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Studies into the mechanism of action of interleukin 3 : purification of interleukin 3 and characterization… Murthy, Sudish C. 1989

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STUDIES INTO THE MECHANISM OF ACTION OF INTERLEUKIN 3: PURIFICATION OF INTERLEUKIN 3 AND CHARACTERIZATION OF ITS C E L L SURFACE RECEPTOR by SUDISH C. MURTHY B.Sc, The University of California at Los Angeles, 1983 M.Sc, The University of British Columbia, 1987 A THESIS SUBMITTED EN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1989 ©Sudish Murthy, 1989 In p resen t i ng this thesis in partial fu l f i lment of the requ i remen ts fo r an a d v a n c e d d e g r e e at the Univers i ty of Brit ish C o l u m b i a , I agree that t he Library shal l m a k e it f reely avai lable fo r re fe rence and s tudy. I fur ther agree that p e r m i s s i o n fo r ex tens ive c o p y i n g of this thesis fo r scholar ly p u r p o s e s may be g ran ted by the h e a d o f m y depa r tmen t o r by his o r her representa t ives . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t i on o f this thesis for f inancial ga in shall no t b e a l l o w e d w i t hou t m y wr i t ten p e r m i s s i o n . D e p a r t m e n t T h e Un ivers i ty of Brit ish C o l u m b i a V a n c o u v e r , C a n a d a Da te ?s /fr? DE-6 (2/88) in p resen t i ng this thesis 'm part ial fu l f i lment o i the fgquif-cfr.er.ti for an a d v a n c e d d e g r e e at the? Univers i ty of Brit ish C o l u m b i a . I agree that the Library shal l m a k e it freely avai lable for re ference and s tudy. I further agree that p e r m i s s i o n for ex tens i ve c o p y i n g of this thesis for scho lar ly p u r p o s e s may b e g ran ted by the h e a d of m y depa r tmen t or by 'vs . o? her representa t i ves , H \r, u n d e r s t o o d that c o p y i n g o r p i ' b i i r a l i o n of this thesis for f inancia l gain shall no? be & i i c w e c w i t hou t m y wr i t t en p§rn -! lss?on. T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r , C a n a d a ii ABSTRACT Hemopoiesis is regulated, in part, by a group of potent, soluble hemopoietic growth factors. One of these growth factors, interleukin 3, stimulates the proliferation and differentiation of both multi-potential hemopoietic stem cells and maturing committed progenitors, and may thus play a central role in regulating hemopoiesis in vivo. Studies of the mechanism of action of this hemopoietic growth factor may therefore yield valuable insights into normal hemopoietic differentiation and provide an understanding of the pathogenesis of hemopoietic malignancies. In order to begin these studies, it was first necessary to devise a simple high yield purification procedure for murine interleukin 3, since published purification protocols were lengthy and resulted in unacceptably low yields. A simple and rapid 3-step procedure was therefore devised which greatly facilitated our studies. The novel aspect of this procedure involved the treatment of B6SUtA^ cells with 0.01% glutaraldehyde which transformed them into mechanically resistant spheres. This made it possible to use these high interleukin 3 receptor-expressing cells as a solid phase reagent suitable for the large scale purification of interleukin 3. Purification, using these fixed cells and two standard purification steps, resulted in a 16,000-fold enrichment and purification to apparent homogeneity of interleukin 3 from serum-free pokeweed mitogen stimulated spleen cell conditioned medium. The effects of pure interleukin 3 on interleukin 3 receptor internalization and re-expression and the relationship between interleukin 3 receptor surface density and growth factor sensitivity were then examined. From these studies, it was demonstrated that surface bound interleukin 3 is rapidly internalized and cleaved at two specific sites, before being completely digested within lysosomes. However, unlike its ligand, the interleukin 3 receptor appears to be recycled to the cell surface without proteolytic cleavage. Furthermore, receptor re-expression is critically dependent upon the extracellular concentration of interleukin 3. More importantly, in the absence of interleukin 3, target cells express extremely high levels of surface receptor and become exquisitely sensitive to interleukin 3. iii Finaly, the structure of the interleukin 3 receptor was examined. Previous atempts to characterize the precise nature of the interleukin 3 receptor have proven particularly dificult as several diferent molecular weight estimates for the receptor have ben forwarded. Through the use of a variety of chemical croslinking agents, this controversy apears to have ben resolved. A model consistent with the data generated sugests that the interleukin 3 receptor is a 140 kd membrane glycoprotein that, upon interleukin 3 binding and chemical croslinking, becomes proteolyticaly cleaved to a 70 kd surface protein. i iv TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS ix Deoii'eaV'O'N CHAPTER I INTRODUCTION 1.1 Principles of Hemopoiesis 1 1.2 Control of Hemopoiesis • 6 1.3 Mechanism of Action of Growth Factors 36 1.4 Interactions Between Hemopoietic Growth Factors 54 1.5 Role of Hemopoietic Growth Factors in Oncogenesis 54 CHAPTER II A SIMPLE AND RAPID 3-STEP PROCEDURE FOR THE PURIFICATION OF MURINE INTERLEUKIN 3 2.1 Introduction 85 2.2 Materials and Methods 87 2.3 " Results -91 2.4 Discussion 106 2.5 References 108 CHAPTER IH INTERLEUKIN 3 DOWN-REGULATES ITS OWN RECEPTOR 3.1 Introduction I l l 3.2 Materials and Methods 113 3.3 Results 116 3.4 Discussion 132 3.5 References 136 CHAPTER IV CHARACTERIZATION OF THE MURINE INTERLEUKIN 3 RECEPTOR 4.1 Introduction 138 4.2 Materials and Methods 139 4.3 Results 142 4.4 Discussion 157 4.5 References 160 CHAPTER V SUMMARY 5.1 Purification of Interleukin 3 161 5.2 Interleukin 3 Receptor Down-Regulation 162 5.3 Characterization of the Interleukin 3 Receptor 163 5.4 Concluding Remarks 164 5.5 References 166 LIST OF TABLES TABLE 1. Purification of PWM_SCCM derived mIL-3 by Absorption onto GDA fixed BeSUtAj cells TABLE 2. Purification of PWM-SCCM derived mIL-3 TABLE 3. Relative Band Intensity as a Function of ^^I-mIL-3 Concentration TABLE 4. Relative Band Intensity as a Function of DSS Concentration Page 97 105 145 148 vi LIST O F F I G U R E S page Figure 1. A simple schematic representing the hemopoietic hierarchy. 7 Figure 2. Structure of the mIL-3 gene and the genesis of two different mIL-3 transcripts (reprinted from reference 119). 16 Figure 3. The GTP-GDP cycle of G proteins (reprinted from reference 322). 42 Figure 4. Schematic representation of the receptor tyrosine kinase subclasses I, II, and III. 48 Figure 5. This schematic represents the hypothesized molecular pathways by which a cell may become activated in response to mitogen binding. 52 Figure 6. Effect of glutaraldehyde treatment of B6SUtAj cells on their ability to absorb mIL-3. 92 Figure 7. Time course of mIL-3 absorption to 0.01% glutaraldehyde fixed B6SUtA1 cells. 93 Figure 8. Effect of pH on the elution of mIL-3 from 0.01% glutaraldehyde fixed ( ) and viable ( yBeSUtAj cells. 94 Figure 9. Time course of mIL-3 elution from glutaraldehyde fixed B6SUtA ^  cells. 96 Figure 10. Sephadex G75 superfine chromatography of concentrated (2.5 ml) Step 1 material from cycles 1-7 (Table 1). 99 Figure 11. Reverse-phase HPLC of Step 2 material on C j g. 100 Figure 12. SDS-Polyacrylamide gel analysis of the mIL-3 fractions from each step in the purification procedure. ^ 101 Figure 13. SDS-Polyacrylamide gel analysis of PWM-SCCM purified mIL-3 following N-glycanase treatment. 102 1 9 S Figure 14. SDS-Polyacrylamide gel analysis of the binding of purified '-"I-mlL-S to B6SUtAj cells in the presence and absence of rmIL-3 and mGM-CSF. 104 Figure 15. Time course of mIL-3 binding to B6SUtAj cells. 117 Figure 16. Internalization of surface bound ^^I-mIL-3. 118 Figure 17. Cellular processing of 12^I-mIL-3 in the presence and absence of methylamine. 119 Figure 18. Short-term re-expression of the mIL-3 receptor. 121 Figure 19. Short-term re-expression of the mIL-3 receptor in the presence of cydoheximide. 122 vii Figure 20. Scatchard analysis of 1 2 5I-mIL-3 binding to B6SUtAj cells from low and high density cultures grown in mIL-3. 124 Figure 21. Scatchard analysis of 12^I-mrL-3 binding to BeSUtAj cells from low and high density cultures grown in mGM-CSF. 126 Figure 22. Scatchard analyses of I 1 2^ m iL-3 binding to low density B6SUtA! cells initially grown in 0.15 nM mIL-3 and then incubated for different lengths of time in the absence of growth factor. 127 Figure 23. Dose response of mIL-3 on ^H-thymidine incorporation into B6SUtAi cells from low and high density cultures grown in mIL-3. 128 Figure 24. Dose response of mGM-CSF on ^H-thymidine incorporation into B6SUtAj cells from low and high density cultures grown in mIL-3. 129 Figure 25. Dose response of mIL-3 on ^H-thymidine incorporation into 32D c3 cells from low and high density cultures grown in mIL-3. 131 Figure 26. Scatchard analysis of 125I-mIL-3 binding to B6SUtAj cells. 142 Figure 27. Time course of DSS crosslinking of 125I-mIL-3 to intact BeSUtAj cells. 143 Figure 28. Crosslinking of various concentrations of 125I-mIL-3 to intact B6SUtAj cells. 144 Figure 29. Crosslinking of 1 2 5I-mIL-3 to B6SUtA1 cells in the presence of varying DSS concentrations. 147 Figure 30. DSS crosslinking of ^2^I-mIL-3 to intact B6SUIAJ cells followed by incubation at37°C. 149 Figure 31. GDA crosslinking of 125I-mIL-3 to intact BeSUtAj cells. 151 Figure 32. DSS crosslinking of 12^I-mIL-3 bound to B6SUtAj plasma membranes and solubilized plasma membranes. 152 Figure 33. a) The postulated heterocrosslinked mIL-3 receptor complex. b) Heterocrosslinking of pl40 with DSP and GDA. 154 Figure 34. a) N-glycanase treatment of ^2^I-mIL-3 crosslinked to intact B6SUtAj cells. b) N-glycanase treatment of ^2^I-mIL-3 crosslinked to B6SUtAj plasma membranes. 156 Figure 35. Model of the mIL-3 receptor. 159 viii LIST OF ABBREVIATIONS ATP , BFU-E cAMP cDNA CML CFU-C CFU-E CFU-G CFU-GEMM CFU-GM CFU-M CFU-Mk CFU-S cpm CSF DAG EGF EPO f FSV G G-CSF GDA GM-CSF FCS h HGF HPLC I FN lg IL-1 IL-2 IL-3 ILA IL-5 11^ 6 IL-7 Ins IP3 kd K D M-CSF MHC mIL-3 min mRNA multi-CSF Ph1 PDGF PI PKC PLC PWM SCCM SDS/PAGE SSV TGFB TNFa adenosine triphosphate burst forming unit-erythroid cyclic adenosine monophosphate complementary DNA chronic myelogenous leukemia colony forming unit-granulocyte colony forming unit-erythroid colony forming unit-granulocyte colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte colony forming unit-granulocyte/macrophage colony forming unit-macrophage-colony forming unit-megakaryocyte colony foiTning unit-spleen counts per minute colony stimulating factor diacylglycerol epidermal growth factor erythropoietin seeding fraction feline sarcoma virus guanine nucleotide binding granulocyte colony stimulating factor glutaraldehyde gTanulocyte/macrophage colony stimulating factor fetal calf serum hours hemopoietic growth factor high pressure liquid chromatography interferon immunoglobulin interleukin 1 interleukin 2 interleukin 3 interleukin 4 interleukin 5 interleukin 6 interleukin 7 inositol inositol trisphosphate kilodalton dissociation constant macrophage colony stimulating factor major histocompatibility complex murine interleukin 3 minutes messenger RNA interleukin 3 probability Philadelphia chromosome platelet-derived growth factor phosphatidylinositol protein kinase C phospholipase C pokeweed mitogen-stimulated spleen cell conditioned medium sodium dodecyl sulphate/polyacrylamide gel electrophoresis simian sarcoma virus transforming growth factor (J tumor necrosis factor a ix A C K N O W L E D G E M E N T S I would like to thank my supervisor, Dr. Gerald Krystal, for his efforts which were without doubt above and beyond the call of duty. He has taught me not only science, but more importantly, patience and compassion, virtues which he himself embodies. v I would also like to thank the members of my graduate committee at the University of British Columbia and the Senior Staff, Post-Doctoral Fellows, students, and technical staff of the Terry Fox Laboratory for their helpful suggestions, relevant discussions, and considerable tolerance. I am also deeply indebted to my good friends here in Vancouver (Randy, Graham, the Daves, Greg, Blake, Dana, etc.), without whose timely distractions would have made my life far more efficient, but far less livable. Let me also acknowledge the love and constant support of my mother and brothers, and the memory of my father. Finally, let me thank my best friend in the whole world for her continuous help, diligent typing, and critical suggestions during the construction of this thesis. Far more importantly, let me voice my deep appreciation and utter amazement at her tremendous commitment and undying devotion to me in the face of less than favorable conditions. I owe you a brilliant future Anita. To DAD, and remember, I'm taller than you too I. CHAPTER I INTRODUCTION: GENERAL CONCEPTS OF HEMOPOIESIS AND HEMOPOIETIC GROWTH FACTORS 1.1 PRINCIPLES OF HEMOPOIESIS 1.1.1 Hemopoietic Stem Cells 2 1.1.2 Committed Progenitors • 4 1.1.3 Morphologically Recognizable Precursors • • 6 1.1.4 Mature End Cells 6 1.2 CONTROL OF HEMOPOIESIS 1.2.1 Regulation of Stem Cells 6 a) Stem Cell Self-Renewal -8 b) Lineage Commitment 9 c) Cell Cycle Activation 9 1.2.2 Stromal Elements and Hemopoietic Control 10 1.2.3 Humoral Regulation of Hemopoiesis 12 a) The Colony Stimulating Factors 13 b) Class I Hemopoietic Growth Factors 14 c) Class II Hemopoietic Growth Factors 23 d) Class III Hemopoietic Growth Factors 27 e) Class IV Hemopoietic Growth Factors 30 f) Class V Hemopoietic Growth Factors 31 1.2.4 In Vivo Activities of Hemopoietic Growth Factors 32 1.2.5 Physiologic Roles of Hemopoietic Growth Factors 34 1.3 MECHANISM OF ACTION OF GROWTH FACTORS 1.3.1 Platelet Derived Growth Factor 37 1.3.2 Early Events Elicited by Growth Factors i....39 a) Guanine Nucleotide-Binding Proteins 40 b) Ion Fluxes 41 c) Protein Kinase C 43 d) Cyclic Nucleotides 45 e) Early Nuclear Events 45 1.3.3 The Meaning of Intracellular Events.... 46 1.3.4 Growth Factor Receptors ; 46 1.3.5 Hemopoietic Growth Factor Mechanism of Action 51 a) Murine IL-3 53 b) Other Hemopoietic Growth Factors 53 1.4 INTERACTIONS BETWEEN HEMOPOIETIC GROWTH FACTORS 54 1.5 ROLE OF HEMOPOIETIC GROWTH FACTORS IN ONCOGENESIS. 54 1.6 THESIS OBJECTIVES , ••• 56 1.7 REFERENCES • -57 2 1.1 PRINCIPLES OF HEMOPOIESIS Hemopoiesis, which first occurs in the yolk sac of the embryo and ultimately in the bone marrow, spleen, thymus, and lymph node of the adult, allows for the constant generation of mature myeloid and lymphoid blood elements [1]. This is essential because of the limited lifespans of many mature blood cell types, which range from a few hours for granulocytes to four months for red blood cells [2]. Thus, in the bone marrow, cells at various stages of maturation are always present and, based on functional and morphological criteria, they can be subdivided into four major developmental compartments [3]. 1.1.1 Hemopoietic Stem Cells Best characterized by their ability to self-renew, their extensive proliferative capacity, and their unremarkable morphology, hemopoietic stem cells comprise the smallest blood cell compartment. The existence of stem cells was originally hypothesized from studies showing that small numbers of bone marrow cells were capable of fully reconstituting the marrow of lethally irradiated mice [4,5]. It was subsequently shown by Barnes et al [6] that only one cell within the donor marrow population was needed to repopulate the recipient. This was done by transplanting chromosomally marked donor stem cells, generated by radiation treatment, into lethally irradiated recipient mice. Upon examination of recipient marrow and lymph tissue, it was found that all tissues carried the same unique chromosomal aberration and thus, were repopulated by the progeny of only one stem cell. Further proof that a single stem cell can repopulate both primary and secondary recipients has been provided by studies with retrovirally-marked cells [14,15]. Till and McCulloch [7] devised the first quantitative assay for murine pluripotent hemopoietic stem cells, the spleen colony assay. They found that intravenously injected stem cells had a propensity to lodge and proliferate within the spleen of lethally irradiated, histocompatible recipients. Thus, quantitation of the resultant macroscopic splenic nodules provided an estimate of the number of colony-forming unit spleen 3 cells or, CFU-S, present in the initial inoculum. Cytological and chromosomal marker studies have since shown that each macroscopic nodule represents a cohort of progeny cells derived from one stem cell [8,9] and the size of each nodule (10^-10^ cells) demonstrates the enormous proliferative capacity inherent within this cell. However, not all splenic nodules are initiated by stem cells [10]. Nodules arising 7-9 days post-transplant appear to represent colonies derived from cells which are neither multi-potential nor self-renewing [10]. Previously, these 'early' colonies were thought to be unilineal at early times and multi-potential and self-renewing at later times, following the 'instructive' differentiative signals provided by the splenic microenvironment [11]. It is now clear that only 'late' colonies (day 12-14) are derived from stem cells (CFU-S), and that the early colonies disappear from the spleen within 72 hours of their formation. Day 12-14 spleen colonies, and not day 7-9 colonies, can successfully reconstitute secondary recipients, demonstrating the pluripotentiality and self-renewal capacity of the CFU-S responsible for late colony formation [12]. Recent data have also suggested a considerable overlap between cells responsible for late colony formation and cells capable of 'long-term' repopulating ability in mice [13]. It should be noted that the spleen colony assay is not without problems. Only a small proportion of injected stem cells seed the spleen, with the remainder lodging elsewhere [12]. The seeding fraction, f, has been estimated to be 0.05-0.15 [12,16], and must be utilized when calculating stem cell number in the inoculum. Another complication is that a single stem cell has been shown, on occasion, to give rise to more than one spleen colony [17]. The existence of human hemopoietic stem cells is supported by several lines of evidence principally derived from the study of hematologic disorders. In chronic myelogenous leukemia (CML), a 9:22 chromosomal translocation generates a truncated chromosome 22 known as the Philadelphia chromosome (Ph*). The Ph^ chromosome is invariably found in most circulating myeloid and B-lymphoid cells. This suggests that these abnormal cells are clonal and are derived from a single neoplastic cell that can give rise to multiple hemopoietic lineages [18,19]. Interestingly, marrow fibroblasts are not part of the malignant clone. Women heterozygous for the X-linked marker enzyme, glucose-6-phosphate dehydrogenase 4 (G6PD), and afflicted with various myeloproliferative disorders, demonstrate a single enzyme isotype in all of their transformed myeloid cells [20-22]. Interestingly, one such female patient with acquired idiopathic sideroblastic anemia was found to have the identical enzyme isotype in both her myeloid and lymphoid tissue [20], suggesting that the initially transformed cell was pluripotential. More recently, molecular probes for polymorphic regions in the X-linked hypoxanthine phosphoribosyl transferase gene have been used to prove that long-term, monoclonal hemopoiesis was of donor origin following allogeneic marrow transplantation [23]. These data strongly support the existence of human hemopoietic stem cells that can yield both myeloid and lymphoid progeny. There is mounting evidence to indicate that, within the hemopoietic stem cell compartment, considerable variation in the proliferative and differentiative capacity of stem cells exists [43]. Worton et al [44] demonstrated heterogeneity within the murine CFU-S compartment, the smallest cells (as assessed by velocity sedimentation [45]) having the greatest self-renewal potential in the spleen colony assay. Other studies indicate that non-cycling CFU-S have a greater self-renewal capacity than those in cycle [43,46]. Increased self-renewal capacity has also been demonstrated for stem cells in marrow versus blood [47], and for stem cells present in mouse fetal liver compared to adult marrow [48]. Moreover, under constant proliferative pressure, all cells within the stem cell compartment lose the ability to self-renew. Experiments where spleen colonies are serially transplanted into lethally irradiated mice show that the generation of new CFU-S by transplants ceases after 3-6 serial passages [49,50]. Thus, heterogeneity in the stem cell compartment can be explained by recognizing that stem cells that have undergone more self-renewal divisions are less likely to be able to self-renew. 1.1.2 Committed Progenitors In the earliest models of hemopoietic differentiation, pluripotent stem cells were thought to give rise directly to morphologically distinguishable precursors [22]. It is now apparent that cells leaving the stem cell compartment undergo extensive amplification before ultimately giving rise to recognizable precursors. 5 Concomitant with cell departure from the stem cell compartment is a loss of self-renewal capacity, and a gradual restriction of cell proliferative capacity. Furthermore, lineage commitment accompanies hemopoietic cell maturation. Thus, the 'committed progenitor' represents a cell in transition, with neither the potential of the stem cell nor the specific morphologic markers of more mature precursors. Since the egress of cells from the stem cell compartment is generally considered to be unidirectional [3,25], committed progenitors can be easily identified by the number and types of progeny they produce. Specifically, when suspended in a semi-solid culture medium (as for clonogenic assays) and furnished with appropriate growth factors, progenitor classes can be identified by the morphologically recognizable, reproducible colonies they generate [26,27]. A progenitor which gives rise to a pure granulocyte colony is termed a colony forming unit-granulocyte (CFU-G or CFU-C), and progenitors yielding pure macrophage colonies are referred to as colony forming units-macrophage (CFU-M). Similarly, CFU-granulocyte/macrophage (GM), CFU-erythroid (E), burst forming unit-erythroid (BFU-E), and CFU-megakaryocyte (Mk) progenitors have been identified. Progenitors with multi-lineage potential have also been observed. Specifically, CFU-granulocyte/erythroid/megakaryocyte/macrophage (GEMM) found in human marrow is not only multi-potential, but occasionally demonstrates self-renewal. Some CFU-GEMM can give rise directly to small 'blast' colonies which subsequently generate CFU-GEMM and other more restricted progenitors upon replating [28]. Lymphoid bipotent and unipotent progenitors have been identified by retroviral marker analysis [15]. Furthermore, cells with lymphoid surface markers have been derived from CFU-GEMM colonies [33,34]. These data suggest that primitive members within the CFU-GEMM compartment may be considered as human analogs to murine CFU-S [35,36]. Committed progenitors can be distinguished from stem cells by a variety of criteria. For example, CFU-C are larger than CFU-S [37] and a greater proportion of CFU-C are in active cell cycle [39]. In addition, CFU-C have little or no capacity for self-renewal [40]. Furthermore, progenitors are responsive to different growth factor molecules than stem cells [31]. Progenitors can also be separated from stem cells by their behavior in vivo. For example, when mice are made polycythemic by hypertranfusion, they show 6 a significant decrease in their CFU-E content, and new red blood cell production ceases. However, these same animals have unchanged numbers of CFU-S [42]. Committed progenitor classes may also be distinguished from each other. Aside from their activity in clonogenic assays, progenitors can be separated based on surface antigen expression [29] and in their responses to cell cycle specific agents [30]. As well, differences in their sensitivity to a wide variety of growth regulatory molecules (including hemopoietic growth factors) can be used to identify progenitors [31,32]. 1.1.3 Morphologically Recognizable Precursors Early autoradiographic studies demonstrated that recognizable precursors were not self-renewing, but simply represented a cohort of cells progressing through a preprogrammed differentiation sequence [22,38]. In the erythroid series, these precursors have a limited proliferative potential, with each cell capable of 5-6 divisions (generating 32-64 terminally differentiated progeny) [22]. 1.1.4 Mature End Cells Fully differentiated, non-dividing blood cells comprise the last compartment. From retroviral marker studies, it has been demonstrated that all the different cell types of this compartment originate from a single pluripotent stem cell [14,15]. A schematic representation of the hemopoietic hierarchy is presented in Figure 1. 1.2 CONTROL OF HEMOPOIESIS 1.2.1 Regulation of Stem Cells Three critical stem cell decisions can be considered as potential regulatory foci [49]:(a) stem cell self-renewal versus restriction of proliferative capacity;(b) lineage commitment;(c) entry into and exit out 7 ULTIMATE STEM CELL ERYTHROID PROGENITORS © —• ''Mil or DufSt I •I* M a l u t • BfU E \ma» frfythroid colony or EMS' l etyihro-rj clusttt I yrri0hooO'«s>s • It • r « n e w « i ( (ffl MEGAKARYOCYTE PROGENITORS I o • C f U M 4>oe mcgaharrocyt tc COtony c atony GRANULOCYTE PROGENITORS © — • * ; COk>r>y jm*H gr«n«A>Cyl>c co+ony G R A N U t O O U S * UACflOPMAGtS Figure 1. A simple schematic representing the hemopoietic hierarchy. Mature progeny of all myeloid and lymphoid lineages originate in the stem cell compartment. 