t i m u l a t i n g f a c t o r and m u l t i l i n e a g e colony-s t i m u l a t i n g f a c t o r production i n T lymphocyte clones. J Immunol 136:1718, 1986. 6. Sehgal PB, Z i l b e r s t e i n A, Ruggieri R-M, May LT, Ferguson-Smith A, S l a t e DL, Revel M, Ruddle F. Human chromosome 7 c a r r i e s the $2 i n t e r f e r o n gene. Proc N a t l Acad S c i USA 83:5219, 1986. 7. P i e r c e JH, D i F i o r e PP, Aaronson SA, Potter M, Pumphrey J , Scott A, I h l e JN. N e o p l a s t i c transformation of mast c e l l s by Abelson-MuLV: abrogation of IL-3 dependence by a nonautocrine mechanism. C e l l 41:685, 1985. 8. Cook WD, Metcalf D, N i c o l a NA, Burgess AW, Walker F. Malignant transformation of a growth factor-dependent myeloid c e l l l i n e by Abelson v i r u s without evidence of an autocrine mechanism. C e l l 41:677, 1985. 9. O l i f f A, Agranovsky 0, McKinney MD, Marty WVS, Bauchwitz R. Friend murine leukemia v i r u s immortalized myeloid c e l l s are converted i n t o tumorigenic c e l l l i n e s by Abelson leukemia v i r u s . Proc N a t l Acad S c i USA 82:3306, 1985. 10. Schrader JW, Crapper RM. Autogenous production as a mechanism f o r transformation of bone marrow-derived c e l l s . Proc N a t l Acad S c i USA 80:6892, 1983. 11. Brown MA, Pi e r c e JH, Watson CJ, Falco J , I h l e JN, Paul WE. B c e l l s t i m u l a t o r y f a c t o r - l / I n t e r l e u k i n - 4 mRNA i s expressed by normal and transformed mast c e l l s . C e l l 50:809, 1987. 12. Young DC, G r i f f i n JD. Autocrine s e c r e t i o n of GM-CSF i n acute m y e l o b l a s t i c leukemia. Blood 68:178, 1986. 13. G r i f f i n JD, Rambaldi A, Vellenga E, Young DC, Ostapovicz D, Ca n n i s t r a SA. S e c r e t i o n of i n t e r l e u k i n - 1 by acute m y e l o b l a s t i c leukemia c e l l s i n 82 v i t r o induces e n d o t h e l i a l c e l l s to secrete colony s t i m u l a t o r y f a c t o r s . Blood 70:1218, 1987. Cheng GYM, K e l l e h e r CA, Miyauchi J , Wang C, Wong G, C l a r k SC, McCulloch EA, Minden MD. Structure and expression of gene of GM-CSF and G-CSF i n b l a s t c e l l s from p a t i e n t s with acute m y e l o b l a s t i c leukemia. Blood 71:204, 1988 

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