8 of the cell cycle [51]. The forces which regulate these processes are either intrinsic (i.e. genetically programmed) or extrinsic (i.e. derived from the local environment). a. Stem Cell Self-Renewal Serial transplantation experiments have demonstrated a limited self-renewal potential for hemopoietic stem cells [49,50]. Since each successive transplant is received by a constant environment (all irradiated syngeneic recipients being the same), it would appear that extrinsic factors are not important determinants associated with the 'decline phenomena' of serially transplanted stem cells. This phenomenon must then be regulated by intrinsic mechanisms. Further support for the importance of intrinsic factors in the regulation of stem cell renewal has come from the observation that the number of CFU-S present in individual spleen colonies is highly variable [12]. Again, since similar extrinsic proliferative pressures exist for all spleen colonies, stem cell intrinsic factors must be responsible for spleen colony CFU-S heterogeneity. To account for this heterogeneity, Till et al [52] have proposed a model of stem cell proliferation and self-renewal based upon stochastic principles. Specifically, stem cells were assigned a probability (p) for self-renewal and another (1-p) for differentiation (with a concomittant loss of self-renewal potential) [52]. Subsequently, computer generated scenarios (employing these probabilities) were found to be consistent with observed results [52]. From these data, it can be concluded that the mechanism governing stem cell renewal has a significant intrinsic component. Studies of genetically-induced macrocytic anemias in mice suggest that both intrinsic and extrinsic factors regulate stem cell proliferation and self-renewal. Stem cells isolated from W/W v mice are incapable of spleen colony formation in either W/W v or syngeneic wild-type mice. However, wild-type stem cells can form spleen colonies in W/W v recipients. Thus, the defect predisposing W/W v mice to anemia is intrinsic to the W/W v stem cell [53]. In contrast, CFU-S from mice of the Sl/Sld genotype are able to form spleen colonies in syngeneic wild-type recipients, but are unable (as are wild-type CFU-S) to 9 colonize spleens of Sl/Sl^ mice. The defect in SI/SI** mice is thus extrinsic to the individual stem cell and resides within the microenvironment [54]. b. Lineage Commitment Two contrasting models to describe lineage commitment have been proposed. Korn et al [55] argue that intrinsic factors guide stem cell differentiation in a random fashion. This model has been substantiated by the corroboration of experimental observations and computer generated data [55]. Another model espouses the importance of 'instructive' extrinsic factors in the regulation of stem cell lineage commitment [56-58]. This so-called 'inductive effect of the microenvironment' has been offered as an explanation to account for the heterogeneity of spleen colonies [57]. It is routinely found that small spleen colonies are often unipotential, while large colonies tend to be multi-potential. If the microenvironment was instructive, then the existence of multi-potential colonies could be rationalized by the ability of larger colonies to overlap with different microenviroments [57]. It was later established, however, that many small unipotent colonies contained progenitors committed to other lineages, and thus, were not unipotent [42]. Accordingly, the theory that the microenvironment regulates stem cell lineage commitment has fallen from favor. c. Cell Cycle Activation Most stem cells are in the resting phase (GQ) of the cell cycle [59]. However, after the application of injurious stimuli (e.g. sublethal irradiation), it has been demonstrated that the proportion of stem cells in active cycle is greatly increased [59]. Moreover, even CFU-S within lead shielded areas of irradiated animals have been shown to be recruited into cycle, suggesting that soluble humoral factors may be responsible for the cycling activity of stem cells [60]. Although these data implicate a global regulatory system for stem cell cycle control, that stem cells located in lead shielded areas more quickly return to their pre-insult quiescent state supports the involvement of local controls as well [60]. As evidence for local 10 control of stem cell cycling, in mice made anemic by phenylhydrazine treatment, marrow CFU-S are activated while spleen-derived CFU-S remain quiescent [61]. These data suggest a role for extrinsic regulation of stem cell cycle activation. A wide variety of pharmacological agents have been shown to induce stem cell cycling [62]. Whether these agents (e.g. isoproterenol, acetylcholine, testosterone, etc.) act directly on stem cells is unclear. More recently, a few peptide growth factors have been shown to directly activate stem cells into cycle (see section 1.2.3). Hemopoietic neoplasia may arise from the abnormal regulation of any one (or all) of these stem cell processes. As suggested earlier, specific stem cell disorders can manifest as both leukemic and preleukemic conditions. Thus, further work is needed to define the nature of the specific stem cell defects that result in malignancy. 1.2 Stromal Elements and Hemopoietic Control In addition to stem cells, progenitors, recognizable precursors and formed elements, bone marrow consists of a non-hemopoietic component, the stroma. The cells of the stromal compartment include: endothelial cells, adipocytes, fibroblasts, adventitial reticular cells, osteoblasts, osteoclasts, and macrophages [63]. Common to most cells of the stroma is a non-hemopoietic origin. Evidence of their distinct origin was provided, in part, by the finding that in CML, stromal cells are not part of the malignant clone (i.e. are Ph* negative) [18,19]. Further studies have indicated that an undifferentiated ubiquitous fibroblast/mesenchymal cell can be rapidly mobilized by bone marrow extracts to begin marrow stroma formation [74,75]. In this regard, it is interesting to note that an embryonic fibroblast cell line, 3T3, can support hemopoiesis [76]. However, there are some cell types within the stromal cell compartment that are derived from hemopoietic tissue. For example, the macrophage, by virtue of its supportive role in erythropoiesis, is classified as a stromal cell [65], yet, is clearly derived from the pluripotent hemopoietic cell compartment [65]. The osteoclast is also of hemopoietic origin [65,67]. 11 Studies of the stroma have established several cell-specific relationships. Macrophages, for example, support erythropoiesis by delivering iron to hemoglobin producing erythroblasts and by phagocytosing extruded nuclei from maturing normoblasts [64] and, perhaps, by secreting erythropoietin [139]. Endothelial cells regulate the entrance of newly formed blood cells into the systemic circulation [64]. Adventitial reticular cells provide a supportive collagen matrix for hemopoietic cells [66] and osteoblasts may function to regenerate marrow stroma following stromal depletion [67]. Aside from its cellular component, the stroma contains high concentrations of matrix molecules. These include collagens, laminin, fibronectin, hemonectin, and proteoglycans. Although the precise function of these molecules has not yet been determined, several interesting observations have been noted. Specifically, collagen (type III) has been found to frequently associate with hemopoietic 'nests' in culture [68]. Furthermore, a recently described adhesion molecule, hemonectin [69], binds firmly only to immature granulocytes and not to more mature cell types. Studies with fibronectin have led to the hypothesis that it mediates mature red blood cell release from bone marrow [70]. Finally, the proteoglycans of the stroma have been demonstrated, by in vitro culture methods, to bind and retain specific hemopoietic growth factors [71]. Although it is not yet possible to assign specific functions to each microcomponent, many studies serve to illustrate the importance of a functioning, intact stroma. Experiments which have characterized the heterogeneity of spleen colonies, although unable to support the theory of direct stem cell instruction (see section 1.2.1), do demonstrate an inductive effect of the microenvironment on hemopoiesis [11]. Specifically, Curry and Trent [11] showed that colonies arising under the spleen capsule, in the vicinity of the septae, or in the marrow spaces, are most often granulopoietic, while spleen colonies arising in the splenic parenchyma are predominantly erythroid. Moreover, stromal transplantation studies have illustrated the importance of the stroma to spleen colony differentiation [72]. For example, transplantation of marrow stroma under the kidney capsule, a region normally unable to support hemopoiesis, has been shown to lead to kidney capsule hemopoiesis [73]. 12 There is no question that bone marrow stroma is critical to normal hemopoiesis. Studies with Sl/Sl" mice demonstrate this clearly [54]. However, the stroma is not solely a stimulator of hemopoiesis, but rather, a regulator of hemopoietic differentiation since it can provide both stimulatory and inhibitory signals. From experiments where mice are given total body irradiation with tibias shielded, examples of simultaneous positive and negative stromal signals have been observed. CFU-S in the shielded areas rapidly enter the proliferative cycle (as did unshielded CFU-S), but then quickly reenter a quiescent state. In contrast, CFU-S in unshielded areas remain in cycle for over 5 days [60]. Thus, local stromal elements are regulating these two populations in very different ways. 1.2 J Humoral Regulation of Hemopoiesis The widespread use of in vitro clonogenic assay techniques has been responsible for the discovery of a group of soluble glycoprotein colony stimulating factors (CSFs) [77]. These factors were initially described as factors capable of stimulating hemopoietic progenitor cell differentiation in semi-solid medium [78]. It soon became apparent that not only were CSFs required for the in vitro maturation of progenitors, but also affected the function of mature hemopoietic cells [79-82]. Several CSFs have now been identified. These include: erythropoietin (EPO), granulocyte (G)-CSF, macrophage (M)-CSF, granulocyte-macrophage (GM)-CSF, and multi-CSF (also known as interleukin 3). Recently the murine [83-87] and human [88-92] genes encoding these factors have been cloned and expressed in suitable bacterial and mammalian systems [94-97]. The current availability of recombinant material has promoted the study of these factors. The CSFs form a subgroup within the much larger set of hemopoietic growth factors (HGFs). The HGF supergroup consists of all factors with demonstrated growth-regulating activity for both myeloid and lymphoid cells. A brief review of HGFs is provided below, with special emphasis given to the CSFs. The role of CSFs in directing myelopoiesis will be highlighted. A synopsis of HGFs and their actions is presented in Appendix I. 13 a. The Colony Stimulating Factors Several key properties of CSFs have been outlined by Metcalf [93]. 11 13 1. All CSFs are glycoproteins that stimulate hemopoietic cells at very low concentrations (10"1 -10" M) and exert maximal stimulation with only 5-10% HGF receptor occupancy. 2. Very little sequence homology exists between CSFs, although modest conservation in the N-terminal regions have been noted [165]. Consequently, all CSFs have distinct surface receptor proteins. 3. CSFs are required continuously to stimulate cell division in responding cells. The concentration of CSF determines both the cell cycle time and the number of progeny produced by responsive progenitors. 4. CSFs are necessary for cell survival in vitro. 5. CSFs can induce irreversible commitment of bipotential cells to a restricted lineage. 6. CSFs are hierarchically arranged, with the multi-potent CSFs able to down-modulate the receptors for the other CSFs [98]. 7. Most CSFs have pleiotropic effects. 8. For complete cell maturation along most lineages, the cooperation and sequential action of several CSFs are required [32]. 9. Unlike other, 'non-hemopoietic' growth factors, only one oncogene, to date, has been linked directly to CSF function (i.e. v-fms, the viral analog of the M-CSF receptor). In fact, all primary, human myeloid leukemias thus far examined are absolutely dependent for proliferation on exogenous CSFs [93]. HGFs can be subdivided into five classes based on their specific biological properties [77]. In the discussion of each provided below, unless specified, biological activities of murine and human factors should be considered interchangable. 14 b. CLASSIHGFs Class I HGFs act on pluripotent cells and immature progenitors. They are not lineage specific and can stimulate the differentiation of immature progenitor cells. Furthermore, these factors are important for the self-renewal and proliferation of hemopoietic stem cells. Murine Interleukin 3 (mIL-3) Initially, an activity contained within the medium conditioned by activated T cells was shown to stimulate the expression of 20-alpha-hydroxysteroid dehydrogenase (20aSDH) in mature T cells [99]. Subsequently, a factor secreted by the WEHI-3B cell line was also shown to have 20aSDH inducing activity [100]. With 20aSDH induction serving as a specific assay, mIL-3 was purified to homogeneity [101]. In a parallel set of experiments, persisting cell stimulatory factor (later to be called panspecific hemopoietin (PSH)), assayed by its ability to stimulate 'persisting' mast cells in culture, was also purified from WEHI-3B conditioned medium [102]. PSH was first described as an activity secreted by activated T cells that enhanced the survival, in vitro, of pluripotent hemopoietic stem cells (CFU-S) [103-105]. Comparison of the amino acid and gene sequences of mIL-3 [87,100] and PSH [112], as well as their biological activities [107], demonstrated complete identity between the factors (although PSH had six additional N-terminal amino acids [102]). Production of mIL-3 is exclusive to T cells. This factor has been shown to be released by Con-A stimulated cloned T-cell hybridomas [108,109], as well as a stimulated T cell lymphoma [110], activated long-term T-helper clones [111], and the thymocyte cell line, EL-4 [112]. Moreover, 20aSDH activity is absent in the spleens of congenitally athymic (nu/nu) mice, but is rapidly induced by a factor present in the medium conditioned by mitogen-stimulated wild-type spleen cells [99]. An exception to the apparently exclusive T cell origin of this factor is the production of mIL-3 by the myelomonocytic leukemia cell line WEHI-3B. Characterization of this line revealed the expression of high levels of the Thy-1 surface antigen [113], a surface marker initially thought to be restricted to the T cell lineage. Thus, it was first 15 hypothesized that the WEHI-3B cel  line might represent an abnormal T cell clone. However, it was later demonstrated that normal myeloid progenitors express Thy-1 antigen when stimulated by mIL-3 [110]. The WEHI-3B line is now considered a myeloid line that mimics the activated state (Thy-1 expression) of other myeloid progenitors [114]. Using cDNA probes, constitutive mDL-3 production by WEHI-3B cells has been shown to be associated with a rearrangement of one of the mIL-3 gene alleles [103]. By restriction enzyme analysis, it has been demonstrated that the rearrangement involves the insertion of sequences approximately 300 base pairs upstream of the 5' end of the gene. Analogous rearrangements have not been observed in either mIL-3-producing T cell lines or activated primary T cell cultures [103]. Fung et al [106] were the first to obtain a cDNA clone for mIL-3. Their strategy involved hybrid selection of mRNAs encoding a factor which could stimulate the growth of mIL-3-dependent cell lines. Rennick et al [96] also isolated a cDNA clone for mIL-3 which encoded a protein of 166 amino acid, including a putative signal peptide of 20 amino acids. While the predicted nascent peptide molecular weight is 15 kd, molecular mass estimates of purified native mIL-3 (SDS/PAGE) have ranged from 25-28 kd [102,115]. The difference between the predicted and apparent size of mIL-3 is presumably due to carbohydrate substitution, since the amino acid sequence suggests that there are four potential glycosylation sites [115]. Murine IL-3 is encoded by a single cellular gene and has been mapped to chromosome 11 by somatic cell hybridization studies and restriction enzyme polymorphisms in back-cross experiments [116]. A clone of the genomic mIL-3 gene has more recently been obtained and sequenced [117]. The gene is composed of four introns and five exons. A 'TATA'-Iike sequence is found 5' to the open reading frame. Interestingly, nine repeats of 14 bases are located in the second intron, and it has been postulated that this sequence arrangement may possesses enhancer-like activity. The structure of the mIL-3 gene allows for the generation of two different mRNA species [119] (Figure 2). In the 'short' mIL-3 mRNA, nucleotides 1-27 represent the 5' untranslated region and the AUG beginning at position 28 is the start codon. The Figure 2. Structure of the mIL-3 gene and the genesis of two different mIL-3 transcripts (reprinted from reference 119). The mIL-3 gene is in the center with its exons (shown as boxes) numbered 1-6. Two different promoters are indicated as PI and P2. In the two different mature mRNAs generated (given at the top and bottom of the diagram), the untranslated regions are shown as the narrow boxes. 17 'long' mIL-3 transcript includes additional 5' sequences which add an extra coding region (exon) to the main body of the gene [141]. This sixth exon is located 14 kilobases upstream of the 'short' transcript promoter. The 'short' mRNA encodes a stretch of eight extremely hydrophobic amino acids within a 27 amino acid leader sequence, and the product of this transcipt represents the secreted form of mIL-3. The significance of the 'long' transcript is unknown. However, since the 'long' transcript encodes an additional charged amino-terminal extension, it has been postulated that the 'long' transcript encodes a membrane bound form of mIL-3, which is anchored to the cell membrane by its amino-terminus [119]. Chemically synthesized analogs of mIL-3 have allowed for the identification of structures critical for bioactivity [118]. These experiments have established that residues 1-16 are not required for bioactivity; this helps explain why PSH and mIL-3 have identical activities [114]. The cysteine residue at position 17 is necessary for activity because it forms a disulphide bond with a cysteine at position 79 or 80 [114]. Another cysteine at residue 140 can form a disulphide bond with the unpaired cysteine at residue 79 or 80, creating a second loop structure [114]. Murine IL-3 can support the growth of a variety of cell lines established from long-term bone marrow cultures [120]. This property has proven particularly useful and has served as the basis for cell proliferation assays. All of these cell lines display an absolute requirement for mIL-3 (or mGM-CSF) and, when this factor is removed from the culture medium, cells lose viability at a rate of half the population every 2-4 h [115]. The properties of these mIL-3-dependent cell lines vary considerably, though most lines have been found to express myeloid markers [120-122]. It has been hypothesized that the establishment of factor-dependent lines requires a transformation-related event [115]. Early studies indicated that a T cell factor could support the in vitro growth of pluripotent hemopoietic stem cells (CFU-S) [104]. This factor was later demonstrated to be mIL-3 [96]. More recently, it has been shown that mIL-3 promotes the survival of CFU-S and increases the proportion of cells in cell cycle [105]. in vitro clonogenic assay data indicate that mIL-3 directly stimulates isolated hemopoietic cells that are capable of giving rise to colonies containing multiple hemopoietic lineages 18 [123]. The multi-lineage, panspecific actions of mIL-3 are perhaps best illustrated in the experiments of Iscove and Roitsch [123]. They conclusively demonstrated that single isolated hemopoietic progenitor cells could generate neutrophils, macrophages, megakaryocytes, mast cells, and erythroid cells when stimulated by mIL-3. Furthermore, mIL-3 has been shown to have significant eosinophil-colony stimulating activity [96]. Of unknown importance is the ability of mIL-3 to induce Thy-1 antigen expression on immature myeloid cells [126]. Relatively high levels of Thy-1 antigen expression are also found on factor-dependent myeloid lines [127]. There is also evidence to suggest that mIL-3 can support the in vitro growth of cells capable of giving rise to thymocytes and T and B cells when injected into irradiated animals [105]. Specifically, when marrow preparations were treated for 6-7 days with mIL-3 prior to inoculation into sub-lethally irradiated recipients, the proportion of donor cells in recipient lymphoid tissues was found to be tremendously enhanced [105]. These results are consistent with the hypothesis that mIL-3 stimulates a pluripotential lymphomyelopoietic stem cell. It remains to be proved whether this observed effect on lymphopoiesis is direct. The action of mIL-3 is not restricted to the stem cell and committed progenitor compartments. Murine IL-3 has also been shown to stimulate the proliferation of mature mast cells [105], and regulate their expression of MHC antigens [124]. In addition, it stimulates peritoneal macrophage proliferation and phagocytosis [125]. Although mIL-3 was initially identified because of its T cell 20aSDH inducing-activity, its effect on mature T cells is unclear [114]. Furthermore, since studies have failed to demonstrate mIL-3 receptors on either thymocytes or T cells [128,129], data indicating that mIL-3 enhances the generation of cytotoxic T cells [130] or suppressor T cells [131] may be the result of indirect effects via the release of T cell factors by other cells when stimulated by mIL-3. A role for mIL-3 in early B cell differentiation has also been proposed. Palacios et al [132] have isolated cell lines which are mIL-3-dependent and share some characteristics in common with the pre-B cell phenotype. The identification of these lines as pre-B is predicated on their expression of a B cell antigen detected by a monoclonal antibody. More detailed examination of one such line has revealed the 19 presence of immunoglobulin gene rearrangements characteristic of the B-lymphocyte lineage [132]. Nonetheless, other data are not consistent with this hypothesis. Spleen cell cultures have not been shown to bind mIL-3 [128] and antigen-stimulated B cells are not responsive to mIL-3. Moreover, the burst of B cell differentiation in murine fetal liver is not accompanied by a detectable rise in mIL-3 levels [115]. The availability of cell lines that require mIL-3 for growth has also allowed for the characterization of the mIL-3 receptor [129]. From a variety of cell lines tested, it was determined that specific A,"I-mIL-3 binding correlated with mIL-3-dependence. Nicola [147] has shown that the equilibrium dissociation constant (KD) determined from kinetic constants differs from the apparent K D determined by Scatchard analysis, and is higher than the concentration required for half-maximal biological effects. His findings suggest that there is no simple relationship between receptor occupancy and biological response and that a biologic response may be observed at low levels of receptor occupancy. Nevertheless, the KQ (Scatchard estimate) of the mIL-3 receptor has been reported to be between 100-1000 pM [147,148]. The molecular weight of the receptor, long thought to be 70 kd, has recently been shown to be 140 kd [149-152, See CHAPTER III]. Human Interleukin 3 (hIL-3) Although attempts to purify a human homologue to mIL-3 had been uniformly unsuccessful, in 1986 a cDNA clone of gibbon IL-3 was used to identify hIL-3 [230]. When compared, only 45% homology (nucleotide sequence) was found between the human and murine genes, although their gene structure was similar [77]. The evolutionary divergence of IL-3 is thus much greater than for any other HGF [77] and it is not yet clear whether the conserved regions between the human and murine forms represent the receptor binding domain of the molecule, although, hIL-3 does not appear to stimulate murine cells. The hIL-3 gene has been localized to chromosome 5 and the mature, native gene product ranges in size from 20-26 kd and reflects heterogeneity of glycosylation [77]. Human IL-3 appears to have the same 20 target cells as does mIL-3 and stimulates both primitive cells within the stem cell compartment and more mature committed progenitors [231]. Of clinical importance is the finding that the hIL-3 gene is deleted in myeloid leukemias presenting with a 5q deletion [232]. Murine Granulocyte/Macrophage-Colony Stimulating Factor (mGM-CSF) Although mGM-CSF is difficult to detect in the tissues of normal mice, it is induced in virtually all tissues after incubation for 8 h in vitro [133]. The most concentrated source of tissue-derived mGM-CSF is medium conditioned by lung tissue [134]. It was from this source that the molecule was initially purified to homogeneity [145] and cDNA clones obtained [86]. When first studied, mGM-CSF isolated from a variety of tissue types appeared to display tremendous heterogeneity. A 200 kd species was purified from muscle tissue, while a 23 kd molecule was derived from lung [133]. However, it soon became obvious that these size descrepancies resulted from either the interaction of mGM-CSF with specific tissue proteins or variable addition of carbohydrate to the single polypeptide chain. After appropriate treatment, all native mGM-CSF species were shown to have a molecular weight of approximately 23-26 kd [137]. N-terminal amino acid sequencing [138] and subsequent studies with the cloned gene [94] revealed no striking homology to any other known protein (including other CSFs). More recently, E. Coli-derived recombinant material has become available [95], and the recombinant (unglycosylated) 14 kd molecule is fully functional both in vitro and in vivo [119]. As with transcription of the mIL-3 gene, two mGM-CSF mRNA species have been isolated, and thus, two protein forms may exist [119]. Another source of mGM-CSF is mitogen-stimulated T lymphocyte conditioned medium [119]--from which mIL-3 can also be obtained. Interestingly, genetic analysis, using an interspecies cross and Southern blot analysis of large DNA fragments separated by pulse field electrophoresis, has shown that the mGM-CSF and mIL-3 genes are less than 250 kilobases apart on chromosome 11 [119]. The tight 21 linkage of these genes might account for their coordinate expression in mitogen-stimulated T cells [119,140]. Murine GM-CSF was initially thought to be restricted in its action, capable of stimulating only the development of CFU-GM colonies in semi-solid medium [142]. However, the availability of recombinant material has allowed for the demonstration that mGM-CSF can also stimulate the proliferation and development of eosinophil, megakaryocyte, and primitive erythroid cells [142], effects once thought exclusive to mIL-3. That mGM-CSF can stimulate the proliferation of multi-potential and erythroid progenitors was first postulated by Metcalf et al [143]. In agar cultures, mGM-CSF alone was unable to stimulate the formation of erythroid colonies. However, when acting in concert with EPO, numerous erythroid colonies were formed [143]. Furthermore, when mouse marrow or fetal liver cells were cultured in the presence of purified mGM-CSF prior to stimulation with activated-T cell conditioned medium, mGM-CSF was found to increase the survival of cells capable of forming erythroid, eosinophil, megakaryocyte, granulocyte/macrophage, and mixed colonies [143]. It is now accepted that mGM-CSF can act on cells within the stem cell compartment. Crosslinking studies with ^ I - m G M - C S F have identified a single cell receptor of 130 kd [145], although, Scatchard analysis consistently reveals the existence of both high affinity (apparent of 20-60 pM) and low affinity (Kp of 1000 pM) classes on a variety of cell types [144]. Another report, however, has confirmed the existence of only the low affinity receptor class [145]. Human Granulocyte/Macrophage-Colony Stimulating Factor fliGM-CSF) Human GM-CSF was first purified to homogeneity from medium conditioned by the human T lymphotropic virus-infected cell line, Mo [81]. A cDNA clone from a Mo library was obtained [91], and subsequently expressed in COS cells. Somatic cell hydrid analysis and in situ hydridization have assigned the hGM-CSF gene to 5q21-32 [166], a region that is deleted in the '5q" syndrome'. The amino acid sequence predicts two N-linked glycosylation sites, and it is the variable glycosylation at these sites that 22 accounts for the heterogeneity of both purified recombinant and native hGM-CSF [91]. Surprisingly, E coli-derived recombinant hGM-CSF is ten times more active in biological assays than native hGM-CSF, suggesting that glycosylation is not important for activity, and perhaps, may actually hinder binding [136]. Although the murine and human GM-CSF genes are 70% homologous, there is no cross-species activity [233]. Human GM-CSF stimulates progenitors of all lineages. Highly purified hGM-CSF directly promotes granulocyte/macrophage and eosinophil colony formation. Furthermore, when EPO is added, BFU-E and CFU-GEMM colonies can also be increased in number [234-236]. Human GM-CSF also has a variety of effects on mature cells. It inhibits neutrophil migration [81] while simultaneously inducing antibody-dependent cytotoxic properties in neutrophils and eosinophils [79,80]. Human GM-CSF also increases neutrophil phagocytic activity [233]. Its effects on mature granulocytes suggest that hGM-CSF is a key mediator of inflamatory and cellular immunity processes. Interleukin 7 (IL-7) Murine IL-3 and mGM-CSF display only weak, if any, direct activity on lymphoid progenitors. Thus, to make this Class I list complete, another factor that specifically stimulates primitive lymphoid cells has been included. Interleukin 7 is a lymphokine that directly stimulates the proliferation of both pro-B and pre-B cell types, but not mature B cells [228]. Pro-B cells are the earliest identified cells committed to the B lineage. Heavy and light chain immunoglobulin genes are in the germline configuration and these cells do not express the B cell-specific, B220 antigen [228]. Pro-B cells differentiate into pre-B cells, which contain rearranged heavy chain immunoglobulin genes that are expressed cytoplasmically. Interleukin 7 is also capable of inducing the proliferation of thymocytes. CD4" CD8" T cell populations, isolated from day 13 fetal thymuses, have been shown to respond to IL-7. Thus, IL-7 can stimulate cells at the earliest stage of intrathymic differentiation thus far identified [229]. The proliferative 23 effect of IL-7 on T cell progenitors is consistent with the finding that the highest level of IL-7 message expression is in the murine thymus [228]. Mature T cells are not sensitive to IL-7. c. CLASS II HGFs Class II factors act on more mature progenitor cells, apparently required only for terminal differentiation steps [77]. Class II HGFs can influence the proliferation, differentiation, and survival of many mature hemopoietic cell types as well. Macrophage-Colony Stimulating Factor (M-CSF) Macrophage-CSF has been purified from serum-free medium conditioned by cultured murine L-cells [153]. Purified M-CSF (also known as CSF-1) is a dimeric glycoprotein of variable molecular weight (45-90 kd) and is composed of two identical subunits [154]. The active dimeric form is maintained by disulphide bridges and mild reducing conditions result in loss of biological activity. Although a substantial portion of the molecule is composed of carbohydrate (40-60%), N-deglycosylated M-CSF retains full biological activity in vitro. The gene for M-CSF has been cloned [155] and, based on the structure of its transcripts, the existence of both secretory and membrane-bound forms has been postulated [156,157]. The predicted molecular weight of the polypeptide backbone of a single subunit is 14.5 kd [154]. Thus far, M-CSF is the only HGF described that is specific for the macrophage/monocyte lineage. However, at high concentrations it can stimulate the more primitive bipotent CFU-C [154], and it can also promote survival of morphologically distinguishable end cells [154]. When acting in concert with other factors, however, M-CSF can also stimulate the proliferation of cells far more primitive than CFU-C [158]. Thus, primitive hemopoietic cells must possess M-CSF receptors [154]. The HGFs necessary to prime primitive cells for M-CSF response are mIL-3 or IL-1. Interleukin 1 is a class III HGF and will be discussed shortly. It is interesting to note here that IL-1 alone has no growth promoting effects on primitive hemopoietic cells [154]. 24 The M-CSF receptor has proven to be particularly interesting in that this 170 kd glycoprotein receptor possesses an intrinsic tyrosine kinase activity that is stimulated by M-CSF binding [159]. Of special note is the apparent homology of this receptor to the viral oncogene v-fms [159]. The v-fms oncogene of the McDonough strain of feline sarcoma virus (FSV) encodes a 140 kd transmembrane glycoprotein [160]. The expression of this oncogene is essential for FSV-induced cellular transformation [161]. That aberrant expression of the normal M-CSF receptor gene might also predispose an individual to malignancy is supported by data derived from patients with '5q~ syndrome'. The M-CSF receptor gene locus has been assigned to the distal long arm of human chromosome 5 [162]. Deletions involving this region of the chromosome have been detected in the 5q" syndrome, a pleiotropic hemopoietic disorder consisting of refractory anemia, mild myeloid hyperplasia, the presence of hyperlobulated bone marrow megakaryocytes, and peripheral thrombocytosis [163]. It has been found that patients hemizygous for the M-CSF receptor gene (i.e. bone marrow cells with one normal and one q" chromosome 5) have a propensity to develop myelogenous leukemia [164]. As noted before, the gene encoding hGM-CSF is located in close proximity to the M-CSF receptor gene and is also deleted in this syndrome [166], and thus, it is difficult to determine which gene deletion, if either, plays a more important role in the pathogenesis of the '5q~ syndrome'. Granulocyte-Colony Stimulating Factor (G-CSF) Initially purified from medium conditioned by lung cells of mice injected with bacterial endotoxin [167], mG-CSF is distinguished from the other murine CSFs by virtue of its ability to stimulate human cells [168]. The human form of G-CSF was the first to be cloned [89]. Subsequently, because of the high degree of homology between murine and human G-CSF, a human cDNA probe was used to obtain a full length mG-CSF clone from a murine fibrosarcoma NFSH cell cDNA library [84]. This clone encoded a polypeptide consisting of a 30 amino acid signal sequence, followed by the mature mG-CSF sequence of 25 178 amino acids (19 kd) [84]. Purified secreted mG-CSF has a molecular weight of approximately 24.5 kd [167]. When mG-CSF is added to agar cultures of mouse bone marrow, granulocytic colony formation is stimulated [169]. At high concentrations of mG-CSF (100 pM), a small proportion of macrophage and granulocyte/macrophage colonies also develop [169]. Purified preparations of mG-CSF also have the unique ability to induce differentiation of murine myeloid leukemia cells [170]. In addition, hG-CSF has been shown to stimulate primitive hemopoietic progenitors [237], and was, in fact, initially purified as 'pluripoietin' by virtue of these effects. It has since been postulated that the pluripotent effects of hG-CSF are indirect, requiring the presence of T cells [238,239]. A recently reported synergy between mG-CSF and hIL-3 may explain the stem cell activating properties of hG-CSF [240]. However, Leary et al [241] still contend that hG-CSF, and not hIL-3, initiates cell cycling of hemopoietic stem cells. Murine G-CSF receptor expression is restricted primarily to granulocytic cells at all stages of differentiation, and the number of receptors per cell is between 300-700 [171]. Only one affinity class has been identified, with an apparent KJ-J for ligand binding of 100 pM [171], and the molecular weight of the receptor has been estimated to be 150 kd [172]. Erythropoietin (EPO) Human EPO was first purified to homogeneity from the urine of aplastic anemia patients [173]. The human EPO gene has been cloned and codes for a 197 amino acid peptide, 27 N-terminal residues of which function as the signal sequence [88]. The murine EPO gene has also been cloned [83] and is highly homologous to the human gene [174]. Transfection of the human EPO gene into COS cells has provided a source of recombinant EPO [77]. The size of this recombinant material is 18.4 kd (native EPO is 34-39 kd). There are three N-linked glycosylation sites at residues 24, 38 and 83, and EPO is glycosylated at O-linked sites as well. The amino acid sequence of EPO includes 4 cysteines, at least two of which participate in disulphide bonding, since activity of the molecule is lost following mild reduction [77]. 26 G e n e t i c a n a l y s i s has revea led a s i ng le h u m a n E P O gene w i t h 5 exons [77] and s tud ies w i t h s o m a t i c c e l l h y b r i d s and in situ h y b r i d i z a t i o n have l o c a l i z e d the gene to 7 q l l - q 2 2 [175] . T h e m u r i n e E P O gene has b e e n a s s i g n e d to c h r o m o s o m e 11 [ 176 ] . B o t h n a t u r a l a n d r e c o m b i n a n t E P O s t i m u l a t e the t e r m i n a l d i f f e ren t ia t i on o f C F U - E and s o m e mature B F U - E [177-179 ] . T h e subset o f E P O - r e s p o n s i v e B F U - E do not requ i re any o ther source o f bu rs t -p romot ing ac t i v i t y ( I L -3 o r G M - C S F ) f o r the i r t e rm ina l ma tu ra t i on . O t h e r less mature B F U - E requ i re bu r s t - p romo t i ng ac t i v i t y to d i f fe ren t ia te in to an E P O - r e s p o n s i v e state [177] . R e c e n t l y , the gene e n c o d i n g the E P O recep to r has b e e n c l o n e d [146 ] . T h e p r e d i c t e d s i z e o f the p ro te in f r o m the sequence o f the E P O receptor c D N A c lone is 55 k d , and is i n d i rect contrasts to the s ize o f the t w o po lypep t ides c r o s s - l i n k e d to ^ I - l a b e l l e d E P O at the sur face o f t ransfected C O S ce l l s (85 k d and 100 kd ) [146] . A t present , the reason fo r th is d i sc repancy is unc lear . W h e n . a p p r o p r i a t e l y e x p r e s s e d , the s i n g l e E P O r e c e p t o r gen& appea rs to c o d e f o r b o t h h i g h a f f i n i t y ( 3 0 p M ) a n d l o w a f f i n i t y ( 2 0 0 p M ) receptor c lasses [146]. In te r leuk in 2 ( IL -2 ) I n t e r l e u k i n 2 , an ac t i va ted T c e l l p r o d u c t , s t imu la tes T c e l l p r o l i f e r a t i o n . B e c a u s e T c e l l c l o n a l p r o l i f e r a t i o n af ter an t i gen c h a l l e n g e is o b l i g a t o r y o n l y fo r i m m u n e respons i veness [220] , I L - 2 does not appear to have a ro le i n the regu la t ion o f m y e l o p o i e s i s . In terest ing ly , the o v e r a l l g e n o m i c o rgan i za t i on o f the I L - 2 gene is r emarkab l y s i m i l a r to the structure o f I L - 4 and G M - C S F genes [221]. T h i s suggests that a f a m i l y o f s i m i l a r pro te ins m a y have e v o l v e d f rom a c o m m o n genet ic p rogen i to r [220]. T h e r e are at least t w o sur face I L - 2 b i n d i n g pro te ins : one is the 55 k d (p55) pept ide that reacts w i t h the an t i -Tac m o n o c l o n a l an t ibody , and the other is a 75 k d (p75) n o n - T a c p ro te in [339] . W h e n expressed i n d e p e n d e n t l y , b o t h p 5 5 a n d p 7 5 r e p r e s e n t l o w a f f i n i t y , a n d i n t e r m e d i a t e a f f i n i t y I L - 2 r e c e p t o r s , r espec t i ve l y ( K D <10"^ M ) ; h o w e v e r , w h e n c o - e x p r e s s e d , bo th p ro te ins assoc ia te to f o r m a h i g h a f f in i ty 27 j IL-2 receptor ( K D of I O " 1 1 M) [339]. Of these three receptor forms, only the high affinity receptor and the low affinity p75 receptor appear able to transduce IL-2 activation signals [340,339]. Interleukin 5 (LL-5) A factor originally derived from the B151 T cell hybridoma [222], T cell-replacing factor (TRF), was first shown to promote IgM secretion by the BCLj B cell line and induce hapten-specific IgG production in vitro and in vivo by antigen-primed B cells [222-224]. When TRF was demonstrated to have identity with eosinophil differentiation factor (EDF) by molecular cloning techniques [225], it was proposed that this factor be called IL-5 [225]. In work by Yamaguchi et al [226], mIL-5 alone was demonstrated to support eosinophil colony formation from both human and murine bone marrow cells. Moreover, mIL-5 was shown to maintain mature eosinophils in liquid culture. As with G-CSF, murine and human IL-5 are quite homologous (77% at the nucleic acid level) and this undoubtedly accounts for their cross-species reactivity [227]. d. CLASS III HGFs This group of HGFs possesses no intrinsic colony stimulating activity, although they are able to enhance the potency of certain class I and II HGFs [77]. Interleukin 1 (IL-1) As alluded to earlier, IL-1 can stimulate primitive hemopoietic progenitors. Functionally, IL-1 activity manifests itself only in the presence of other, later-acting CSFs. Synergy has been demonstrated between IL-1 and M-CSF, IL-3, GM-CSF and G-CSF [180-182]. That this effect is mediated through the indirect release of IL-6 has not been ruled out. Two distinct forms of IL-1 have been identified, IL-1 a and IL-1 (3. The amino acid sequences for human E L -la and p are only distantly related, with only 70 of 271 amino acid residues shared (26% homology) [183]. However, half of the conserved positions occur in the 28 17.5 kd, biologically active, C-terminal portion. Moreover, both forms appear to bind to the same receptor, and have similar, if not identical, biological activities [184,185]. Also of note is that neither IL-lct nor IL-ip are synthesized with a hydrophobic signal sequence, leading to the hypothesis that IL-1 may be released primarily by damaged cells [183]. Interleukin 1 was initially characterized by its ability to induce the proliferation of T lymphocytes [186], and can elicit a variety of other biological responses as well. These stimulatory effects include: bone marrow resorption [187], fibroblast proliferation [188], acute phase protein release from hepatocytes [189], cartilage breakdown [190], and fever [191]. More recently, IL-1 has been shown to induce a variety of mesenchymal cell types to secrete several different growth factors [192-194], including CSFs [195-198]. Taken together, the spectrum of activities stimulated are consistent with a role of IL-1 as a mediator of inflamation. Interleukin 4 (IL-4) Interleukin 4 was first described as a cofactor for the proliferation of B cells stimulated through the cross-linking of their membrane lg by anti-Ig antibody [199], since B cell proliferation was not stimulated by either component alone. Since then, IL-4 has been purified to homogeneity [200], and cDNA clones of the gene obtained [201,202] and expressed in various systems. This 20 kd glycoprotein binds with high affinity ( K D of 200 pM) to membrane receptors found on most hemopoietic cell lines [203-205]. The murine IL-4 receptor has been identified as a 60-70 kd protein [203,204], while the human receptor is 140 kd [205]. Interleukin 4 has a broad range of effects on myeloid cells. It collaborates with mIL-3 to promote mast cell division [206] and can replace mIL-3 in synergizing with EPO to stimulate erythroid colony formation from BFU-E [207]. Furthermore, IL-4 stimulates macrophages to differentiate into a cytotoxic phenotype [208]. 29 Interleukin 6 (IL-6) Although IL-6 is listed as a class III HGF, the properties of this molecule allow it to be grouped into any of the three classes thus far described. From molecular cloning studies [211,212] it was learned that a factor involved in the terminal differentiation of B cells to antibody-producing plasma cells [209] (B cell stimulatory factor), secreted by fibroblasts induced by poly (I)-poly (C) (interferon f32) [210] and that possessed both myeloma-plasmacytoma growth factor [217] and hepatocyte stimulatory [218] activities was the same molecule. Subsequently, this pleiotropic factor was named IL-6 [213]. Further studies have demonstrated that IL-6 has amino acid sequence homology with another CSF, G-CSF [214], and that the organization of the IL-6 gene shares considerable structural homology with the G - C S F gene. This has led to the speculation that IL-6 has colony stimulating activity [215]. This hypothesis has been validated recently by Wong et al who showed that IL-6 synergistically supports the formation of several different types of hemopoietic colonies, including those derived from early blast cells, and, directly stimulates the proliferation of granulocyte/macrophage progenitors [216]. The IL-6 receptor has recently been cloned by Yamasaki et al [219]. The receptor consists of 468 amino acids, including a 19 amino acid signal peptide and a stretch of 90 amino acids that form a structure similar to the unit domain structure within the immunoglobulin superfamily [219]. The cytoplasmic domain (82 amino acids) lacks a tyrosine kinase consensus sequence. TC-1 Synergistic Activity A n activity that synergizes with M-CSF is produced by the TC-1 cell line. These murine adherant fibroblast-like cells were originally derived from liquid marrow culture of C57BL/6J mice [262]. The synergistic activity in TC-1 conditioned medium can be separated from M-CSF (which is also produced by the line) using anti-M-CSF antibody affinity columns [182]. Although not as yet purified to homogeneity, TC-1 synergistic activity appears to be a glycoprotein (binds to Con-A) and has an apparent molecular weight of 60 kd (as assessed by gel filtration). Interestingly, this activity shares many biochemical and 30 biological characteristics with IL-1, and is only distinguished from the latter by its ability to bind Con-A [182]. e. CLASS IV HGFs Class IV HGFs can only stimulate hemopoiesis through indirect, non-synergistic events. Many of these factors induce stromal cell production of other CSFs. Platelet-Derived Growth Factor (PDGF) Although reviewed in greater detail in section 1.3.1, PDGF warrants mention here because of its stimulatory effects on hemopoietic cell cycling [242]. This glycoprotein is produced by a variety of cell types, including endothelial cells and tissue macrophages [243,244]. PDGF secretion from these cells is induced by endotoxin, tumor necrosis factor-a and phorbol esters, while PDGF release from platelets is stimulated by platelet aggregation [244]. The effects of PDGF on hemopoietic cells are mediated by induction of GM-CSF production in mesenchymal cells within the stroma [245]. Tumor Necrosis Factor Alpha (TNFct) TNFa was first described as a serum factor responsible for the effects (including hemorrhagic necrosis) of endotoxin [246]. Further characterization of TNFa revealed that, although it induced hyperplasia of the reticuloendothelial system, it also inhibited the growth of neoplastic cell lines in culture and did not affect the growth of mouse embryonic fibroblasts [246]. TNFa has now been purified [247] and its gene cloned [248]. The molecular weight of monomeric TNFa is 17 kd [247], though the molecule is thought to exist as a dimer or trimer in vivo [249]. The non-hemopoietic activities of T N F a include: stimulation of bone resorption [250], enhancement of procoagulant activity [251], and induction of class I major histocompatibility expression on endothelial cells and fibroblasts [252]. Recently, cachectin, a factor derived from endotoxin-stimulated 31 murine macrophages that inhibits lipoprotein lipase activity in cultured adipocytes [253], has been purified to homogeneity [254], N-terminally sequenced, and found to be highly homologous, if not identical, to TNFa. A human serum factor, lymphotoxin, which also possesses TNFct activity, has recently been cloned [255]. Although not identical to TNFa, lymphotoxin demonstrates significant homology (46%) [256] and has now been designated as TNFp [257]. The relationship between TNFa and hemopoiesis stems from the production of TNFa by activated monocytes [258] and mitogen-stimulated peripheral blood leukocytes [259]. The released TNFa, in a manner analogous to IL-1 and PDGF, then induces the expression of G-CSF and GM-CSF genes in stromal cells [260,261]. f. CLASS V HGFs Class V HGFs represent a group of growth regulatory molecules that are distinguished by their profound inhibitory effects on hemopoiesis. Because stimulating activities are far more amenable to study, there are few hemopoietic inhibitory factors that are well characterized. One such inhibitory factor has been described by Lord et al [263] as a factor able to decrease the proportion of CFU-S engaged in DNA synthesis. Further experimentation demonstrated that this factor prevented CFU-S entry into S phase [264]. Complete purification of this ill-defined factor has not yet been reported. Studies of numerous other inhibitory activities tend to follow this same pattern, with promising results in biological assays and subsequent failure in biochemical characterization [142]. Clearly, hemopoietic inhibitory molecules do exist and more conclusive data demonstrating direct effects of inhibitory factors are required. Nonetheless, there are two inhibitory molecules that have been well studied. Tumor Growth Factor Beta TGFb* The discovery that many different cell types synthesize T G F p [265-267] and that virtually all cells have surface receptors for T G F p [268-271] stimulated speculation that this molecule might be a key 32 regulator, capable of acting by paracrine or autocrine mechanisms. TGFP is a 25 kd dimer that is comprised of two identical disulphide-linked 112 amino acid peptides [272,273]. The near-complete evolutionary conservation of the TGFP gene (human and murine TGF differ by one amino acid [274,275]) is further testament to the biologic importance of this molecule. Initially, TGFp was described as having profound inhibitory effects pn primary cultures of hepatocytes [271], embryonic fibroblasts [278], bronchial epithelial cells [279], and on both neoplastic and non-neoplastic cell lines [280-282]. However, more recently TGFP has been shown to function as a potent inhibitor of hemopoiesis as well [276,277]. The initial evidence to suggest that TGFP could inhibit hemopoietic cell proliferation was the demonstration that many factor-dependent, murine myeloid cell lines could be inhibited by TGFp [283]. Subsequently, it was shown that TGFP could inhibit the proliferation of primary hemopoietic cell cultures [276,277]. Interferons Interferons (IFNs) are a family of polypeptides that possess antiviral activity and can alter the growth behavior of a variety of normal and malignant cells in vitro and in vivo [284]. They are produced by many different cell types in response to induction by a variety of agents, including viral infection. IFNs are able to inhibit the growth of cycling cells and also of quiescent cells that are re-entering the Gj -phase of the cell cycle [285]. Furthermore, p-IFN can inhibit the proliferation of primitive myelopoietic progenitors by suppressing c-myc expression [286]. 1.2.4 In Vivo Activities of HGFs The availability of mammalian and/or bacterial forms of recombinant HGFs has made possible large scale animal and human trials with a variety of different HGFs. An initial problem encountered in these investigations was the rapid clearance of HGFs from the circulation [77]. The introduction of infusion pumps and/or multiple injection regimens has alleviated this problem. 33 A single injection of mIL-3 (into a mouse) leads to a rapid recruitment of quiescent stem cells into active cell cycle [287]. Continuous administration of mIL-3 results in dramatic increases in CFU-S and committed progenitor compartments and causes the adult murine spleen to become hyperplastic and myelopoietic [288,289]. Phase I trials involving administration of hIL-3 to patients with advanced progressive neoplasms or bone marrow failure have recently been conducted [290]. In the nine patients evaluated, substantial increases in WBC, absolute segmented neutrophil, eosinophil, monocyte, reticulocyte and platelet counts were observed [290]. Of these effects, the thrombopoietic effect of hIL-3 was the most dramatic. As observed with mIL-3, administration of mGM-CSF expands both CFU-S and committed progenitors [142]. Continuous intravenous infusion of GM-CSF into normal monkeys dramatically raises leukocyte counts within 1-3 days of the onset of administration [291]. Of greater significance is the finding that GM-CSF given to a pancytopenic, immunodeficient monkey with a simian virus-induced aplasia and undetectable levels of leukocytes, causes white cell counts to reach supranormal levels [291]. Similarly, G-CSF causes a dose-dependent increase in neutrophil counts in primates [77]. Clinical settings where HGF therapy may prove beneficial are list below [292]. Reduction of cancer treatment morbidity. Myelosuppression and febrile neutropenia are common clinical syndromes following high-dose chemotherapy. The administration of such potent granulopoietic stimulators as IL-3, GM-CSF, or G-CSF can reduce the occurrence of these side-effects and may also decrease toxicity levels of various chemotherapeutic agents (i.e. raise the maximum tolerated dose). Recruitment of malignant cells into S phase. By virtue of direct or indirect stem cell activating properties, administration of certain HGFs might enhance the susceptibility of malignant cells to the cytotoxic activity of cycle-specific agents. Bone marrow transplantation. The time between transplantation and recovery of blood cell counts is a critical period, in which patients are vulnerable to a variety of opportunistic, virtually untreatable infections. HGF therapy may promote graft recovery,-thus shortening the window of patient risk. 34 Differential induction. Human G-CSF can induce the differentiation of leukemic cell lines in vitro. Administration of such a factor in vivo might terminate self-renewal and uncontrolled proliferation of the malignant hemopoietic clone. Bacterial sepsis. Any HGF that increases neutrophil counts can serve as an important adjuvant to therapy. More specifically, GM-CSF can directly activate macrophages and will therefore augment the cellular immune response. G-CSF may also enhance host defenses [293]. Phase I/II trials have begun for hTL-3, GM-CSF, G-CSF, and EPO. Specific diseases which might be amenable to HGF therapy include: idiopathic and cyclic neutropenia, aplastic anemia, AIDS, hairy-cell leukemia, chronic-lymphocytic leukemia and refractory anemias (i.e. 5q- syndrome, etc.). Erythropoietin, although only a terminally acting HGF (class III), has proven particularly useful clinically. Phase I/II trials with EPO have been completed, and patients with end-stage renal disease appear to benefit greatly from EPO therapy [294,295]. The clinical use of HGFs is not without problems. A recent study of transgenic mice, with continuously elevated GM-CSF production, indicates a number of systemic abnormalities [296]. In addition, Dedhar et al [338] have recentiy shown that hGM-CSF can stimulate the proliferation of two osteogenic sarcoma cell lines, a breast carcinoma cell line, a simian virus 40-transformed marrow stromal cell line, and normal marrow fibroblast precursors. Their findings raise the possibility of adverse side effects of hGM-CSF therapy in patients whose malignant cells may be directly stimulated by hGM-CSF. 1.2.5 Physiologic Roles of Hemopoietic Growth Factors It is difficult to assign physiologic roles for HGFs. Although intravenous administration provides some insight into their biological activities in vivo, the value of many of these trials (save for EPO) should not be over estimated. It is curious that in normal healthy animals (including humans), the serum concentrations of many HGFs are below the level of current detection. Thus, the data obtained from in 35 vivo administration studies and/or transgenic animals [296] is derived from a grossly perturbed system, with serum concentrations of HGFs several orders of magnitude above normal. What then is the true in vivo function of IL-3, GM-CSF, G-CSF, etc.? Interestingly, in the best available in vitro model of hemopoiesis, long-term bone marrow culture [297], medium conditioned by murine marrow stromal cells contains little, if any, colony-stimulating activity [297]. Furthermore, CSF gene expression has been difficult to demonstrate in marrow stromal cells of hemopoietically active cultures [142]. To help explain this apparent enigma, it has been hypothesized that, in fact, CSFs are produced by stromal cells, but in such low concentrations that they escape detection [142]. That hemopoietic cells require only 5-10% HGF receptor occupancy for maximal stimulation is consistent with this hypothesis. Moreover, because direct contact of the hemopoietic cells with the stroma is required to stimulate hemopoiesis [297] and altered transcripts for mIL-3 and mGM-CSF genes have been discovered [119], there is evidence to support the existence of membrane bound HGFs, which would provide a particularly efficient delivery system and reduce the need for soluble CSFs. Amid this uncertainty, recent proposals argue in favor of a key regulatory role for HGFs. For example, it has been suggested that CSFs are important mediators of resistance to acute infections [298]. Also, Emerson and Gale [299] suggest that HGFs produced by T cells regulate hemopoiesis following bone marrow transplantation, since T cell-depleted marrow is less successfully engrafted. Presumably, by removing donor T cells (and irradiating the recipient), production of HGFs by the few infused stromal cells is insufficient to support engraftment. Finally, abnormal HGF production may be responsible for hemopoietic disturbances observed in graft-versus-host disease [300]. These examples illustrate that the overall function of many HGFs may be to provide for immune regulation of the hemopoietic system. In normal animals, intrinsic stochastic processes alone may control hemopoietic differentiation, while HGFs, specifically CSFs, might function only in a permissive (survival) role. However, in times of stress, activation of the immune system by Class III and IV HGFs (IL-1, TNFa, etc.) might induce dramatic elevations in CSF concentrations with subsequent elevation of blood cell 36 counts. Induction of specific CSFs (e.g. GM-CSF) would permit the activation of lineage-specific differentiation pathways (e.g. granulocytic/monocytic) needed to combat specific injuries (e.g. sepsis). In this model, the selective action of intrinsic (stochastic) and extrinsic (HGFs) forces serve to regulate the hemopoietic system. 1.3 MECHANISM OF ACTION OF GROWTH FACTORS Studies into the mechanism of action of HGFs have only recently been initiated. However, during the past several years, data derived from studies with 'non-hemopoietic' growth factors have provided the framework for a model of the early events involved in mitogenesis. Since many of the mitogenic signal transduction pathways defined by these studies may eventually be found to be utilized by HGFs, a brief review of these pathways is provided. It is now well established that a group of serum-derived, tissue specific, polypeptides are essential non-nutritive substances that provide regulatory signals for the control of proliferation of cells both in vivo and in vitro [343]. These growth regulatory molecules, collectively termed growth factors, have been demonstrated in vivo to participate in the development of the embryo [344] and to function in tissue renewal and injury repair in the adult [345]. Quiescent cultures of cells deprived of growth factor in vitro have been shown to re-enter the cell cycle upon addition of tissue specific growth factors [346]. Growth factors are delivered to their target tissues by a variety of mechanisms [343]. Among several delivery systems, perhaps the most interesting is autocrine growth factor production, whereby the target cell produces its own growth factor [347]. Although this delivery system participates in normal growth control in vivo, autocrine production of growth factor has also been identified as a mechanism of neoplastic transformation [347]. The ability of a growth factor to stimulate cell proliferation is dependent upon expression of high affinity surface receptors specific for that particular growth factor. When bound by their ligand, it is then 37 the responsibility of the cohort of activated cellular receptors to transduce the growth signal into the cell. Although they differ by virtue of their binding specificities, a peculiar feature common to most growth factor receptors is that they are members of a family of tyrosine-specific protein kinases. Kinase activity in a polypeptide is certainly not a novel finding, since the existence of serine and threonine-specific kinases was established several years ago [371]. What makes growth factor receptors so unique among kinases is that until their discovery, only certain enzymes encoded by some RNA tumor viruses had been shown to have tyrosine-specific kinase activity [348]. Furthermore, the tyrosine kinase activity of these viral proteins was shown to be essential for the acute transforming ability of the viruses [348]. It has now been established that growth factor receptor tyrosine kinase activity is essential for normal receptor function [349]. Platelet-derived growth factor (PDGF) and its cell receptor typify many growth factor/growth factor receptor characteristics, and thus, will be described in detail here. The PDGF system is particularly interesting since PDGF, unlike many other growth factors, has been demonstrated to stimulate DNA synthesis and cell division of target cells (e.g. Swiss 3T3 fibroblasts) in the absence of any other growth factors [346]. Thus, a review of this system will serve to provide a basis for the regulation of normal cellular growth and possibly suggest mechanisms for improperly regulated, malignant growth. 1.3.1 PDGF PDGF was initially described as a mitogen present in serum derived from platelet rich plasma (and not from platelet poor plasma) that supports the growth of fibroblasts in vitro [350]. Further work demonstrated that this same mitogen was, in fact, sequestered within circulating platelets [351]. PDGF was subsequently purified from platelet-rich blood [352,353] and characterization revealed it to be a heat stable glycoprotein with a molecular weight ranging from 28-35 kd (due to heterogeneity in glycosylation [354]). Further studies demonstrated that PDGF was a dimer consisting of di sulphide-linked 17 kd subunits. The mature dimeric PDGF molecule can be constructed from two nonidentical 17 kd subunits (A 38 and B) or homodimers of either [355-357]. Claesson-Welsh et al [358] have cloned a PDGF receptor which, when expressed in Chinese hamster ovary (CHO) cells, binds BB-PDGF with high affinity, AB-PDGF to a lesser extent, and does not bind AA-PDGF. Although there is significant sequence homology between the A and B chains (40%) [357], much more surprising is the virtual identity between the B chain and part of the transforming protein, p28sis, of the simian sarcoma virus (SSV) [359]. It is likely that v-sis represents a transduced, mutated B chain gene [357]. Aside from a deletion at the 5' end of the v-sis gene, v-sis is 94% homologous to c-sis [357]. The v-sis gene product acquires a 5' stretch of hydrophobic amino acids (analogous to a signal sequence) by fusion with a viral envelope gene [360]. Subsequent dimerization and processing results in the formation of p28sis, which is virtually identical in structure to BB-PDGF [357]. The mechanism by which p28 s i s induces cell transformation was controversial until recently. Although endowed with a surrogate leader sequence by virtue of v-sis-env fusion, p28 s i s has been shown by Robbins et al [361] to remain cell associated. Very little PDGF-like activity could be recovered from culture supernatants of SSV-transformed cells. Yet there is considerable evidence to suggest that p28 s l s functions primarily as a PDGF agonist. First, successful SSV transformation is restricted to PDGF receptor positive cells [357]. Furthermore, antibodies to p28 s i s have been shown to induce revertant phenotypes in SSV transformed cell populations [362]. Finally, an agent that prevents PDGF receptor occupancy (suramin) converts SSV-transformed cells to the wild-type phenotype [363]. Although these data support the hypothesis that p28 s i s is secreted by SSV infected cells and stimulates the same cells through the membrane PDGF receptor, the extremely low PDGF receptor surface expression on SSV-transformed cells [364] suggests another mechanism. Keating and Williams [366] have presented evidence to suggest that the autocrine loop of SSV-transformed cells is mediated through cytosolic, rather than surface, PDGF receptors. After demonstrating that partially processed forms of the PDGF receptor (i.e. those likely to be encountered in intracellular membrane compartments) are capable of normal receptor function [365], these investigators provide convincing data that it is these incompletely processed, 39 intracellular receptor forms that participate in the transduction of mitogenic signals in SSV-transformed normal rat kidney (NRK) cells [366]. More specifically, low molecular weight PDGF receptor precursors have been shown to be autophosphorylated on their tyrosine residues in SSV-transformed cells, whereas similar precursor molecules are not autophosphorylated in untransformed cells, even in the presence of exogenous PDGF. Also, all kinase active forms of the PDGF receptor in SSV-transformed cells are trypsin resistant (i.e. are intracellular), while receptor forms that are activated in untransformed cells by the addition of PDGF (i.e. only mature receptors) are susceptible to trypsin digestion. Clearly, for SSV-transformed NRK cells, autocrine stimulation provided by p28 s i s is effected through intracellular PDGF receptors. It would be interesting to determine if a similar mechanism of intracellular receptor activation is operative in PDGF autocrine loops of non-neoplastic cells. Placental cytotrophoblasts express high levels of c-sis transcripts, display high affinity PDGF receptors, and respond to PDGF [367]. Similarly, vascular smooth muscle cells produce and respond to a PDGF-like growth factor activity [368]. Here, the autocrine loop may function to promote smooth muscle hyperplasia, but may also direct the development and differentiation of these cells [369]. 1.3.2 Early Events Elicited by Growth Factors Subsequent to growth factor receptor activation, a cascade of temporally and spatially coordinated intracellular events leads to DNA synthesis. Within seconds of PDGF addition, phosphatidylinositol 4,5 bisphosphate (PI-4.5-P2), located on the inner leaflet of the plasma membrane, is hydrolyzed by phospholipase C to yield two biologically active second messengers, inositol 1,4,5 trisphosphate (Ins-1,4,5-P3) and diacylglycerol (DAG) [370]. Ins-1,4,5-P3 diffuses into the cytosol and stimulates the release of calcium from the endoplasmic reticulum [370], and perhaps other intracellular stores. DAG, which is lipid soluble, remains associated with the inner leaflet of the membrane and activates the calcium and phospholipid-dependent protein kinase C [371]. 40 The biological importance of the hydrolysis of PI-4.5-P2 by phospholipase C has recently been questioned [372]. It has been shown that DAG and inositol phosphates can be generated by the action of phospholipase C on phosphatidylinositides other than PI-4,5-P2 [373]. Furthermore, there is a kinetic inconsistency between the short half-life (one minute) of Ins-1,4,5-P3 generated from PI-4.5-P2 and the sustained elevation (several minutes) of intracellular calcium stimulated by PDGF [372]. Moreover, Hanburg and Rozengurt [374] have provided evidence that PDGF can elevate calcium by a mechanism not involving Ins-1,4,5-P3. What then, is the substrate for phospholipase C, if not PI-4,5-P2? Auger et al [372] argue that the hydrolysis of PI-3.4-P2 provides the second messengers responsible for the sustained intracellular calcium increase stimulated by PDGF. They hypothesize that the transient increase in Ins-1,4,5-P3 elevates calcium levels for the first minute, then Ins-1,3,4-P3 (and/or Ins-1,3,4,5-P4) provides the sustained signal for calcium elevation. Interestingly, the PI-3' kinase responsible for the formation of PI-3.4-P2 (and PI-3,4,5-P3) associates with the PDGF receptor and can be precipitated from detergent extracts with anti-phosphotyrosine antibodies [372]. The close association of the PI-3' kinase to the PDGF receptor and the possibility that the kinase can be regulated by tyrosine phosphorylation [372] may allow PDGF to regulate the activity of the PI-3' kinase, and therefore, second messengers that transduce mitogenic signals. a. Guanine nucleotide-binding proteins (G proteins) G proteins carry and amplify signals from activated membrane receptors to effector enzymes and channels [322]. Among those proteins regulated by G proteins are adenylate cyclase (coupled through P-adrenergic and serotonin receptor activation [316,317]), cGMP phosphodiesterase (through rhodopsin [318]), potassium channels (through muscarinic receptors [319]), and phospholipase C (through IgE and f-Met peptide chemotactic receptors [320,321]). Interestingly, the receptors coupled by G proteins share a common 'seven membrane-spanning' structure. Since phospholipase C activation appears to be necessary 41 for PDGF induction, a role for G proteins as essential mitogenic signal transducing agents can be postulated. G proteins isolated from a variety of cells share a common design [322]. They consist of three distinct polypeptides: a guanine nucleotide-binding a chain (39-52 kd), a P chain (35-36 kd), and a y chain (8 kd). Depending upon the state of the bound guanine nucleotide, G proteins cycle between active and inactive conformations (Figure 3). When GDP is bound, the a chain (Ga-GDP) associates with the p and Y chains (Gp^) to form the G a p y complex. This complex is inactive and is membrane associated. However, when GTP is bound, the a chain (G a-GTP) dissociates from Gp^ and begins effector function. The hydrolysis of bound GTP to GDP by Ga-GTP GTPase activity terminates the activated state. G proteins are activated by receptors that are integral membrane glycoproteins. Presumably, ligand binding initiates a conformational change in the receptor that triggers G protein activation on the inner leaflet of the plasma membrane [322]. It is interesting to note that both stimulatory (Gs) and inhibitory (Gj) G proteins have been identified. For activated G g proteins, only the G s a - G T P subunits have been shown to act as effectors; however, for Gj proteins, both G i a - G T P and G-^y can directly inhibit the function of enzyme systems (e.g. adenylate cyclase) [322]. That G s and Gj proteins can be activated by membrane receptors specific for different ligands demonstrates the potential for multi-hormonal control of numerous cell processes, including mitogenesis. b. Ion Fluxes As stated above, the addition of PDGF to human fibroblasts promotes a rapid increase in intracellular calcium levels, which reaches a concentration of two or three times that found in quiescent cells [375]. Due to the action of inositide phosphates on intracellular calcium stores, the rapid increase in calcium can occur even in the absence of extracellular calcium [376]. Mitogens which do not stimulate inositide phosphate production, increase intracellular calcium levels by a channel-mediated calcium influx 42 G-GDP Inactive state Figure 3. The GTP-GDP cycle of G proteins (reprinted from reference 322). The intrinsic GTP hydrolytic activity of the G a subunit rapidly returns the G protein to the inactive state. 43 across the plasma membrane [377]. For these mitogens, the stimulated-rise of cytosolic calcium is dependent upon the presence of extracellular calcium. This mechanism has not been shown to be utilized by PDGF. PDGF, as well as many other growth factors, increases the fluxes of sodium, potassium, and hydrogen ions across the plasma membrane [346]. Sodium ion entry is mediated through the activation of an amiloride-sensitive sodium/hydrogen ion antiport [378]. The antiporter increases intracellular sodium ion concentration and causes cytoplasmic alkalinization through expulsion of hydrogen ions [379]. The increased intracellular sodium ion concentration causes a secondary stimulation of the sodium/potassium ion ATPase, such that intracellular potassium ion concentrations are increased and sodium ions are actively extruded [378]. Studies by Pouyssegur et al [380] have clearly demonstrated that growth factor stimulation of the amiloride-sensitive sodium/hydrogen ion antiport is a regulatory signal required for mitogenesis and not simply a secondary mechanism for the extrusion of protons generated by growth factor induced increases in cellular metabolism. It has been shown that mutant fibroblasts lacking the antiport exchange failed both to elevate their cytosolic pH and initiate DNA synthesis when stimulated with mitogens at neutral pH [380]. The mechanism by which PDGF activates the antiporter is by increasing the affinity of the antiporter for internal protons [381]. More recent studies have suggested that it is the direct activation of protein kinase C (PKQ by growth factors that subsequently leads to activation of the sodium/hydrogen ion antiporter [382]. Diacylglycerol and PKC-activating agents, such as phorbol esters, increase amiloride-sensitive sodium ion uptake [383], and produce rapid increases in cytoplasmic pH [384]. Growth factors which do not stimulate phosphatidylinositol turnover (i.e. EGF) may activate the antiporter by another mechanism, possibly mediated through intracellular calcium [376]. c. Protein Kinase C The second messengers generated by inositol phospholipid hydrolysis, DAG and Ca"1"^ (mediated via IP3), cooperate to activate PKC. Since these messengers are transient in nature (DAG is rapidly 44 converted back to phosphatide acid and then to membrane phospholipid or metabolized into arachidonic acid), PKC is only briefly activated; however, within its short-lived active phase, PKC efficiently couples extracellular signals to serine and threonine-specific membrane and cytosolic protein phosphorylations. Furthermore, the consequences of PKC activation may persist for extended periods of time, depending upon the level of various intracellular phosphatases [323]. The activation of PKC normally depends on the presence of C a + 2 , DAG, and phospholipids. Interestingly, since DAG dramatically increases the affinity of PKC for C a + 2 , PKC activation can be achieved without a net increase in intracellular C a + 2 concentration [324]. Thus, PKC activity can be considered to be biochemically dependent on C a + 2 , yet, physiologically independent of changes in C a + 2 concentration [323], although the presence of increased cytosolic C a + 2 will synergize with DAG to activate PKC. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol-13-acetate) have a molecular configuration that mimics DAG and directly activate PKC both in vivo and in vitro [325,326]. Similarly, phorbol esters increase the affinity of PKC for Ca"1"2, resulting in full activation of PKC at physiologically normal concentrations of calcium. Of the phospholipids required for activity, phosphatidyl serine is absolutely essential, whereas other phospholipids (e.g. phosphatidylinositol, phosphatidylcholine, etc.) are inert [323]. Through the use of phorbol esters and calcium ionophores, it has been demonstrated that PKC activation must be accompanied by calcium mobilization to elicit full cellular responses. Potential physiologic roles of PKC [reviewed in 323] involve all organ systems. Of relevance here is that PKC, in the presence of high intracellular calcium levels, acts to promote DNA synthesis in resting lymphocytes, and when suboptimal concentrations of IL-1 are added, rapid induction of the IL-2 receptor [327,328], IL-2 release [328], and T cell proliferation ensue. Suboptimal concentrations of insulin can also synergize with PKC to stimulate proliferation of 3T3 cells [329]. Finally, phorbol ester-stimulation of PKC results in the expression of several genes [330-333] and suggests that PKC either directly or indirectly activates 45 nuclear messengers. Selected cellular substrates for PKC include growth factor receptors [334], G proteins [335], cytoskeletal proteins [336], and the src oncogene product, pp60s r c [337]. d. Cyclic Nucleotides Sustained increases in levels of cyclic adenosine monophosphate (cAMP) constitute a growth promoting signal for Swiss 3T3 cells [385]. PDGF can stimulate the accumulation of cAMP in target cells [386]; however, this alteration of cAMP metabolism induced by PDGF occurs via an indirect mechanism [387]. PDGF-generated DAG is metabolized to arachidonic acid, much of which is then converted to E-type prostaglandins. The E-type prostaglandins leave the cell and induce cAMP production by an autocrine stimulation of their own receptor [346]. Thus, cAMP elevation may be another regulatory signal that PDGF stimulates. There is no evidence, however, to suggest that all growth factors induce elevated cAMP levels. e. Early Nuclear Events PDGF stimulates the expression of the cellular oncogenes c-fos and c-myc in quiescent fibroblasts [388]. The induction of c-fos expression precedes the expression of c-myc. These cellular oncogenes may themselves regulate other growth controlling genes to amplify the mitogenic signal delivered by PDGF. It has recently been suggested that many polypeptide growth factors may regulate gene expression directly without the aid of second messengers. There is evidence to suggest that several growth factors (including PDGF) can directly bind chromatin [389]. The binding is specific and reversible, and occurs on DNAsell sensitive regions of chromatin [389]. A mechanism by which growth factors may be delivered to the nucleus is through a receptor shuttle system, whereby endocytotic vesicles carry occupied growth factor receptors to the nucleus [390]. Further work is required to substantiate this hypothesis, since PDGF has been shown to bind specific nuclear chromatin sequences in cells which do not express PDGF receptors [389]. 46 1.3.3 The Meaning of Intracelular Events Rozengurt [346] has proposed that intracellular events stimulated by mitogens can be divided into regulatory signals and obligatory events. Regulatory signals represent induced cellular processes that mediate the activity of specific mitogens. Obligatory events are common pathways shared by all mitogens. For PDGF, regulatory signals include: calcium mobilization, phosphatidylinositol hydrolysis, cytoplasmic alkalinization, protein kinase C activation, elevation of cAMP, and receptor tyrosine kinase activity. All of these events are necessary, though not sufficient, for PDGF induction of mitogenesis. Furthermore, all of these processes may not be essential to the function of other growth factors (e.g. EGF has not been shown to stimulate polyphosphatidylinositol turnover [376]). Similarly, other regulatory signals may be utilized by other growth factors (see section 1.3.5). Obligatory events are much more difficult to define, but may involve activation of nuclear regulatory proteins (c-fos/c-myc?). Mitogenesis appears far more complicated than the simple sum or cooperation of the regulatory signals listed above. Escobedo and Williams [391] have constructed a mutant PDGF receptor (lacking the kinase insert domain) which, when transfected into receptor negative fibroblasts and appropriately activated with exogenous PDGF, activates many of the established regulatory signals associated with PDGF induction (phosphatidylinositol turnover and diacylglycerol generation, receptor autophosphorylation, ion fluxes etc.), yet, does not stimulate mitogenesis. From this work it is clear that the growth factor receptor is the key cellular signal transducing agent. It is the activation of the receptor which subsequently sets into motion all of the regulatory signals discussed. The pivotal importance of the growth factor receptor in growth control can be appreciated if it is realized that some retroviral transforming genes are the transduced avian and mammalian genes encoding these receptors. 1.34 Growth Factor Receptors Many growth factor receptors have been shown to be tyrosine kinases. This family includes the cell surface receptors for PDGF, insulin, insulin-like growth factor I, EGF, and M-CSF, along with the putative 47 receptors neu/HER2 and c-kit. From structural studies of these receptors, the family of receptor tyrosine kinases can be subdivided into three distinct groups (Figure 4) [392]. Subclass I: Monomeric receptors with two cysteine-rich repeated sequences in the extracellular domain. Examples of subclass I receptors include the EGF receptor and the putative neu/HER2 receptor. Subclass II: Heterotetrameric receptors composed of two a and P subunits that are disulphide linked. The a subunit is responsible for ligand binding, while the P subunit contains the kinase activity. There is one cysteine-rich region in each a subunit, and thus, two such regions per heterotetramer. Representative members include the insulin and insulin-like growth factor I receptors. Subclass III: Monomeric receptors that lack the cysteine rich units found in the extracellular domains of subclass I and II. Instead, subclass III receptors have distinct, short cysteine rich repeating units flanked by specific sequences. These extracellular repeats comprise the ligand binding region of the receptor. The kinase domain of subclass III receptors is interrupted by a 40 amino acid insertion that is unique to this receptor class. The PDGF receptor is a member of subclass III (as are the M-CSF and c-kit receptors). The PDGF receptor consists of six distinct domains [reviewed in 392]. 1. Extracellular 2. Transmembrane 3. Cytoplasmic 4. Juxtamembrane 5. Tyrosine kinase 6. Carboxy-terminal tail The function of each of these domains can be inferred from site directed mutagenesis studies of other receptor tyrosine kinases [393]. Furthermore, Keating et al [394] have established that ligand activation of the PDGF receptor induces a phosphorylation-dependent change in PDGF receptor conformation. Although the extent of the conformational change remains to be determined, specific 48 II III iss S S I ss EGF-fl neu/HER2 y y y l-R IGF-1-R PDGF-R c-fms/CSF-1-R c-kit Figure 4. Schematic representation of the receptor tyrosine kinase subclasses I, II, and III. Reprinted from Yarden and Ullrich [55]. The PDGF receptor is a representative member of subclass III. 49 antibodies have been made which can react directly with activated PDGF receptors, yet do not bind the unstimulated receptor conformation [394]. Of particular importance to PDGF receptor function is the tyrosine kinase domain. As stated previously, the receptor kinase activation is a regulatory signal utilized by PDGF. It is not, by itself, sufficient to stimulate mitogenesis [391], though it appears to be necessary [395]. Interestingly, site-directed mutagenesis of the PDGF receptor (to abrogate the kinase activity) impairs the ability of the receptor to transduce mitogenic signals. However, this mutant receptor can still undergo ligand-induced internalization, down-regulation, and lysosomal degradation [395]. Kinase-independent receptor events may be different for other receptor tyrosine kinases. Kinase defective EGF receptors fail to undergo ligand-induced internalization in CHO cells [396], whereas, mutant insulin receptors behave similarly to mutant PDGF receptors [397]. Perhaps the most rapidly phosphorylated substrate of the receptor tyrosine kinase is the receptor itself. Although characterization of PDGF receptor autophosphorylation is currently in progress, studies with the EGF receptor have shown that there are several autophosphorylation sites in the carboxy-terminal domain of the receptor [392]. Autophosphorylation at these tyrosine residues has been implicated in a regulatory role [398-399]. Schlessinger [400] argues that the binding of EGF stabilizes its receptor in a dimeric conformation, allowing for receptor-receptor interaction and stimulation of kinase activity. Crosslinking experiments support the contention that EGF receptors dimerize [401]. Similarly, insulin binding induces receptor aggregation which might alter the conformation of the (3 subunit [402]. Currently, data are insufficient to suggest an analogous clustering mechanism for PDGF kinase activation. The search for biologically relevant exogenous substrates for receptor tyrosine kinases has been relatively unsuccessful. Thus far, only a few proteins have been identified. The PDGF receptor has been shown to phosphorylate a 36 kd peripheral membrane protein, calpactin, that binds phospholipids and calcium [403]. Another, less abundant cytosolic protein, p42, has also been shown to be tyrosine 5 0 phosphorylated in response to PDGF induction [404]. However, the tyrosine phosphorylation of p42 is modulated by PKC (a serine/threonine specific kinase) and may involve a complicated cascade of events initiated by an activated PDGF receptor kinase [404]. One of the major obstacles to identifying exogenous kinase substrates may be their low cytosolic concentrations. From studies with the EGF receptor, regulation of receptor tyrosine kinase activity is mediated by PKC. The phosphorylation of a regulatory site (threonine-654) by PKC induces the down regulation of the protein tyrosine kinase activity [405] and the attenuation of the mitogenic response to EGF [406]. Interestingly, phorbol esters, which directly activate PKC, also induce EGF receptor down-modulation [406]. PDGF also down-regulates and decreases the tyrosine kinase activity of the EGF receptor on cells where receptors for both growth factors are present [407]. The effects of PDGF on the EGF receptor are similar, though not identical, to those elicited by phorbol esters (i.e. PKC) [408]. However, PDGF 'transmodulation' of the EGF receptor can be demonstrated in cells lacking PKC [409], although it may involve other calcium-dependent pathways [392]. Evidence has not yet been presented to show that PDGF receptor kinase activity can be regulated. By virtue of their key position in growth control of the cell, receptor tyrosine kinases are ideal candidates for oncogene products. Although the transduction of cellular genes by retroviruses is rare, it is unusual that no retrovirus containing a PDGF receptor homologue has been identified, since PDGF may be considered the most potent of the growth factors by virtue of its ability to activate, by itself, quiescent cells [349]. Moreover, the genes encoding two known cellular growth factor receptors (EGF and CSF-1) have been transduced by retroviruses and have conferred acute transforming potential to these viruses. However, there is recent data that supports the hypothesis that inappropriate expression of the PDGF receptor leads to malignant transformation [41]. An anaplastic thyroid carcinoma cell line, C643, demonstrates aberrant expression of the B-subunit binding PDGF receptor isotype, which might confer a growth advantage to the cells and permit tumor development. Further site directed mutagenesis studies are needed to determine the extent of the oncogenic potential of the PDGF receptor. 51 PDGF is a key growth regulator of a variety of cell types. Although the role of this potent mitogen in vivo requires further characterization, stimulation of proliferation of endothelial cells and fibroblasts in wound repair has been demonstrated [345]. The existence of the viral transforming protein, p28 s i s, illustrates the function of PDGF-like substances in oncogenesis. The PDGF receptor is a member of the receptor tyrosine kinase family, and presumably, by virtue of its enzymatic properties, rapidly and efficiently transduces mitogenic signals across the plasma membrane. It is difficult to connect the cascade of intracellular events elicited by PDGF to receptor activation due to the lack of endogenous receptor substrates which have been isolated. It is possible that the kinase insert mutant receptor constructed by Escobedo and Williams [391] may lose specificity for an endogenous substrate which is a key intermediate in an undiscovered regulatory pathway. Further studies of this kinase insert mutant receptor and isolation of intracellular receptor substrates are required to advance the understanding of the mechanism of action of PDGF and other growth factors. A model summarizing early events of cell activation is presented in Figure 5. However, from this simplified schematic, the complex interactions between biochemical pathways cannot be fully appreciated. 1.3.5 HGF Mechanism of Action Studies of HGF mechanisms of action have been greatly facilitated by the development of factor-dependent cell lines, as experiments with primary cell cultures have been exceptionally difficult to interpret due to the tremendous heterogeneity of the cell populations and the lack of adequate cell purification techniques. As mentioned earlier, a major disadvantage of factor-dependent cell lines is that these cells rapidly lose viability in the absence of their HGF. It is therefore difficult to obtain a measure of the baseline or unstimulated state for virtually any parameter examinable, and consequently, survival functions of HGFs can not easily be distinguished from direct growth promoting activities. Even so, the use of cell lines has allowed for valuable insights into the mechanisms of action of various HGFs. 52 F i g u r e 5 . T h i s s c h e m a t i c r e p r e s e n t s t h e h y p o t h e s i z e d m o l e c u l a r p a t h w a y s b y w h i c h a c e l l m a y b e c o m e a c t i v a t e d i n r e s p o n s e t o m i t o g e n b i n d i n g 53 a. Murine IL-3 From work by Whetton et al [301,302], one of the fundamental processes for which mIL-3 seems to be required is the maintenance of intracellular ATP levels for primary cellular metabolism. Murine IL-3 is thought to regulate this function at the level of the glucose transporter. Since it had been demonstrated previously that the glucose transport protein could be activated by PKC catalyzed phosphorylation [303], Farrar et al's [304] observation that mIL-3 stimulated PKC translocation from the cytosol to the membrane is consistent with the model that mIL-3 regulates glucose uptake (and ATP generation) by indirectly altering the phosphorylation of the glucose transporter. This hypothesis predicts that other agents that can activate PKC in mIL-3-dependent cell lines will be able to mimic mIL-3 activity. Accordingly, phorbol esters have been shown by several groups to maintain these factor-dependent lines in culture in the absence of mIL-3 [305-307]. A corollary to this hypothesis is that PKC inhibition (by long-term exposure to phorbol esters) should decrease the mIL-3 responsiveness of target cells. This, however, has not yet been demonstrated. Interestingly, mIL-3 stimulates neither phosphatidyl inositol breakdown nor diacylglycerol generation [308], requirements previously thought essential for PKC activation [309]. Recent evidence suggests that mIL-3 stimulates the tyrosine-specific phosphorylation of several cellular proteins [310,311]. Of note is the observation that a 140 kd membrane protein is tyrosine phosphorylated [149,151]. Studies by Sorensen et al [149,151] indicate that this 140 kd protein is the mIL-3 receptor. There is no data, as yet, to suggest that the mIL-3 receptor is itself a tyrosine kinase. b. Other HGFs Very little is known about the mechanism of action of other HGFs. Hamilton et al [342] have recently demonstrated that M-CSF rapidly stimulates glucose uptake into quiescent bone-marrow derived macrophages. Although their studies indicate that M-CSF activates a cytochalasin B-inhibitable glucose transporter, evidence is lacking to suggest the involvement of PKC. The involvement of tyrosine phosphorylation in M-CSF action is clear, since of all the HGF receptors thus far characterized, the M-CSF 54 receptor is the only known tyrosine kinase. Recent data provided by Farrar et al [341] suggest that IL-2 acts through the stimulation of inositol phospholipid turnover resulting in the activation of PKC and calcium mobilization. Furthermore, in IL-2 responsive cells, the direct activation of PKC by phorbol esters mimics the action of IL-2, and stimulates y interferon gene transcription as well as the temporal expression of the c-fos, c-myc, and c-myb cellular oncogenes [341]. Finally, GM-CSF has been demonstrated to stimulate a variety of phosphorylation events [312]. 1.4 INTERACTIONS BETWEEN HEMOPOIETIC GROWTH FACTORS The relationships between the mechanisms of action of different HGFs have not yet been determined. Although both mIL-3 and M-CSF stimulate tyrosine specific phosphorylations, it is unknown whether common intermediates are phosphorylated, although a 90 kd cytosolic protein has been recently shown to be tyrosine phosphorylated in response to both mIL-3 and mGM-CSF [312]. An argument that favors intracellular interactions between CSF receptors has been forwarded by Walker et al [98]. They have shown that a hierarchical down-modulation of CSF receptors exists, whereby mIL-3 down-modulates all other CSF receptors, while M-CSF has virtually no down-modulation potential. Down-modulation is not down-regulation per se, but rather, is the ability of one CSF to activate the cellular receptors specific for other CSFs, thereby stimulating their internalization. The cellular mechanism of down-modulation is completely unknown. It is interesting to speculate that phosphorylated intermediates may be involved. 1.5 ROLE OF HGFs IN ONCOGENESIS From the data reviewed here, it is clear that hemopoietic cell proliferation and differentiation is finely regulated by the local (paracrine) release of a miriad of HFGs and hemopoietic inhibitory molecules. The aberrant expression of an HGF gene in a cell that is also responsive to that HGF will upset this 55 intercellular regulatory network, and may ultimately lead to perpetual cell stimulation through an autocrine mechanism. Experimental evidence supports this contention. When recombinant retroviruses containing the coding sequence of either mIL-3 or mGM-CSF are used to infect previously immortalized, yet growth factor-dependent cells, the infected cells rapidly acquire factor-independence and tumorigenicity [313]. Therefore, in the presence of an immortalizing defect, HGF genes are able to act as transforming oncogenes. When identical viral constructs are introduced into primary hemopoietic cell cultures, the cells acquire factor-independence, but the growth of cells is accompanied by terminal differentiation, not leukemogenesis [142]. There is considerable data suggesting that aberrant mIL-3 gene activation and autocrine loop formation is responsible for the development of the myelomonocytic leukemia, WEHI-3B. As reviewed earlier, mIL-3 production by WEHI-3B cells is constitutive (and due to the insertion of a promoter) rather than inducible, and is not accompanied by the secretion of other HGFs. Thus, production of mIL-3 by these cells is completely different than by T cells. Furthermore, the expression of the Thy-1 antigen by WEHI-3B is consistent with the notion that WEHI-3B is the malignant counterpart of a neutrophil-macrophage progenitor activated by mIL-3 [114]. That WEHI-3B cells neither respond to mIL-3 nor express mDL-3 surface receptors can be attributed to secondary genetic changes that have arisen during in vitro passage, since it has been reported that WEHI-3B cells were once responsive to mIL-3 [314]. An aberrant response to inhibitory factors may be another mechanism by which hemopoietic malignancy can develop. Eaves et al [315] have found that multi-potential stem cells from CML patients do not respond to a growth inhibitory molecule produced in long-term marrow cultures in vitro. Thus, CML stem cells have acquired a proliferative advantage over their normal counterparts that might permit their clonal expansion [142]. Since the chromosomal translocation (9:22) found in CML patients results in the overexpression and increased enzymic activity of the cellular oncogene, c-abl, it would be interesting to determine whether the loss of inhibitory molecule responsiveness is connected with the inappropriate expression of this oncogene. 56 1.6 THESIS OBJECTIVES Although the functions and mechanisms of action of a number of 'non-hemopoietic' growth factors have been elucidated in great detail, very little of the biology and biochemistry of hemopoietic growth factor-target cell interactions is understood. Since a role for HGFs in hemopoiesis has clearly been established, the need for such an understanding is clear. Moreover, in light of recent evidence suggesting that aberrant HGF regulation may be involved in the genesis of hemopoietic malignancy, there is considerable urgency to begin studies into the mechanisms of action of HGFs. By virtue of its pleiotropic effects within the hemopoietic system, mIL-3 may to be a key regulator of hemopoiesis. A detailed study of this HGF may therefore provide insight into the regulation of pluripotent hemopoietic stem cells and more committed progenitors. Accordingly, three specific objectives were undertaken. 1. Purification of mIL-3. When these studies were initiated, mIL-3 was not commercially available and existing purification protocols were lengthy and resulted in poor yields. Thus, the first objective was to devise a simple, high yield, purification protocol for mIL-3. 2. Interleukin 3 receptor biology. Once sufficient quantities of mIL-3 could be obtained, the mechanism of action of mIL-3 at the receptor level was investigated by examining mIL-3 receptor expression, intracellular processing, and receptor recycling under various growth conditions. 3. Characterization of the mIL-3 receptor. 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Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C deficient human fibroblasts. J Biol Chem 262:6832, 1987. 85 CHAPTER n A SIMPLE AND RAPID 3-STEP PROCEDURE FOR THE PURIFICATION OF MURINE INTERLEUKIN 3 2.1 INTRODUCTION Murine interleukin 3 (mIL-3) is a 21-36 kD glycoprotein [1] that stimulates the proliferation and differentiation of a variety of pluripotent [2] and committed [3-6] hemopoietic progenitor cells, as well as more differentiated mast cells and macrophages [7,8]. The only known physiological source of mIL-3 is the activated T cell [9], although several malignant murine T [10] and myeloid [11,12] cell lines have also been found to produce mIL-3. Like many other hemopoietic growth factors, mIL-3 is rarely found in biological fluids at detectable concentrations, and even in conditioned media has not been found at levels above 3 ng/ml [13-15]. In the past, this posed a serious obstacle to the purification and characterization of different hemopoietic growth factors. Although this has been largely overcome by gene cloning and expression, there is still a need for pure native hemopoietic growth factors in order to compare their spectrum of biological activities and in vivo half-lives with those of recombinant molecules. Previously reported procedures for purifying native mIL-3 have required 5-8 steps and have been limited by poor yields of from 4-10% [10,13-15]. In this paper a simple and rapid 3 step procedure for obtaining 5-10 pg of pure mIL-3 from 1 liter of medium conditioned by normal activated mouse spleen cells is described. Starting with pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCCM), prepared in the absence of any added protein, except insulin, the first step involves a novel target cell absorption technique. When followed by Sephadex G75 gel filtration and Cjg-HPLC, this yielded 2 mIL-3 species with molecular weights of 19.5 kd and 16.5 kd, and gave an overal yield of 16%. The simplicity and advantages of this target cel absorption-based protocol sugest a potential aplication to the purification of other ligands. 87 2.2 MATERIALS AND METHODS Materials. Pure E. coli derived recombinant mIL-3 (rmEL-3) and E. coli derived recombinant granulocyte macrophage colony stimulating factor (rmGM-CSF) were kindly supplied by Biogen, Geneva, Switzerland. (i2^I)-sodium iodide (100 mCi/ml; carrier-free) in NaOH solution, pH 7-11, and (methyl-JH)-thymidine (1 mCi/ml;2 Ci/mmol) were purchased from Amersham. Pokeweed mitogen (PWM), was obtained as a lyophilized powder from Gibco Laboratories, insulin (bovine pancreas, 1-5500) from Sigma, and glutaraldehyde (GDA) 50% (w/w) from BDH Chemicals. Other reagents, unless otherwise indicated, were purchased from Sigma. Cells. The mIL-3 dependent cell lines, B6SUtA and 32D Clone 3 (c3), were obtained from Joel Greenberger (Worchester,.MA) [16] and maintained in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS), 100 u/ml penicillin G, 100 pg/ml streptomycin and 5% PWM-SCCM. The B6SUtA cell line was initially derived from the non-adherent cell population of a murine long-term bone marrow culture [16], and represents a permanent factor-dependent multipotential hemopoietic progenitor cell line. The B6SUtAj subline is a high mIL-3 receptor expressing derivative (cloned in methylcellulose), and has been used previously [17]. B6SUtAj cells respond to a variety of hemopoietic growth factors [18] whereas 32D c3 cells have as yet only been found to respond to mIL-3 [19]. To prepare GDA fixed B6SUtAj cells, BeSUtAj cells were washed 3 times with PBS and incubated at 5 x 10^  cells/ml in PBS containing 0.01% GDA (unless otherwise indicated) for 40 min with continuous gentle rocking at 4°C. The cells were then pelleted, washed 3 times with PBS and used immediately or stored at 4°C. Preparation of Serum-free PWM-SCCM. Spleen cells from B6C3Fj mice were suspended at a final concentration of 4 x 10^  cells/ml in RPMI 1640 supplemented with 0.3% PWM, 100 pg/ml insulin, and 0.1 mM 2-mercaptoethanol (2-ME) as the only additives and then incubated at 37°C for 4-5 days. The cells were then removed by centrifugation and the supernatant passed through a 0.45 urn filter. 88 Proliferation Assay. To measure mIL-3 induced proliferation, 32D c3 cells were washed twice with RPMI 1640 and then aliquoted in 96 well microtitre plates at 2 x 104 cells per well in 100 ul of RPMI 1640 with 20% FCS plus factor. After 22 h at 37°C, 1 uCi of 3H-thymidine (2 Ci/mmole) was added to each well and incubation was continued for a further 2 h prior to harvesting the well contents onto glass fiber filters and assaying for radioactivity using a Beckman LS7500 liquid scintillation counter. One unit of activity was defined as the amount of mIL-3 that gave a half-maximal response in this assay. mIL-3 Absorption Procedure. 5 x 109 of GDA (0.01%) fixed BeSUtAj cells were first incubated with 145 ml of PWM-SCCM for 2 h at 4°C, then washed twice with ice cold PBS, and finally rocked in 50 ml of 0.1 M glycine, 0.15 M NaCl, pH 2.5, for 5-10 min at 23°C (unless specified otherwise) on an Adams nutator to release bound mIL-3. The cells were pelleted and the supernatant brought to pH 7.0 with 10 ml of 0.2 M sodium phosphate, 0.15 M NaCl, 1% Tween 20, pH 7.6. The supernatant was then filtered (0.2 um, Nalgene) and stored at 4°C. The cells were immediately washed with ice cold PBS and reused for absorption with another 145 ml of PWM-SCCM. After seven cycles of mIL-3 absorption and release, the accumulated supernatants were pooled and concentrated using an Amicon ultrafiltration unit and a YM-10 membrane (Step 1). Gel Filtration Chromatography. Concentrated Step 1 mIL-3 (2.5 ml) was applied to a 124 ml gel bed of Sephadex G75 superfine (1.5 cm x 70 cm) previously equilibrated with PBS containing 0.02% Tween 20. The column was eluted at 4°C with equilibration buffer at 10 ml/h and 2 ml fractions collected, assayed and bioactive fractions combined and Amicon concentrated to 2.5 ml (Step 2). Reverse-Phase High Pressure Liquid Chromatography (RP-HPLC). Step 2 material was acidified to pH 2.1 with trifluoroacetic acid (TFA) and applied directly to a uBondapak C i g column (Waters) equilibrated with 0.1% TFA (Buffer A). Buffer B was 0.1% TFA in 60% acetonitrile. One ml fractions were collected at 1 ml/min into tubes containing 50 ul of 1 M Hepes, pH 8.0, 1% Tween 20. Elution was carried out using a 3 stage linear gradient of increasing acetonitrile concentration starting with a 5 min gradient to 30% acetonitrile, continuing with a 60 min gradient to 39% acetonitrile and finishing with a 5 min gradient 89 to 6 0 % ace ton i t r i l e . F r a c t i o n s c o n t a i n i n g b i o a c t i v i t y were p o o l e d and the ace ton i t r i l e l y o p h i l i z e d a w a y (Step 3). C e l l B i n d i n g S tud ies w i t h ' ^ - l a b e l l e d m I L - 3 . T o assess s p e c i f i c b i n d i n g to v i a b l e o r f i x e d B 6 S U t A j c e l l s , pure E . c o l i de r i ved r m I L - 3 o r step 3 P W M - S C C M der i ved m I L - 3 was iod ina ted u s i n g the m o d i f i e d C h l o r a m i n e T m e t h o d o f P a s l a s z y n s k i and Ih le [20] . L a b e l l e d m I L - 3 was separa ted f r o m f ree l 2 ^I b y c h r o m a t o g r a p h y t h r o u g h a 5 m l P 2 ( B i o R a d ) c o l u m n equ i l i b ra ted w i t h P B S c o n t a i n i n g 0 . 0 1 % ge la t i n . T y p i c a l l y , l 2 ^ I - m I L - 3 prepara t ions re ta ined > 9 0 % o f the i r b i o l o g i c a l ac t i v i t y and had s p e c i f i c ac t iv i t ies b e t w e e n 0.01 and 0.1 p C i / n g . F o r c e l l b i n d i n g s tud ies w i t h E . c o l i d e r i v e d l 2 ^ I - r m I L - 3 , 1 0 ^ v i a b l e o r fixed B 6 S U t A i c e l l s w e r e w a s h e d and s u s p e n d e d i n b i n d i n g b u f f e r ( P B S c o n t a i n i n g 1% B S A , 0 . 1 % ge la t i n , 0 . 0 5 % N a N 3 ) , w i t h 1 2 5 I - r m I L - 3 ± a 2 0 - f o l d excess o f un labe l l ed r m I L - 3 i n a to ta l v o l u m e o f 100 u l . A f t e r r o c k i n g g e n d y fo r va r i ous t imes at 4 ° C , c e l l suspens ions we re l aye red o v e r 2 5 0 p i a l iquo ts o f a 1.5:1 d i b u t y l ph tha la te :d ioc t y l phthalate o i l m i x tu re i n 0.5 m l m i c r o f u g e tubes and m i c r o f u g e d fo r 2 m i n . T h e tubes we re then f r o z e n at - 7 0 ° C , the t ips cut and c o u n t e d i n a B e c k m a n G a m m a 5 5 0 0 coun te r . T o release bound i 2 ^ I - r m I L - 3 , ce l ls were washed tw ice w i t h b i nd i ng buf fer , once w i t h 0.15 M N a C l , and then resuspended i n 1 m l o f 0.1 M g l y c i n e , 0 .15 M N a C l ( p H 2-4) o r 0.1 M s o d i u m phospha te , 0 .15 M N a C l ( p H 5 -7 ) . A f t e r g e n t l y r o c k i n g f o r v a r i o u s t imes at 2 3 ° C , the c e l l s we re pe l l e t ed and the s u p e m a t a n t s coun ted ! T h e ce l l s we re washed once i n P B S and then a lso counted . F o r c e l l b i n d i n g s tud ies w i t h p u r i f i e d l 2 ^ I - l a b e l l e d m I L - 3 , l a b e l l e d m I L - 3 ( 2 0 0 , 0 0 0 c p m ) w a s a d d e d to 2 x 1 0 ^ B 6 S U t A j ce l l s i n 100 u l o f b i n d i n g bu f f e r i n the p resence and absence o f e i the r 1 p g r m E L - 3 (1000 - fo ld excess) o r 1 p g r m G M - C S F . A f t e r 2 h at 4 ° C , the ce l l s were washed 3 t imes i n b ind ing b u f f e r and then r esuspended i n S D S / P A G E s a m p l e bu f f e r and e lec t ropho resed o n S D S - p o l y a c r y l a m i d e gels [21]. N - G l y c a n a s e D i g e s t i o n . F o r N - g l y c a n a s e s tud ies , 100,000 c p m o f step 3 p u r i f i e d l 2 ^ I - m I L - 3 i n 5 0 p i o f 0.2 M s o d i u m phospha te , p H 8.2, 0 . 0 5 % N P 4 0 , 10 m M phenanth ro l ine , were incuba ted w i t h 0.1 o r 1 uni t o f N - g l y c a n a s e f o r 2 h at 3 7 ° C . A f t e r th is t ime , the s a m p l e s were m a d e 2 % w i t h S D S , 5 % w i t h 2 - M E , 90 10% with glycerol and boiled for 1 min. Similar treatment of i2^I-labelled bovine serum albumin demonstrated that the commercial preparation of N-glycanase used for this study was free of detectable protease activity. Samples were electrophoresed on SDS-polyacrylamide gels. 91 2.3 RESULTS Binding of mIL-3 to Glutaraldehyde Fixed B6SUtA 1 Cells. Other studies demonstrated that the BeSUtAj subline expressed, under mEL-3 starved conditions, approximately 100,000 receptors/cell (CHAPTER IV, Figure 26). This high level of receptor expression suggested their potential use for purifying mIL-3 from conditioned medium by an absorption and specific release strategy. However, preliminary attempts with fresh cells proved unsuccessful because they lysed during the procedure. To prevent this B6SUtA^ cells were fixed with G D A , in a manner similar to that reported by Crapper et al [22]. Preliminary experiments showed that exposure of the cells to high concentrations of G D A led to a significant loss of binding activity (50% with 0.08% GDA) whereas cells fixed with 0.01% G D A retained 97% of their 1 2 5 I-rmIL-3 binding capacity (Figure 6). These latter cells also resisted mechanical disruption as assessed by repeated vigorous vortexing and centrifugation at 800 g whereas few intact cells remained when unfixed cells were similarly treated. Figure 7 shows the increase in specific binding (i.e., total binding minus non-specific binding) of ^ 2 ^I-rmIL-3 to 0.01% G D A fixed B 6 S U t A ! cells as a function of incubation time. Nonspecific binding was consistendy less than 10% of total binding. The amount of mIL-3 bound at 4°C was >90% of maximum within 2 h, similar to the binding kinetics of viable B6SUtAj cells (CHAPTER III, Figure 15) and to that reported for G D A fixed R6-X cells by Crapper et al [22]. Release of mIL-3 Absorbed to Fixed B6SUtAj Cells. To determine the optimal conditions for specifically eluting bound mIL-3, aliquots of fixed cells were first allowed to saturably bind ^2^I-rmIL-3 for 2 h at 4°C, and then washed and resuspended in buffered-saline solutions ranging in pH from 2.0 to 7.0. After 5 min at room temperature, the cells were pelleted, and both pellets and supernatants counted. More than 90% of the bound ^2^I-rmIL-3 could be reproducibly eluted from fixed cells at a pH below 3 (Figure 8) and this activity could again be bound to newly prepared fixed cells. Interestingly, it was more difficult to release bound 1 2^I-rmIL-3 from viable cells (Figure 8). To establish the minimum time required for 92 Figure 6. Effect of glutaraldehyde treatment of B6SUtAj cells on their ability to absorb mIL-3. Total binding is shown. Increasing the concentration of GDA above 0.01 % results in a gradual loss of mIL-3 binding capacity. 93 Figure 7. Time course of mIL-3 absorption to 0.01% glutaraldehyde fixed B6SUtAj cells. Total binding is shown (non-specific binding of mIL-3 was always less than 10%). The kinetics of mIL-3 binding are similar when unfixed cells are used. 94 Figure 8. Effect of pH on the elution of mIL-3 from 0.01% glutaraldehyde fixed (A) and viable (A) B^SUtAj cells. Total binding is shown. At a pH of 2.5, >90% of mIL-3 absorbed onto fixed cells can be eluted, while only 60% can be eluted from unfixed cells. 95 efficient release of absorbed mIL-3, time course studies were performed. As shown in Figure 9, fixed cells released >90% of absorbed 1 2 5I-rmIL-3 within 5 min of incubation at 23°C in 0.1 M glycine, 0.15 M NaCl,pH2.8. Purification of mIL-3 from PWM-SCCM. Preliminary studies carried out using methylcellulose assays confirmed a previous report [23] that the colony stimulating activity of PWM-SCCM, prepared serum-free in a medium containing only 100 ug/ml insulin and 2-ME, was equivalent to that present in PWM-SCCM containing 10% FCS or 1% BSA [24]. Subsequently, more specific bioassays, using 32D c3 cell proliferation, confirmed the presence of a high level of mIL-3 (of specific activity approximately 125 U/mg). This material was absorbed to fixed B6SULAJ cells and eluted as described above. However when the procedure was scaled up to use 5 x 109 cells and the elution volume kept low (i.e., 50 ml) to try and simultaneously concentrate the released mIL-3, the yield of mIL-3 eluted was unacceptably low, probably due to a substantial innate buffering capacity of the cells themselves. However, by simply decreasing the pH of the glycine-saline buffer from pH 2.8 to pH 2.5, it was again possible to elute >90% of the absorbed activity. Both fixed cells and depleted PWM-SCCM could be re-used in repeated cycles (Table 1), although the fixed cells did undergo some lysis and were no longer useful after 8 cycles. Interestingly, the material isolated from the first cycle had the lowest specific activity of the first seven cycles, presumably because the first low pH treatment eluted a significant amount of contaminating proteins absorbed to the cells during their growth in 20% FCS. In the next 6 cycles the specific activity of the eluted mIL-3 remained constant at approximately 5000 U/mg. By the eighth cycle, cell depletion and cell debris resulted in a significant decrease in the amount and specific activity of mIL-3 eluted. As indicated in Table 2, the first 7 cycles resulted in the absorption and release of a total of 38,000 U from the 100,000 U originally present in the 1015 ml of PWM-SCCM subjected to absorption. 96 Figure 9. Time course of mIL-3 elution from glutaraldehyde fixed B6SUtAj cells. By 10 min at pH 2.8, >90% of surface bound mIL-3 can be successfully recovered from the cells. 97 TABLE 1. Purification of PWM-SCCM derived mIL-3 by Absorption onto GDA fixed B6SUtAj Cells Cycle Number Units Specific Activity Released* (Units/mg**) 1 5200*** 4000 2 5200 4800 3 5100 5200 4 5000 5200 5 4800 5800 6 5000 5000 7 4700 4400 8 3200 2800 Units assessed by JH-thymidine incorporation into 32D-c3 cells (see Methods). Protein assayed by the Coomassie Brilliant Blue procedure [31]. Each value is the mean of duplicates. 98 G75 Chromatography of Step 1 Material. Pooled material from cycles 1-7 were concentrated using an Amicon ultrafiltration unit with a YM-10 membrane. This material was then chromatographed on a Sephadex G75 superfine column in the presence of 0.02% Tween 20 to reduce losses through non-specific sticking [15,25]. Figure 10 shows the protein and activity profdes of eluted fractions. Fractions with activity were pooled and concentrated for reverse-phase HPLC. This G75 chromatography step resulted in a 5-fold purification with an 85% recovery of activity. Reverse-Phase HPLC-Cjg Chromatography. Reverse phase HPLC was the final step in the purification procedure (Figure 11). This gave an additional 80-fold increase in mIL-3 specificxactivity and a yield of approximately 50%. Analysis of mIL-3 Purity. Aliquots from each stage in the purification procedure were i2^I-labelled and subjected to SDS-PAGE (Figure 12). The starting PWM-SCCM, and Step 1 and 2 material were electrophoresed under reducing conditions. However, both the l2^I-labelled Oane 4) and unlabelled Step 3 material, used for sectioning and proliferation analysis, were run under non-reducing conditions to retain biological activity. Autoradiograms of these gels revealed only 2 l2^I-labelled species, both with mEL-3 activity. Since the Step 3 material was run under non-reducing conditions (lane 4), the 2 bands seen ran slightly faster than the corresponding bands in the Sephadex G75 (Step 2) pool (lane 3). this is in agreement with previous reports [1] and reflects intrachain disulfide bridges [26]. Under reducing conditions these 2 bands ran as a 19.5 kd and 16.5 kd species (Figure 12). To examine the relationship between the 2 purified bands, N-glycanase treatment was undertaken. From Figure 8, treatment with 0.1 U or 1 U of N-glycanase for 2 h at 37°C resulted in the disappearance of the 19.5 kd band and an increased intensity of the 16.5 kd band. Since intermediate bands were not observed with lower N-glycanase concentrations, and assuming that glycosylation does not significandy alter the electrophoretic mobility of mIL-3, a single 3 kd N-linked carbohydrate moiety is most likely present on the 19.5 kd species, while the 16.5 kd species should represent N-deglycosylated mIL-3. The fact that litde, if any, residual 19.5 kd material was observed after N-glycanase treatment and that it was 99 Figure 10. Sephadex G75 superfine chromatography of concentrated (2.5 ml) Step 1 material from cycles 1-7 (Table 1). Fractions were assayed for absorption at 280 nm (A) and activity in the 32D c3 cell proliferation assay (•). The region marked "pool" was taken for HPLC. 100 1.0 0 10 20 . 30 40 50 60 70 F r a c t i o n N u m b e r Figure 11. Reverse-phase HPLC of Step 2 material on C 1 8 . The three stage acetonitrile gradient is shown as a thin solid line. Protein was monitored continuously by absorption at 215 nm (—) and mIL-3 activity determined from various dilutions of the 1 ml fractions collected, following lyophilization and resolubilization, using the 32D c3 cell proliferation assay (#). The solid bar refers to the active fractions pooled (Step 3). 101 Figure 12. S D S - P o l y a c r y l a m i d e ge l ana l ys i s o f the m I L - 3 f rac t ions f r o m e a c h step i n the p u r i f i c a t i o n p r o c e d u r e . l 2 ^ I - l a b e l l e d P W M - S C C M s ta r t i ng m a t e r i a l , p H 2.5 eluate and Sephadex G 7 5 p o o l , co r respond ing to samples 1, 2 and 3 respect ive ly were e l e c t r o p h o r e s e d under r e d u c i n g c o n d i t i o n s . S a m p l e 4 , c o r r e s p o n d i n g to l 2 ^ I - l a b e l l e d C j g e lu ted m a t e r i a l , was run u n d e r n o n - r e d u c i n g c o n d i t i o n s as w a s an i d e n t i c a l , u n l a b e l l e d C j g e lu ted samp le w h i c h w a s u s e d , subsequen t l y , for c e l l p ro l i f e ra t i on ana lys is by shak ing gel s l ices ove rn igh t at 4 ° C w i t h 0.5 m l o f R P M I 1640 c o n t a i n i n g 0 . 1 % B S A and then assay ing at d i f fe ren t d i l u t i ons in the 3 2 D c3 c e l l assay. 'XT - label led mo lecu la r weight markers ( A m e r s h a m , UK) were e lec t rophoresed in lane M . 102 PWM-SCCM Ecoli N-glycanase mIL-3 M miL-3 r - vu I 200-92.5— 6 9 — 46 — 3 0 — Figure 13. SDS-Polyacrylamide gel analysis of P W M - S C C M purified mIL-3 following N-glycanase treatment. A l l samples were electrophoresed under reducing conditions. Undigested 1 2^I-labelled Cjg-HPLC purified material is shown in the left most lane. Purified E. coli derived rmIL-3 typically gives two bands, the major larger molecular weight band being full length unglycosylated mIL-3. N-glycanase treatment of P W M - S C C M purified mIL-3 was carried out as described in Materials and Methods. 1 4C-labelled molecular weight markers were electrophoresed in lane M . 103 apparently all converted into a 16.5 kd species is supportive of the notion that the 19.5 kd band represents a single molecular species. In addition, the fact that N-glycanase treated Step 3 material still ran slightly slower than full length [27] rmIL-3 (Figure 13) suggests that a low level of 0-linked sugar might be present on mIL-3 produced by normal activated T cells. As a further test of the identity of the 2 purified proteins, l2^I-labelled Step 3 material was added to B6SUtA1 cells in the presence and absence of excess amounts of either rmIL-3 or rmGM-CSF. SDS-PAGE of cell-bound radioactivity revealed that rmIL-3 specifically inhibited the binding of both the 19.5 kd and 16.5 kd mIL-3 species, while rmGM-CSF did not (Figure 14). The fact that a 1000 fold molar excess of rmIL-3 completely inhibited the binding of both Il2^-mIL-3 species, while rmGM-CSF at the same molar ratio had no effect, is consistent with both mIL-3 species in the Step. 3 preparation being pure. A summary of the purification scheme is shown in Table 2. A 16,000-fold purification was achieved from serum-free PWM-SCCM. The overall yield was 16% and from 1 liter of conditioned medium, 8 pg of mIL-3 was purified. The entire purification protocol proved to be easily reproducible with yields consistently between 14-20%. When larger numbers of cells were used for the absorption procedure (>5 x 10 )^, a problem of increasing non-specific absorption to cells was encountered. Thus, there was a limit to the degree that the purification procedure could be scaled up. 104 2 0 0 -92.5 — 6 9 — 4 6 — 30 Figure 14. SDS-Polyacrylamide gel analysis of the binding of purified 1 2 5 I - m I L - 3 to B 6 S U t A j cells in the presence and absence of rmIL-3 and m G M - C S F . 1 2 5 I -labelled C j g purified mIL-3 was added to B6SUtA| cells in the presence and absence of a 1000 molar excess of rmIL-3 or m G M - C S F , added at the same molar ratio as excess rmIL-3. After 2 h at 4°C, the cells were washed three times and then boiled for 1 min in SDS sample buffer and electrophoresed. 105 Table 2. Purification of PWM-SCCM derived mIL-3 Step Volume Total Protein Total Units* Specific Overall (ml) (mg) Activity Yield Units/mg (%) PWM-SCCM 1015 800** 100,000 125 100 Absorbed onto Cells - - 38,000 - 38 1. pH2.5Eluate 420 6.9** 35,000 4,900 35 2. Sephadex G75 superfine 3 1.14** 30,000 26,000 30 3. C 1 8 - H P L C 6 0.008*** 15,700 2 x 106 16 * units assessed by thymidine incorporation into 32D-c3 cells ** protein assayed by Coomassie Brilliant Blue procedure [31 ] *** protein assayed by the soluble Silver assay procedure [32] 106 2.4 DISCUSSION A simple three step protocol has been devised for the purification of mIL-3 from PWM-SCCM. The novel aspect of this purification procedure is the initial step which utilizes absorption to fixed mIL-3 receptor-positive cells. Under the conditions used, this step provided botii a 40-fold purification and a 2.5-fold concentration of mIL-3. While this is the first report where fixed cells have been used for the purification of a growth factor, absorption to cells has been used by several investigators to identify previously uncharacterized growth factors [28] and to determine receptor numbers on different cell populations [22,29]. For example, Swain and Dutton used GDA fixed BCLj tumor cells to absorb out and thus demonstrate the existence of a B cell growth promoting activity, BCGF, from the medium conditioned by the Dennert cell line C.C3.11.75. [28]. Later, Palacios and Garland used cell absorption to establish that mIL-3 receptors were present on a mouse pre-B cell line, Ea3 [29]. More recendy, Crapper et al [22] established that GDA-fixed mIL-3 dependent R6-X or CBA P cells could bind and release mIL-3 in a pH-dependent manner. Recently, Ziltener et al [1] used polyclonal and monoclonal antibodies to mIL-3 to show that WEHI-3B cells, transfected Cos 7 cells and two T cell clones each produce several forms of mIL-3 and that the forms differ in their degree of N-linked glycosylation. SDS-PAGE analysis of the mIL-3 derived from the T cell clones, for example, revealed the presence of three broad bands with molecular weights of 32-36 kd, 27-31 kd and 21.5-22.5 kd. In contrast, the mIL-3 isolated by Cuder et al [15] from PWM-SCCM consisted of 2 protein species, one of 24 kd and one of 19 kd. Since only two low molecular weight species of mIL-3 from PWM-SCCM were purified here, it is conceivable that higher molecular species may have been selectively lost during purification. This is consistent with the observation of a second minor and discarded peak of activity in the HPLC profile Figure 11). Another possible explanation for the absence of purified higher molecular weight forms is that they may not be produced in sufficient 107 quantities by activated primary T cell cultures. An examination of the cDNA for mIL-3 reveals 4 potential sites for N-linked glycosylation [30] and based on the results described here and by others [1,15], it seems most likely that all 4 sites can be N-glycosylated giving rise to 19.5 kd, 24 kd, 28 kd and 32 kd species. Lastly, the N-glycanase studies suggest that there may be a low level of previously unreported 0-linked glycosylation on normal T cell-derived mIL-3. In conclusion, it has been demonstrated that fixed cells can be useful for purifying at least one of the hemopoietic colony stimulating factors. It is conceivable that similar techniques may be applied to other cells to purify as yet uncharacterized regulators of cell growth and differentiation. One possible avenue in this regard might be the addition of conditioned media to COS cells transfected with receptor-like oncogenes as an approach to the identification of their specific ligands. 108 2.5 R E F E R E N C E S 1. ZiltenerHJ, Fazekas de St Groth B, Leslie KB, Schrader JW. Multiple glycosylated forms of T cell-derived interleukin-3 (IL-3). J Biol Chem 263:14511,1988. 2. Garland JM, Crompton S. A preliminary report: preparations containing interleukin-3 (IL-3) promote proliferation of multipotential stem cells (CFUS) in the mouse. Exp Hematol 11:757,1983. 3. Iscove NW, Roitsch CA, Williams N, Guilbert LT. Molecules stimulating early red cell, granulocyte, macrophage, and megakaryocyte precursors in culture: similarity in size, hydrophobicity, and charge. J Cell Physiol Suppl 1:65,1982. 4. Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Fitch F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynski E, Dy M, Lebel B. Biologic properties of homogeneous interleukin-3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, P cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J Immunol 131:282, 1983. 5. Ihle JN, Lee JC, Rebar L. T cell recognition of Moloney leukemia virus proteins. III. T cell proliferative responses against gp70 are associated with the production of a lymphokine inducing 20 alpha-hydroxysteroid dehydrogenase in splenic lymphocytes. J Immunol 127:2565, 1981. 6. Palacios J. Cloned lines of interleukin 2 producer human T lymphocytes. J Immunol 129:2586, 1982. 7. Clark-Lewis I, Crapper RM, Leslie K, Schrader JW. P cell stimulating factor; Molecular and biological properties. In Cellular and Molecular Biology of Lymphokines, ed. C. Sorg, A. Schipl. New York: Academic Press, 1985. 8. Crapper RM, Vairo G. Hamilton J, Clark-Lewis I, Schrader JW. Stimulation of bone marrow-derived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, resisting cell-stimulating factor. Blood 66:859,1985. 9. Ihle JN, Weinstein Y. Immunological regulation of hematopoietic/lymphoid stem cell differentiation by interleukin 3. Adv in Immunol 39:1, 1986. 10. Urdal DL, Mochizuki D, Conlon PJ, March CJ, Remerowski ML, Eisenman J, Ramthun C, Gillis S. Lymphokine purification by reversed-phase high performance liquid chromatography. J Chromatogr296:171, 1984. 11. Ymer S, Tucker QJ, Sanderson CJ, Hapel AJ, Campbell HD, Young IG. Constitutive synthesis of interleukin-3 by leukemia cell line WEHI-3B is due to retroviral insertion near the gene. Nature 317:255,1985. 12. Humphries RK, Abraham S, Krystal G, Lansdorp P, Lemoine F, Eaves CJ. Activation of multiple hemopoietic growth factor genes in abelson virus-transformed myeloid cells. Exp Hematol 16:774, 1988. 109 13. Ihle JN, Keller J, Henderson L, Klein F, Palaszynski E. Procedures for the purification of interleukin 3 to homogeneity. J Immunol 129:2431, 1982. 14. Clark-Lewis I, Kent SBH, Schrader JW. Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells. J Biol Chem 259:7488,1984. 15. Cutler RL, Metcalf D, Nicola N, Johnson GR. Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium. J Biol Chem 260:6579,1985. 16. Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci USA 80:2931, 1983. 17. Murthy SC, Sorensen PHB, Mui AL-F, Krystal G. Interleukin-3 down-regulates its own receptor. Blood 73:1180, 1989. 18. Sorensen PHB, Mui AL-F, Murthy SC, Krystal G. Interleukin-3, GM-CSF and TPA induce distinct phosphorylation events in an IL-3 dependent multipotential cell line. Blood 73:406,1989. 19. Greenberger JS, Eckner RJ, Sakakeeny M, Marks P, Reid D, Nabel G, Hapel A, Ihle JN, Humphries RK. Interleukin-3-dependent hematopoietic progenitor cell lines. Fed Proc 42:2767, 1983. 20. Palaszynski EW, Ihle JN. Evidence for specific receptors for interleukin-3 on lymphokine-dependent cell lines established from long-term bone marrow cultures. J Immunol 132:1872, 1984. 21. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970. 22. Crapper RM, Clark-Lewis I, Schrader JW. Analysis of the binding of a hemopoietic growth factor, P-cell-stimulating factor, to a cell surface receptor using quantitative absorption of bioactivity. Exp Hematol 13:941, 1985. 23. Nakeff A, Lezama RV. Regulation of megakaryocyte proliferation. Blood 58(Suppl 1):384,1981. 24. Eaves CJ, Krystal G, Eaves A C . Erythropoietic Cells. In: Baum SJ (ed) Bibliotheca Haematologica, Current Methodology in Experimental Hematology, No 48. S. Karger, Basel, pp 81-111,1984. 25. Krystal G, Pankratz HRC, Farber NM, Smart JE. Purification of human erythropoietin to homogeneity by a rapid five-step procedure. Blood 67:71, 1986. 26. Clark-Lewis I, Hood LE, Kent SBH. Role of disulfide bridges in determining the biological activity of interleukin-3. Proc Natl Acad Sci USA 85:7897,1988. 27. Kindler V, Thorns B, de Kossodo S, Allet B, Eliason JF, Thatcher D, Farber N, Vassalli P. Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin-3. Proc Natl Acad Sci USA 83:1001,1986. 110 28. Swain SL, Dutton RW. Production of a B cell growth-promoting activity, (DL) BCGF, from a cloned T cell line and its assay on the BCLj B cell tumor. J Exp Med 156:1821,1982. 29. Palacios R, Garland J. Distinct mechanisms may account for the growth-promoting activity of interleukin-3 on cells of lymphoid and myeloid origin. Proc Natl Acad Sci USA 81:1208, 1984. 30. Fung MC, Hapel AJ, Ymer S, Cohen DR, Johnson RM, Campbell D, Young IG. Molecular cloning of cDNA for murine interleukin-3. Nature 307:233, 1984. 31. Bradford M. A rapid and sensitive method for the quantitation of microgram amounts of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248, 1976. 32. Krystal G. A silver-binding assay for measuring nanogram amounts of protein in solution. Anal Biochem 167:86, 1987. Ill CHAPTER m INTERLEUKIN 3 DOWN-REGULATES ITS OWN RECEPTOR 3.1 INTRODUCTION Several reports have appeared within the last 3 years which shed some light on the mechanism of action of mJJL-3. Specifically, mIL-3 appears to stimulate protein kinase C translocation [1], elevation of intracellular ATP [2] and glucose [3] levels, and membrane and cytosolic tyrosine and serine specific protein phosphorylations [4-6]. However, key to the further understanding of the mechanism of action of mIL-3 is a more detailed characterization of its cell surface receptor. One important issue not addressed in previous reports is whether mIL-3 receptor occupancy and subsequent cellular processing of the receptor complex regulates receptor expression and cell sensitivity to mIL-3. In other systems, it appears that ligand induced receptor internalization is an important regulatory focus not only for receptor down-regulation and hormone desensitization, but also for transduction of mitogenic signals [7]. Thus, a study of mIL-3 receptor internalization and processing may yield important insights into the mechanism of action of mIL-3. In this chapter, the fate of surface bound recombinant mIL-3 has been examined. Furthermore, changes in mIL-3 receptor expression following short and long term exposure of factor-dependent cells to recombinant mIL-3 or mGM-CSF have also been studied. The result from these investigations suggest that mIL-3 is rapidly internalized and specifically cleaved before being completely degraded in lysosomes, while internalized receptors appear to be recycled back to the cell surface. The rate of re-expression of mIL-3 receptors on the cell surface is dependent upon the initial mIL-3 concentration used for receptor down-regulation (i.e., the higher the level of mIL-3 used, the slower the rate of re-expression of receptors). Murine GM-CSF, however, has no apparent effect on mIL-3 receptor expression in short term studies. From long-term exposure studies, cells exponentially growing in mIL-3, under normal culture conditions, 112 have been shown to express low numbers of mIL-3 receptors and are relatively insensitive to mIL-3 in cell proliferation assays. However, cells either grown to mIL-3 exhaustion or grown in mGM-CSF, have 10-fold higher mIL-3 receptor numbers and are far more sensitive in proliferation assays to mIL-3. This last finding should prove especially useful in facilitating mIL-3 receptor purification. 113 3.2 MATERIALS AND METHODS Materials. Pure E. coli derived recombinant mIL-3 [8] and recombinant mGM-CSF [9] were kindly supplied by Biogen, Geneva, Switzerland. Pure COS cell derived recombinant mIL-3, which was used solely for the mIL-3 intracellular processing studies, was produced by Drs. Rob Kay and Keith Humphries (TFL, Vancouver, Canada) and purified in our laboratory. (12^I)-sodium iodide (100 mCi/ml; carrier-free) in NaOH solution, pH 7-11, (methyl-3H)-thymidine (1 mCi/ml; 2 Ci/mmol), and 3H-leucine (1 mCi/ml; 190 Ci/mmol) were purchased from Amersham. Other reagents, unless otherwise indicated, were purchased from Sigma. Cells. See CHAPTER II. In some instances cells were grown in either 0.3 nM mlL-3 or 0.3 nM mGM-CSF, in lieu of PWM-SCCM. Preparation of radiolabelled mIL-3. Radio-iodination of mIL-3 was performed by the chloramine T method as described by Palaszynski et al [10] with one modification. Radiolabelled mIL-3 was separated from free iodide by gel filtration through a 5 ml Sephadex G-25 (Pharmacia) column equilibrated with PBS containing 0.01% gelatin. Radioactive fractions (0.2 ml each) were collected at 10 ml/hand the first peak pooled and made 0.1% with BSA and stored in 50 ul aliquots at -70°C. Typically, 1 2 5I-mIL-3 preparations retained > 90% of their biological activity, and had specific activities between 0.01 and 0.1 uCi/ng. Internalization studies with mIL-3. B6SUtAj cells were washed twice in 20% FCS/RPMI, suspended at 2 x 10^ cells/ml in the same medium and 100 ul aliquots dispensed into Falcon 3911 Microtest III flexible assay plates. To each well was added 3 nM ^I-mIL-3 and the plates were rocked for 2 h at 4 ° C The plates were centrifuged at 400 x g for 3 min in a Beckman TJ-6 centrifuge and the cells in each well washed 3 times with ice cold 20% FCS/RPMI, resuspended in 100 ul of pre-warmed cell line medium and incubated at 37°C in a humidified atmosphere of 5% CO2, 95% air. At various times, 114 cells were transferred to 5 ml Falcon tubes containing 3 ml of either PBS or 0.1 M glycine, 0.15M NaCl, (pH 2.0). After 5 min at room temperature, the tubes were centrifuged (400 x g, 5 min) and cell pellets counted in a Beckman Gamma 5500 counter. Intracellular Processing of mIL-3. B6SUtAj cells were suspended at 2 x 10^  cells/ml in cold 20% FCS/RPMI and 100 ul aliquots added to flexible assay plate wells. i2^I-mIL-3 (3 nM) was added to each well and plates were rocked for 2 h at 4°C. Cells were then washed 3 times in 20% FCS/RPMI, resuspended in 100 pi of cell line medium ± 10 mM methylamine and incubated at 37°C. At various times, cells were transferred into 1.5 ml microfuge tubes and centrifuged at 2000 fpm for 2 min in an Eppendorf centrifuge 5415. Cell pellets were suspended in 50 pi of SDS sample buffer containing 5% mercaptoethanol, boiled for 5 min, and electrophoresed on SDS polyacrylamide gels [11]. Gels were then fixed, dried for autoradiography and exposed to Kodak X-OMAT film at -70°C. Cell proliferation assays. To measure factor-induced proliferation, cells were washed twice with unsupplemented RPMI and then incubated at 37°C in a final volume of 0.1 ml with 20% FCS/RPMI in Linbro U-shaped microtitre plate wells at 2 x cells/ml in the presence and absence of either mIL-3 or mGM-CSF. After 22 h, 20 pi of a 3H-thymidine stock containing 50 pCi/ml (2 Ci/mmole) in RPMI was added to give a final concentration of 1 pCi/well. After a further 2 hour incubation at 37°C, the well contents were harvested onto glass fiber filters using a micro-harvester (Richter Scientific, Vancouver, Canada) and radioactivity was measured with a Beckman LS7500 liquid scintillation counter. Short-term mIL-3 receptor expression studies. B6SUtAj cells were washed and resuspended in 20% FCS/RPMI at 2 x 106 cells/ml and seeded in flexible assay plates at 100 pi per well. To each well was added various concentrations of either mIL-3 or mGM-CSF to give final volumes of 110 pi. The plates were incubated at 37°C for 2 h, and then the cells were washed 3 times with 20% FCS/RPMI, resuspended in 100 pl/well of 20% FCS/RPMI ± 10 pg/ml cycloheximide and returned to the incubator. Following times from 30 min to 6 h at 37°C, plates were cooled to 4°C, and supplemented with 3 nM i2^I-mIL-3/weli. After rocking on an Adams Nutator for 2 h at 4°C, cells were washed three times with 115 binding buffer (1% BSA, 0.1% gelatin, 0.02% sodium azide in PBS). After the final wash, 50 ul of 2% agarose was added to each cell pellet, and the agarose was allowed to polymerize at 4°C. For time course studies, the same binding procedure was carried out at both 4°C and 37°C. The wells were then cut from the plates and counted in a Beckman Gamma 5500 counter. To measure the ability of cycloheximide to inhibit protein synthesis, 2X105 BeSUtAj cells in 100 ul of cell line medium were incubated at 37°C for various time periods ± 10 ug/ml cycloheximide. Cultures were then made 40 uCi/ml with %-leucine and incubated at 37°C for one hour. Cells were then washed 3 times with ice-cold PBS, resuspended in 0.5 ml 1% BSA, supplemented with 0.5 ml 10% TCA, vortexed and kept on ice for 15 min. Suspensions were then filtered through 0.45 um cellulose acetate disks (Sartorius). After the disks were washed with 5% TCA, they were dried and counted. Long-term mIL-3 receptor expression studies. B6SUtAi and 32D cl 3 cells were seeded into 75 cm 2 Falcon flasks at either 1 x 104 or 5 x 105 cells/ml in 20% FCS/RPMI supplemented with 0.3 nM mIL-3 or 0.3 nM mGM-CSF. After 3 days of growth at 37°C, the cells were harvested for Scatchard analyses and proliferation assays. For Scatchard analysis, 2-fold dilutions of 125I-mIL-3 in binding buffer (starting with 10 nM) were carried out in flexible 96 well assay plates ± 20-fold excess unlabelled mrL-3 to correct for non-specific binding. Cells were then added in binding buffer to give a final concentration of 1 x 10^ cells/ml in a total volume of 100 ul. After 2 h of rocking at 4°C, plates were washed 3 times with binding buffer and 50 ul of 2% agarose added to each well. After polymerization, the wells were excised and counted in a Beckman Gamma 5500 counter. 116 3.3 RESULTS Binding and internalization of mIL-3. As a first step in delineating the effect of mIL-3 on mIL-3 surface receptor expression, the rate of binding and internalization of mIL-3 was measured. For the binding experiments, exponentially growing B6SUtAj cells were incubated with saturating concentrations of l2^I-mIL-3 (3 nM) in the presence and absence of excess unlabelled mIL-3 for various times at 4°C and 37°C (Figure 15). As expected, binding was more rapid at 37°C, but even at 4°C was essentially complete by 2 h (which is similar to the binding kinetics generated with GDA fixed cells-Figure 7). To study internalization, B6SUtAj cells were pre-incubated with saturating concentrations of l2^I-mIL-3 for 2 h at 4°C. The cells were then incubated at 37°C in cell line medium and the time-dependent decrease in surface-associated radioactivity was monitored by resistance to acidic buffer treatment (Figure 16). Simple dissociation of l2^I-mIL-3 from the cell surface receptor into the medium did not occur at detectable levels during this time, as measured by release into PBS. The internalization of surface bound mIL-3 was half maximal by 15 min, and >90% complete by 60 min. Intracellular processing of mIL-3. To investigate the intracellular degradation of mIL-3, SDS/PAGE was performed on total cell extracts following internalization of both E. coli and COS cell derived l2^I-mIL-3. As can be seen from Figure 17a, E. coli derived l2^I-mIL-3, bound at 4°C, remained intact for at least one hour after shifting up to 37°C. However, by 3 h at 37°C, the initial E. coli derived mIL-3, which appeared as a single band at 16 kd, was somewhat diminished and 2 new bands appeared, a major one at 15 kd and a minor band at 12 kd. By 6 h of incubation at 37°C, the original ligand was further diminished and the 12 kd fragment was now the major breakdown product. Longer incubations resulted in the decreased intensity of all three bands and accumulation of lower molecular weight fragments which migrated with the dye front. However, the addition of methylamine, a potent lysosomal inhibitor [12], inhibited the formation of these breakdown products (Figure 17a). Using COS cell-derived 5-i 0 1 2 3 4 5 6 Time (hr) Figure 15. Time course of mIL-3 binding to B6SLUA! cells. B6SUtAj cells were incubated with saturating concentrations of 12^I-mIL-3 (3 nM) for various times at 4°C and 37°C in the presence of 0.02% NaN 3 . Total binding at 4°C (A), non-specific binding at 4°C (A), specific binding at 4°C (O), specific binding at 37°C (•). 118 Figure 16. Internalization of surface bound 1Z3I-miL-3. BeSUtAj cels were incubated with saturating concentrations of 12I^-mIL-3 (3nM) for 2 h at 4°C and then washed and cultured in 20% FCS/RPMI with 5% PWM-SCM at 37°C for various times. Surface bound 12I^-mIL-3 is represented as the ratio betwen pH 2 elutable cpm and total cpm present. Values are the means of triplicate determinations. 119 E COli mIL-3 b) COS mIL-3 + MA IL-3 1 6 1 1 6 +MA 2 0 0 -IL-3 1 3 6 ' 3 6 1 9 2 5 _ 200-69-92.5-4 6 -69-Figure 17. Cellular processing of lz:5I-mIL-3 in the presence and absence of methylamine. After 12*I-mIL-3 was allowed to bind to B6SUtAj cells for 2 h at4°C, cells were incubated for 1 h, 3 h. and 6 h at 37°C ± 10 mM methylamine and cell extracts analyzed by SDS/PAGE on a 10-20% gradient gel. In (a), E coli derived 12^I-mIL-3 was used whereas in (b), COS cell derived l25I-mBL-3 was udlized. The left most lane in each panel demonstrates the purity of the l 2 ^I-mIL-3 preparations used for this study. The numbers at the top of both panels refer to the h at 37°C and MA is methylamine. 120 ^I -mlL-S , which more closely resembles the native glycosylated growth factor, a similar reduction in molecular weight is seen by 6 h of incubation at 37°C, and this degradation was also inhibited by the addition of methylamine (Figure 17b). However, because of the heterogeneity in glycosylation of COS cell derived mIL-3, it is difficult to ascertain the molecular weight(s) of the immediate cleavage product(s). mIL-3 receptor expression following short-term exposure to mIL-3. To further investigate the regulation of mIL-3 receptor expression, the effect of a short-term exposure to mIL-3 on the rate of mIL-3 surface receptor expression was examined. For these experiments B6SUtA j cells were preincubated with various concentrations of unlabelled mIL-3 for 2 h at 37°C. At various times subsequent to this preincubation period, cells were assessed for mIL-3 receptor expression by their ability to bind 125j_ mEL-3. Using 2 x 105 B6SUtA| cells, mIL-3 levels of >5 nM totally blocked any 125I-mJJL-3 binding at time zero, indicating that all the receptor sites were temporarily inaccessible (Figure 18). However, although mIL-3 concentrations >5 nM were capable, at time zero, of completely blocking mIL-3 surface binding, the rate of surface re-expression of these binding sites was dependent upon the concentration of mIL-3 used during the preincubation period. For example, mIL-3 concentrations < 10 nM allowed for the complete re-expression of mIL-3 receptors within 6 h, while preincubation with 20 nM mIL-3 resulted in only a 50% re-expression by this time. A 50% decrease of surface binding site expression appeared to be the maximum effect that could be obtained since concentrations >20 nM IL-3 were found to yield no greater inhibition. Cells were also preincubated with various concentrations of mGM-CSF. The effect of one concentration (10 nM) on mIL-3 receptor expression is shown in Figure 4. This concentration, as well as both ten-fold higher or ten-fold lower concentrations of mGM-CSF had no effect on mIL-3 receptor expression during the 6 h period examined. To address the question of whether internalized mIL-3 receptors were degraded together with internalized mIL-3 or recycled back to the cell surface, receptor re-expression studies were conducted in the presence and absence of cycloheximide. As can be seen from Figure 19, cells re-expressed the same 121 2 4 Time (hours) Figure 18. Short-term re-expression of the mIL-3 receptor. B6SUtA! cells were preincubated with several concentrations of either mIL-3 or mGM-CSF for 2 h at 37°C. After washing, the cells were incubated at 37°C and assayed at various times for mEL-3 receptor expression (l2^I-IL-3 binding). Inhibition of binding is defined as the ratio of experimental binding values to control (mock preincubated) values. Murine IL-3 concentrations were 20 nM (•), 10 nM (•), 5 nM (•) and 0.15 nM (•), and the mGM-CSF concentration was 10 nM (O). Values are the means of duplicate determinations. 122 Time (hr) Figure 19. Short-term re-expression of the mIL-3 receptor in the presence of cycloheximide. B6SUtAj cells were preincubated with 5 nM mIL-3 for 2 at 37°C, then washed and placed in medium ± 10 pg/ml cycloheximide. At various times, cells were examined for mIL-3 receptor expression. Without cycloheximide (•), with cycloheximide (A). Also included is the effect of 10 pg/ml cycloheximide on receptor expression in the absence of preincubation with 5 nM mIL-3 ( A ) . Superimposed on the same graph is the effect of 10 pg/ml cycloheximide on protein synthesis (•). Values are the means of duplicate determinations. 123 number of internalized binding sites on their cell surfaces whether cycloheximide was present or not. The effect of cycloheximide on protein synthesis is also shown in Figure 19. Even though cycloheximide inhibited protein synthesis by 80% witiiin 1 h, re-expression of internalized receptors continued to increase from 1 h to 6 h, at which dme it was >90% complete. mIL-3 receptor expression following long-term exposure to mIL-3. To examine the effect of mIL-3 and mGM-CSF on mIL-3 receptor expression in a long-term model, it was necessary to have, as a control, cells growing in the absence of these growth factors. To approximate this condition, B6SU1A] cells were seeded at high (5 x 105 cells/ml) and low (1 x 104 cells/ml) densities in 20% FCS/RPMI containing 0.3 nM mIL-3 or 0.3 nM mGM-CSF and cultured at 37°C. By 3 days, cultures initiated at high densities (with either growth factor) had completely exhausted the growth factor in tiieir medium as assessed by the lack of bioactivity in high density culture supernates (i.e., the high density culture supernates contained less than 0.3 pM mIL-3 or less than 12 pM mGM-CSF, as determined by ^H-thymidine assays using high density cells in the presence and absence of exogenously added growth factor to rule out the presence of inhibitors in the culture supernates). These cells began to die on day 4 if not resupplemented with growtii factor. Day 3 high density cells, however, were still >95% viable (trypan dye exclusion) and served as a source of cells grown in the "absence of factors". Cells seeded at low densities in either factor continued to grow in log phase and, after 3 days, culture supernates still contained 0.125 ± 0.025 nM mIL-3 or mGM-CSF. These cells represented a population of cells continually exposed to high levels of either mIL-3 or mGM-CSF. Cells from high and low density cultures grown in mIL-3 were examined for mIL-3 receptor expression by Scatchard analysis (Figure 20). Scatchard plots revealed a single class of receptors with an apparent Kpj of 1 nM in both cell populations. However, the number of receptor sites was 10-fold higher on the high density cells (>100,000 sites available on high density cells vs. <10,000 sites on low density cells). This tremendous difference in receptor density could not be explained by a change in cell size since FACS analysis of the two cell populations, using orthogonal light scatter, revealed that the high density 124 Figure 20. Scatchard analysis of *2^I-mIL-3 binding to B6SUtA2 cells from low and high density cultures grown in mIL-3. Low density cultures (O) were found to express < 10,000 receptors/cell while high density cultures (•) expressed > 100,000 receptors/cell. Values are the means of triplicate determinations. Supernates from the low density cultures contained 0.125±0.025 nM mIL-3, and those from high density cultures contained <0.3 pM mIL-3. 125 cells were, in fact, slightly smaller than the low density cells. The receptor density difference could also not be explained by partial receptor occupancy in the low density cell population since B6SUtAj cells, preincubated with 0.15 nM mIL-3, showed less than a 10% reduction in available binding sites (Figure 18). It thus appeared that the mIL-3 present in the cultures of low density cells was actively down regulating mIL-3 surface receptor expression. To rule out the possibility that simple cell density alone was regulating mIL-3 receptor expression and, also, to examine the effect of growth in mGM-CSF on mIL-3 receptor expression, Scatchard analyses were performed on cells grown to the same high and low densities in mGM-CSF (Figure 21). The results demonstrated that high and low density cells in mGM-CSF had similar levels of mIL-3 receptor expression (> 100,000 sites/cell with an apparent of 1 nM). To further corroborate these findings and to study the kinetics of up-regulation of the mIL-3 receptor, low density cells growing in 0.15 nM mEL-3 were washed free of mIL-3 and incubated at 37°C in 20% FCS/RPMI for various time periods before assaying the cells for mIL-3 receptor expression. Scatchard plots revealed that B6SUtA j cells start to up-regulate their mIL-3 receptors within 1 hour of mIL-3 removal and reach plateau levels (> 100,000 receptors/cell) by 18 h (Figure 22). In many other growth factor systems, the level of receptor expression correlates with the biological responsiveness of target cells to growth factor [13]. To examine the possible biological consequences of high and low mIL-3 receptor expression on responsiveness to mIL-3, cells grown in mIL-3 from both high and low density cultures were assayed for their sensitivity to mIL-3 and mGM-CSF. Figure 23 shows that in 24 h 3H-thymidine proliferation assays, BeSUtAj cells grown to high density were 30-fold more sensitive to mIL-3 than cells grown to low density, with a 50% maximal activity at 0.03 ng/ml (3 pM) for high density cells, vs. 0.75 ng/ml (75 pM) for low density cells. The difference in both mIL-3 receptor number and mIL-3 sensitivity in these cell populations in no way affected their proliferation in response to mGM-CSF, as shown in Figure 24. In this case, both cell populations responded with half-maximal activities at close to 0.9 ng/ml of mGM-CSF. Figure 21. Scatchard analysis of lz:5I-mIL-3 binding to B6SU1A1 cels from low and high density cultures grown in mGM-CSF. Cels from both low (O) and high density (•) cultures were found to expres > 10,00 receptors/cel. Values are the means of triplicate determinations. Supernates from the low density cultures contained 0.125±0.025 nM GM-CSF, and those from the high density cultures contained < 12 pM GM-CSF. 127 0.14 0 100 50 200 250 l 2 5 l - I L - 3 B O U N D ( p M ) Figure 22. Scatchard analyses of 1 1 0 mIL-3 binding to low density B6SUtAj cells initially grown in 0.15 nM mIL-3 and then incubated for different lengths of time in the absence of growth factor. Cells taken at zero time (O) expressed < 10,000 receptors/cell; those taken at 1 h (A), 25,000 receptors/cell; those at 12 h (•), 50,000 receptors/cell; while those taken after 18 h in the absence of growth factor expressed approximately 100,000 receptors/cell (•). In all cases the K D remained at approximately 1 nM. 128 uo( 03 o 100 Q . o a c • 80 a> c 60 E sz r-i 40 X n E 20 E X CO o CT" -Figure 23. Dose response of mIL-3 on ^H-thymidine incorporation into B6SUtAj cels from low and high density cultures grown in mIL-3. The diference in sensitivity betwen low (o) and high density (•) populations, as asesed by the 50% maximal incorporation value, is aproximately 30-fold. Values are the means of duplicate determinadons ± S.E.M. 129 Figure 24. Dose response of mGM-CSF on ^H-thymidine incorporation into B6SUtA j cels from low and high density cultures grown in mIL-3. From 50% maximal incorporation values, low (O) and high density (•) cels show litle diference in their sensitivities to mGM-CSF. Values are the means of duplicate determinations ± S.E.M. 130 Another factor-dependent cell line, 32D c3, was similarly cultured for 3 days in mIL-3 and assayed for mIL-3 sensitivity to determine if the effect of mIL-3 on B6SUtA j cells was unique. Figure 25 shows that analogous results were obtained with this cell line since high density 32D c3 cells (50% maximal activity at 0.15 ng/ml) were 5-fold more sensitive to mIL-3 than low density cells (50% maximal activity at 0.79 ng/ml). 131 Figure 25. Dose response of mIL-3 on •}H-thymidine incorporation into 32D c3 cells from low and high density cultures grown in mIL-3. High density cells (•) appear to be 5-fold more sensitive to mIL-3 than low density cells (O). Values are the means of duplicate determinations ± S.E.M. 132 3.4 DISCUSSION Growth factor binding to specific membrane bound receptors has been shown to be a major site of regulation of growth factor response. The subsequent down-regulation of receptors following exposure to a given specific growth factor has been demonstrated for numerous growth factor systems and has been associated both in vitro and in vivo with growth factor desensitization [12-15]. To gain further insight into the mechanism of action of mIL-3, the fate of mIL-3 and its cell surface receptor as well as changes in mIL-3 receptor expression following acute or chronic stimulation of factor-dependent cells with either mIL-3 or mGM-CSF have been investigated. From binding and acid treatment studies it appears that mIL-3 is rapidly internalized and specifically processed (Figures 16,17a, 17b). The products of the proteolytic processing begin to appear 3 h after internalization at 37°C, the first cleavage product being a molecule truncated by approximately 1 kd. This finding may have some bearing on the two distinct forms of mIL-3 that have been purified from WEHI-3 conditioned medium [16,17]. The two are identical in amino acid sequence, although one is shorter by six amino acids at its N-terminal. This is probably a result of post-translational cleavage since both forms appear to be coded for by the same gene [18,19]. The relationship between the two species is not fully understood, though the full length form may be more active [16]. The E. coli-derived unglycosylated recombinant mIL-3 used in this study is the full length sequence of mIL-3 [8]. The early cleavage events, seen with both E. coli and COS cell derived material, and the subsequent degradation steps to TCA soluble material appear to take place within lysosomes since they are inhibited by methylamine. Unlike its ligand, it appears from cycloheximide studies that internalized mIL-3 receptors are not degraded but are recycled back to the cell surface (Figure 19). Alternatively, although less likely, mIL-3 receptors may indeed be degraded but a large intracellular pool of mIL-3 receptors may exist that is not 133 exhausted during the 6 h exposure to cycloheximide used in this study. Moreover, from short-term re-expression studies (Figure 18), it appears that the mIL-3 concentration in the medium determines the rate of cell surface receptor re-expression. This is not surprising as other growth factors regulate their cell surface receptor densities in a similar fashion [12,13]. For example, it was found that increasing the extracellular concentration of insulin increased the down-regulation of its receptors on BC3H-1 cells (to a maximum of 50%) by specifically increasing the rate of internalization. However, in this system, a threshold effect exists, such that concentrations < 10 nM mIL-3 induce only a transient alteration in receptor expression, whereas concentrations > 20 nM IL-3 inhibit re-expression by 50% for at least 6 h. The data from long-term exposure studies also suggest that mIL-3 down-regulates its receptor. It has been demonstrated that B6SUtA| cells grown to low density in mIL-3 exhibited a 10-fold reduction in mIL-3 receptor expression compared to cells grown in mIL-3 depleted medium (high density cells) (Figure 20). To ensure that this was not a cell density phenomenon, B6SUtAj cells were grown in mGM-CSF to both low and high densities and assayed by Scatchard analysis for mIL-3 receptor expression (Figure 21). Cells from botii populations expressed equally high levels of mIL-3 receptors (> 100,000 receptors/cell) presumably due to an absence of mIL-3 induced down-regulation. Kinetic studies carried out with low density cells grown in mIL-3 and then starved for growth factor revealed that up-regulation from approximately 10,000 to 100,000 mIL-3 cell surface receptors takes approximately 18 h at 37°C (Figure 22) To investigate if mIL-3 receptor expression correlated with B6SUtAj cell sensitivity to mDL-3, 24 h proliferation assays were performed with cells from low and high density cultures grown in mIL-3 (Figure 23). It was found, as expected from studies of other growth factor systems, that the high receptor expression seen in high density cells correlated with a 30-fold increase in mIL-3 sensitivity when compared to low density cells. Furthermore, another mEL-3-dependent cell line, 32D c3, responded in a similar fashion when cultured in an identical manner (Figure 25). Given these results, it becomes particularly difficult to assign accurate specific activities to preparations of mIL-3 or perhaps other 134 hemopoietic growth factors. With low and high density B6SU1A1 cells grown in mIL-3, a unit of bioactivity (as defined by 50% maximal incorporation) could easily be defined as being equivalent to either 75 pM (low density cells) or 3 pM (high density cells). The fact that the biological response to mIL-3 (i.e., 50% maximal responses at 75 pM and 3 pM for low and high density cells, respectively) is occurring far below the apparent (1 nM) suggests, in the absence of any evidence for two affinity classes for mIL-3, that very few ligand molecules are required to be bound to elicit a biological response. If the same absolute number of molecules are needed for both the low and high density cells, this would explain why the high density cells give a 50% maximal response at a lower mIL-3 concentration. No effect of mGM-CSF on mIL-3 receptor expression has been found, possibly confirming, in part, work by others [20]. However, in this study, Walker et al [20] reported that down-modulatory effects of CSFs were observable in primary cell cultures. Thus, data provided here (generated with an immortalized line) can not be used to substantiate the claims made by Walker et al. In short-term assays, mGM-CSF could neither compete directly with 12^I-mIL-3 for receptor sites, nor alter the steady state level of mIL-3 receptor expression. For purposes of mIL-3 receptor purification, it would appear then that mGM-CSF is the obvious choice as the factor in which to grow cells which are both mIL-3 and mGM-CSF responsive, as no difficulty with mIL-3 receptor down-regulation would be encountered and cells could be harvested while in healthy log phase growth. In conclusion, equilibrium binding of mIL-3 to cells is reached within 30 min at 37° and surface-bound mIL-3 appears to be rapidly internalized (>90% within 1 h at 37°) and degraded in lysosomes (by 6 h). In short-term studies, mIL-3 receptor reexpression rate varies with the exogenous concentration of mIL-3 and is essentially complete by 5 h when down-regulated in the presence of <20 nM mIL-3. Moreover, in these studies, mIL-3 receptor reexpression did not require <fo=7>de novo<fo=5> protein synthesis. Under standard culture conditions, concentrations of mIL-3 as low as 0.15 nM profoundly down-regulate surface receptors, however, as cells begin to exhaust the mIL-3 in their medium, receptor 135 reexpression ensues. Within 18 h of the removal of mIL-3 from their surround, BeSUtAj cells express maximal numbers of mIL-3 receptors. From the data generated within, it would appear that mIL-3 expression is dependent not only on the concentration of mIL-3, but also the time exposed to mIL-3. Thus, even low concentrations (i.e. those required to maintain cells in log phase growth) can effect marked receptor down-regulation if cells are permitted time to 'adjust' to their environment Finally, a correlation between reduction in m I L r 3 surface binding sites and a decrease in cell sensitivity to mIL-3 has been demonstrated. 136 3.5 REFERENCES 1. Farrar WL, Thomas TP, Anderson WB. Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C. Nature 315:235, 1985. 2. Whetton AD, Dexter TM. Effect of haematopoietic cell growth factor on intracellular ATP levels. Nature 303:629, 1983. 3. Whetton AD, Bazill GW, Dexter TM. Haemopoietic cell growth factor mediates cell survival via its action on glucose transport. EMBO J 3:409,1984. 4. Sorensen P, Mui A, Murthy S and Krystal G. Interleukin-3, GM-CSF and TPA induce distinct phosphorylation events in an IL-3 dependent multipotential cell line. Blood 73:406,1989. 5. Koyasu S, Tojo A, Miyajima A, Akijama T, Kasuga M, Urabe A, Schreurs J, Arai K, Takaku F and Yahari I. Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of M r 150,000 in multi-factor-dependent myeloid cell lines. EMBO J 6:3979, 1987. 6. Morla AO, Schreurs J, Miyajima A and Wang JYJ. Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in Interleukin-3-dependent murine cell lines. Mol Cell Biol 8:2214-2218,1988. 7. Glenney JR, Chen WS, Lazar CS, Walton GM, Zokas L M , Rosenfeld MG, Gill GN. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell 52:675, 1988. 8. Kindler V, Thorns B, de Kossodo S, Allet B, Eliason JF, Thatcher D, Farber N, Vassalli P. Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3. Proc Nad Acad Sci USA 83:1001,1986. 9. Delamarter JF, Mermod JJ, Liang C-M, Eliason JF, Thatcher D. Recombinant murine GM-CSF from E coli has biological activity and is neutralized by a specific antiserum. EMBO J 4:2775, 1985. 10. Palaszynski EW, Ihle JN. Evidence for specific receptors for interleukin 3 on lymphokine-dependent cell lines established from long-term bone marrow cultures. J Immunol 132:1872,1984. 11. Laemmli VK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970. 12. Heldin CH, Wasteson A, Westermark B. Interaction of platelet-derived growth factor with its fibroblast receptor. J Biol Chem 257:4216,1982. 13. Gavin JR, Roth J, Neville DM, De Meyts P, Buell DN. Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc Nad Acad Sci USA 71:84, 1974. 137 14. Heaton JH, Kreh NL, Gelehrter TD. Regulation of insulin an insulin-like growth factor (IGF) responsiveness by IGFs in rat hepatoma cells. Endocrinology 118:2555,1986. 15. Wehrenberg WB, Serfert H, Bilezikjian L M , Vale W. Down-regulation of growth hormone releasing factor receptors following continuous infusion of growth hormone releasing factor in vivo. Neuroendocrinology 43:266,1986. 16. Clark-Lewis I, Kent SBH, Schrader JW. Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells. J Biol Chem 259:7488,1984. 17. Ihle JN, Keller J, Henderson L, Klein F, Palaszynski E. Procedures for the purification of interleukin 3 to homogeneity. J Immunol 29:2431,1982. 18. Yokota T, Lee F, Rennick D, Hall C, Arai N, Mosmann T, Nabel G, Cantor H, Arai K. Isolation and characterization of a mouse cDNA clone that expresses mast-cell growth-factor activity in monkey cells. Proc Nad Acad Sci USA 81:1070, 1984. 19. Fung MC, Hapel AJ, Ymer S, Cohen DR, Johnson RM, Campbell D, Young IG. Molecular cloning of cDNA for murine interleukin-3. Nature 307:233, 1984. 20. Walker F, Nicola NA, Metcalf D, Burgess AW. Hierarchical down-modulation of hemopoietic growth factor receptors. Cell 43:269,1985. 138 CHAPTER IV CHARACTERIZATION OF THE MURINE INTERLEUKIN 3 RECEPTOR 4.1 INTRODUCTION Recently, a number of different molecular weight assessments for the mIL-3 receptor have been reported. Nicola and Peterson have reported two mIL-3 receptors of 60 and 70 kd [2]. Park et al also found two mIL-3 receptors, but these were approximately 70 and 115 kd [3]. May and Ihle reported a single mIL-3 receptor of 65-70 kd [4] as has Sorensen et al [1], while Schreurs et al presented data suggesting the presence of three distinct mIL-3 binding proteins of 70, 115 and 150 kd [5]. Palaszynski et al recently demonstrated 3 crosslinked proteins of 70, 130 and 150 kd [6]. Lastly, Isfort et al [8], without the aid of chemical crosslinking agents, have reported the specific interaction of mIL-3 with a 140 kd protein. In this chapter we have used a number of homobifunctional chemical crosslinking reagents and N-glycanase to further characterize the structure of the mIL-3 receptor and we propose a model for receptor structure based on our data that may help explain some of the molecular weight discrepancies previously reported. 139 4.2 MATERIALS AND METHODS Materials. Pure E.coli derived recombinant mIL-3 [7] was kindly supplied by Biogen, Geneva, Switzerland. (12^I)-sodium iodide (100 mCi/ml; carrier-free) in NaOH solution, pH 7-11, was purchased from Amersham. Disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), dithiobis (succinimidylpropionate) (DSP), and ethylene glycolbis (succinimidylsuccinate) (EGS) were from the Piece Chemical Company. Glutaraldehyde (GDA) was obtained as a 50% (w/w) solution from BDH Chemicals. Other reagents, unless otherwise indicated, were purchased from Sigma. Cells. See CHAPTER II. Preparation of radiolabelled mIL-3. See CHAPTER III. Scatchard analysis. Starting with an initial concentration of 12 nM, 2-fold serial dilutions of 12^I-mIL-3 in binding buffer (1% BSA, 0.1% gelatin, 0.02% sodium azide in PBS) were carried out in flexible Falcon 3911 Microtest III assay plates with and without a 20-fold excess of unlabelled mIL-3 to correct for non-specific binding. Cells were then added in binding buffer to give a final concentration of 1 x 10** cells/ml in a total volume of 100 ul. After 2 h of rocking at 4°C, plates were washed 3 times with binding buffer and 50 ul of 2% agarose added to each well. After polymerization, the wells were excised and counted in a Beckman Gamma 5500 counter. Chemical crosslinking of ^2^I-mIL-3 to intact cells, plasma membranes and solubilized plasma  membranes. For studies with intact cells, B6SUtA1 cells were washed twice with PBS containing 0.1% BSA, resuspended at 2 x 10^  cells/ml in PBS containing 1% BSA and gently rocked at 4°C for 2 h with 12^I-mIL-3 (3 nM unless otherwise indicated). The cells were then washed twice with 4°C PBS and 50 ul aliquots, at 2 x 10^ cells/ml, added to 0.5 ml microfuge tubes. Crosslinkers were added to give final concentrations of 400 uM (unless otherwise indicated) and the samples gently rocked at 4°C for 1 h. The cells were then either washed twice with PBS and incubated for a further 1 h at 37°C with or without 140 sodium azide, methylamine or bacitracin or simply pelleted, resuspended in SDS sample buffer and boiled for 1 min in preparation for SDS-PAGE. For studies with plasma membranes, cells were suspended in ice-cold homogenization buffer (10 mM Tris-Cl, pH 7.4, 100 kallikrien inhibitor units/ml aprotinin, 40 ug/ml leupeptin, 1.0 mM freshly added PMSF) and homogenized on ice by 30 strokes of a Wheaton homogenizer. The homogenates were then passed 3 times through a 26 gauge needle and centrifuged at 800 g for 5 min at 2°C to pellet nuclei and unbroken cells. The supernatants were layered onto 41% sucrose cushions and centrifuged at 50,000 g for 1 h at 4°C using a SW41 rotor in a Beckman L8-80M ultracentrifuge. The material at the interface was then centrifuged at 80,000 g for 1 h at 4°C and resuspended at 40 mg/ml in homogenization buffer containing 0.2 M sucrose and used immediately or stored at -70°C until use. For crosslinking experiments, the plasma membranes, at a final concentration of 2 mg/ml in PBS containing 1% BSA were rocked in microfuge tubes with 12^I-mIL-3 (3 mM) at 4°C for 2 h. Membranes were then pelleted at 16,000 x g in an Eppendorf 5415 microfuge and washed twice with PBS, resuspended at 2 mg/ml in PBS and crosslinked at 4°C for 1 h in 50 ul aliquots with the same concentrations of crosslinkers used with intact cells. Samples were then microfuged at 16,000 g for 15 min and boiled for 1 min in 40 ul of SDS sample buffer for SDS-PAGE. For studies with solubilized plasma membranes, plasma membranes at a final concentration of 2.9 mg/ml in an 0.5% NP40 buffer containing 50 mM Hepes, pH 7.4, 100 mM NaF, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 4 mM EDTA and freshly added 100 KlU/ml aprotinin, 1 mg/ml leupeptin and 0.5 mM PMSF, were rocked for 1 h at 4°C. An equal volume of the same buffer without NP40 was added and, after 5 min rocking at 4°C, the preparation was centrifuged at 16,000 x g in a microfuge. The supernatant was then supplemented with ^2^I-mIL-3 (3 nM) and rocked at 4°C for 3 h. Crosslinkers at the same concentration described above were added to 50 ul aliquots and the samples rocked for 1 h at 4°C. They were then made 2% with SDS, 5% with 2-ME, 10% with glycerol and boiled for 1 min in preparation for SDS-PAGE. Samples were electrophoresed on 7.5% polyacrylamide gels. 141 Dried gels were exposed to Kodak X-Omat AR film at -70°C. Autoradiograms were scanned on a Hoefer GS 300 densitometer (Hoefer Scientific Instruments) and peak areas were analyzed by a SP4100 computing integrator (Spectra-Physics). N-glycanase treatment of receptor complexes. For N-glycanase studies with intact cells, B6SUtAj cells were incubated with 3 nM i2^I-mIL-3 and crosslinked with DSS as described above. 2x10^ crosslinked cells were then lysed and solubilized in 50 ul of 0.5% SDS, 100 mM 2-ME and boiled for 1 min. Plasma membranes from B6SUtAj cells were incubated with 3 nM i2^I-mIL-3 and crosslinked with DSS as described above. The membranes were then washed with 150 mM NaCl and briefly treated with 100 mM glycine, 150 mM NaCl, pH 3.0 to remove uncrosslinked l2^I-mIL-3. After 5 min at 4°C, crosslinked membranes were washed with PBS, suspended in 70 pi of 0.5% SDS, 100 mM 2-mercaptoethanol (2-ME) and boiled for 3 min. Both the intact cell and plasma membrane samples were then made 0.17% SDS, 40 mM 2-mercaptoethanol, 200 mM sodium phosphate, pH 8.6, 10 mM 1,10 phenantiiroline hydrate, 1.25% NP40, and 1 unit of N-glycanase (Genzyme Corporation) was added. To stop the reaction, samples were made 4% SDS, 10% glycerol, 5% 2-ME and boiled for 3 min. 1 2 5 I -BSA was used as a control to test for protease activity in the N-glycanase preparation. Since a 24 h incubation of l 2 ^I-BSA with 2 units of N-glycanase resulted in no proteolytic degradation of 1 2^I-BSA, the N-glycanase stock was considered free of detectable protease activity. SDS-PAGE analysis and autoradiography was performed as described above. 142 4.3 RESULTS Scatchard Analysis. As a first step in characterizing mIL-3 receptors on B6SUtAj cells, Scatchard analyses were performed with ^I-mIL-3. These studies revealed that there was only a single affinity class (apparent K D ~1 nM) and that this cell line expressed approximately 100,000 receptors/cell (Figure 26) under mIL-3 exhausted conditions. Crosslinking of ^I-mIL-3 to B6SUtAj cells. Although data from Scatchard analyses revealed only one affinity class, upon crosslinking of 12^I-mIL-3 to intact B6SUtAj cells, 2 protein species were observed, their apparent molecular masses being 70 kd (p70) and 140 kd (pl40) (after subtracting the 15 kd molecular weight of E.coli-derived 12^I-mIL-3). It should be noted that a high molecular weight complex (>200 kd) has also been observed on some gels, although its significance is unknown. The ratio of the 2 radiolabeled complexes remained constant regardless of the incubation time with 12^I-mIL-3 (Figure 27, lanes 1 through 5) and both could be specifically competed by excess unlabelled mIL-3 (Figure 27, lane 6). Because of the insensitivity inherent in Scatchard analyses, the possibility still existed that p70 and pl40 represented 2 distinct mIL-3 receptor classes with a difference in K D of less than one order of magnitude. To address this, a variety of *2^I-mIL-3 concentrations were used in DSS crosslinking studies with intact B6SUtAj cells. Since a receptor is 50% occupied by its ligand at its K D , decreasing the concentration of the ligand might allow for the selective binding and crosslinking of 12^I-mIL-3 to the receptor with die higher affinity. However, as can be seen from the autoradiogram presented in Figure 28 and corresponding densitometric scan shown in Table 3, the relative intensities of pl40 and p70 appear to remain unchanged over a wide range of 12^I-mIL-3 concentrations used for binding. This suggested that p70 and pl40 had similar affinities for mIL-3 and substantiated the Scatchard data. To this point the data were consistent with three models; i.e., p70 and pl40 were two distinct receptor proteins with similar affinities for mIL-3; p70 was the ligand binding subunit and, when 143 Figure 27. Time course of DSS crosslinking of 1Z5I-mIL-3 to intact BeSUtAj cells. Cells were incubated with 125I-mIL-3 (3 nM) at 4°C. At set times, cells were washed and crosslinked with DSS. Lanes 1-4 are 1, 2, 4 and 6 h binding times, respectively. Lane 6 is a 6 h binding interval to which a 20-fold excess of unlabelled mIL-3 was added. Lane 5 represents a 30 min binding point at 37°C. 144 5 .25 .125 .06 .03 .015 .008 200 — 92.5 — 69 ***** ; r. ., 46 — 41 30 — Figure 28. Crosslinking of various concentrations of 1Z3I-mIL-3 to intact B6SUtAj cells. Cells were incubated with a variety of concentrations (given above each lane) of labelled mIL-3 for 2 h at 4° C, then washed and crosslinked with DSS. The concentrations used for crosslinking are given in mM. 145 TABLE 3. Relative Band Intensity as a function of 1 Z 3I-mIL-3 Concentration I-mIL-3 Concentration (nM) p70(%) p!40(%) 1 75 25 0.5 79 21 0.25 71 29 0.125 71 29 0.063 71 29 0.032 78 22 146 crosslinked with another protein, formed pl40 or; that pl40 was the mIL-3 receptor and was cleaved to form p70. To discriminate amongst these models, it was reasoned that if pl40 represented a dimeric complex that included p70, then the formation of pl40 (relative to p70) should be favored as crosslinker IOC concentration was increased. However, when lz,JI-mIL-3 crosslinking studies were conducted over a wide range of DSS concentrations, p70 predominated at high DSS concentrations, while pl40 was the major species at low concentrations (Figure 29 and Table 4). These data did not favor the hypothesis that pl40 was a dimeric complex which included p70. Since DSS stocks were dissolved in DMSO, the concentration of DMSO in each sample was kept constant, and thus, this effect was not due to increasing DMSO concentrations. A similar shift in p70/pl40 could be obtained by keeping the crosslinker concentration fixed and varying the cell concentration, i.e., as the cell concentration was raised, the crosslinker/cell ratio dropped and pi40 became more prominent. To further test the three models, cross-linked samples were incubated under various conditions prior to SDS-PAGE analysis. As can be seen in Figure 30, when intact B6SUtAi cells were shifted up to 37°C after standard l z , JI-mIL-3 binding and crosslinking, an increase in the relative intensity of p70 was observed. This has also recently been reported by Isfort et al [8]. Interestingly, this temperature induced decrease in pl40/p70 could be prevented by sodium azide, bacitracin or methylamine, but not by ATP. Moreover, when 0.02% sodium azide was present during the standard ^2^I-mIL-3 binding (2 h, 4°C) and DSS crosslinking (1 h, 4°C) periods, there was a marked increase in pl40/p70 (compare Figure 30a, lanes 1 and 6). These changes were most consistent with the third model and suggested that pl40 was associated with an azide, bacitracin and methylamine inhibitable protease. That a protease was involved was suggested by the finding that the 37°C induced decrease in pl40/p70 could be prevented by first heating the sample in SDS sample buffer (Figure 30b, lane 6). This shift in pl40/p70 with incubation at 37°C was in apparent contradiction to the earlier finding that pl40 and p70 co-developed with time (Figure 27). However, this discrepancy suggested that the act of crosslinking itself might play a role in allowing protease cleavage. 147 2 0 0 -46-F igu re 29. C r o s s l i n k i n g o f l z ; 5 I - m I L - 3 to B 6 S U t A j ce l l s i n the p resence o f v a r y i n g D S S concent ra t ions . 1 2 ^ I - m I L - 3 was bound to ce l l s fo r 2 h at 4 ° C , then c r o s s l i n k e d to c e l l s w i t h s e v e r a l d i f fe ren t concen t ra t i ons o f D S S ( g i v e n at the top o f e a c h lane i n m M ) . In the absence o f c r o s s l i n k e r (0 m M ) , ne i t he r p 7 0 n o r p l 4 0 are c r o s s l i n k e d . 148 TABLE 4. Relative Band Intensity as a function of DSS Concentration DSS Concentration (mM) p70(%) p!40(%) 2 57 43 1 54 46 0.5 49 51 0.25 39 61 0.125 36 64 0.063 23 77 0.032 0 100 149 Figure 30. a) DSS crosslinking of l z ; > I - m I L - 3 to intact B6SUtAj cells followed by incubation at 37°C. Cells were incubated for 2 h at 4°C with 3 nM 1 2 5 I -mIL-3 , crosslinked with 400 p M DSS for 1 h at 4°C (lane 1) and then shifted up to 37°C for 1 h in the absence (lane 2) or presence of 0.02% sodium azide (lane 3), 10 mM methylamine Gane 4), or 1 mM ATP (lane 5). Lane 6 is identical to lane 1 except the sample contained azide throughout. b) In this overlapping experiment, lanes 1 through 4 are equivalent to lanes 1 through 4 in Fig. 3a, the sample electrophoresed in lane 5 contained 1 mM bacitracin during the 37°C incubation and that in 6 was boiled for 1 min in 2% SDS, 5% 2-ME prior to the 1 h 37°C incubation. 150 To investigate further the nature of the mIL-3 receptor, the effect of different length crosslinkers was examined. Because of the effect of crosslinker concentration on the pl40/p70 ratio, extreme care was taken to ensure that all crosslinkers were tested at the same molar concentration. The 5-carbon crosslinker, glutaraldehyde (GDA) crosslinked 1 2^I-mIL-3 to only p70 (Figure 31). This result could be explained by the ability of G D A to induce a conformational change within the receptor that favored proteolysis. Alternatively, due to distance constraints and a position shift that might place a p70 fragment into very close proximity to *2^I-mIL-3, the 5-carbon crosslinker might only be able to bridge the 1 2^I-mIL-3-p70 gap, and not crosslink an uncleaved pl40 to 1 2^I-mIL-3. Interestingly, incubation with sodium azide during crosslinking with G D A did not permit the appearance of pl40. Crosslinking of 1 2^I-mIL-3 to plasma membranes. To determine whether tiie putative cleavage of pl40 to p70 was occurring at the cell surface, crosslinking studies were performed with plasma membranes prepared from BeSUtAj cells. It was found that DSS crosslinking of 1 2^I-mIL-3 to plasma membranes yielded only p70 (Figure 32, lane 1). This was the case even when the binding and crosslinking periods were shortened to 15 min each or when a variety of protease inhibitors (leupeptin, aprotinin, PMSF, and EDTA) were present. However, if the binding and crossslinking were carried out in the presence of 0.02% sodium azide, both species were obtained and in a ratio similar to that seen with intact cells (Figure 32, lanes 2 & 3). Most importantly, to obtain tiie pl40 band, it was crucial to have the sodium azide present during the 1 h, 4°C crosslinking step. If it was present throughout the preparation of plasma membranes from intact cells and also during the 2 h 4 ° C incubation with 1 2 ^I-mIL-3, but not during crosslinking, pl40 was not seen (Figure 32, lanes 3 & 4). These results suggested that cleavage did indeed occur at the cell surface, since it took place with plasma membranes, and that the protease was more active in the plasma membrane preparation (since only p70 was typically seen) tiian in intact cells. In addition, because sodium azide was only necessary during crosslinking for the appearance of pl40, it suggested that perhaps the crosslinking per se altered the conformation of pl40 such that it became a,substrate for a closely 151 .03 .06 1.25 2.5 5 10 20 40 C 2 0 0 -9 2 . 5 -6 9 -4 6 -Figure 31. GDA croslinking of lz;5I-mIL-3 to intact B6SLHA! cels. A variety of concentrations of GDA (given above each lane in mM) were used to croslink previously bound 12I^-mIL-3 to intact B6SUtAi cels. Only p70 apears to be croslinked by GDA. 152 Figure 32. DSS crosslinking of 1 Z 3I-mIL-3 bound to B6SUIAJ plasma membranes and solubilized plasma membranes. Plasma membranes were either prepared from cells, bound with ^2^I-mIL-3 and DSS crosslinked in the absence of 0.02% sodium azide (Jane 1); prepared without azide but bound and crosslinked in azide (lane 2); prepared, bound and crosslinked in azide (lane 3); prepared and bound but not crosslinked in azide (lane 4); prepared but not bound nor crosslinked in azide (lane 5). Solubilized membranes from azide prepared plasma membranes but bound and crosslinked in the absence of azide (lane 6); were treated as in lane 6 and then shifted up to 37°C for 1 h (lane 7); were prepared, bound and crosslinked in the presence of azide (lane 8); were prepared, bound and crosslinked in the absence of azide (lane 9). M indicates molecular weight markers. 153 associated protease and that sodium azide inhibited this cleavage by acting on the protease or by direcdy interfering with crosslinking. Crosslinking of 1 2^I mIL-3 to solubilized receptors. To gain further insight into the physical relationship of the putative protease with the mIL-3 receptor, plasma membranes were solubilized with NP40 and the solubilized receptor preparation incubated with 12^I-mIL-3 and then crosslinked with DSS (Figure 32, lanes 6-9). Unlike with plasma membranes, both p70 and pl40 were observed whether sodium azide was present or not, suggesting the protease was not as active as in plasma membranes. However, some protease activity was still evident since shifting up to 37°C after crosslinking caused pl40 to disappear (Figure 32, lane 7). Heterocrosslinking of 12^I-mIL-3 to its receptor. To direcdy demonstrate that p70 was a component of pl40, a heterocrosslinked pl40 species was generated, using GDA and the disulfide containing crosslinker, DSP. If p70 was indeed a cleavage product of pl40, the use of these two crosslinkers might allow for the selective extraction of p70 from pl40 under reducing conditions (see the postulated heterocrosslinked mIL-3 receptor complex in Figure 33a). Accordingly, heterocrosslinked cells were solubilized in SDS sample buffer and electrophoresed using SDS-PAGE under non-reducing conditions. After tiie unfixed gel was dried and autoradiographed, the radiolabelled pl40 band was excised, eluted, and lyophilized. P140 was then re-electrophoresed under both reducing and non-reducing conditions (Figure 33b). Lane 1 is the heterocrosslinked pl40 species re-electrophoresed under non-reducing conditions. It is clear that pl40 represents the only labelled species under non-reducing conditions. Under reducing conditions (lane 3), labelled IL-3 was released from the complex and travels with the dye front (not pictured), and a small amount of pl40 remained unreduced. More importantly, a small but significant amount of p70 appeared as a consequence of the reduction of pl40. Lane 2 is a control showing that all pl40 complexes, crosslinked with only DSP, do not release their 12^I-mIL-3 under identical reducing conditions. More importantly, p70 was not recovered from this sample. Figure 33. a) The postulated heterocrosslinked mIL-3 receptor complex. b) Heterocrosslinking of pl40 with DSP and GDA. Cells were incubated with 3 nM 125I-mIL-3 for 2 h at 4°C, then washed and crosslinked with . mM DSP and 0.6 mM GDA. Samples were electrophoresed on an 8-12% gradient gel under non-reducing conditions, and, after the unfixed gel was dried and autoradiographed, the pl40 species was excised, eluted, and lycphilized. Purified pl40 was then re-electrophoresed on an 8-12% gradient gel under the following conditions: lane 1, heterocrosslinked pl40 under non-reducing conditions; lane 2, DSP crosslinked pl40 under reducing conditions; lane 3, heterocrosslinked pl40 under reducing conditions. 155 N-deglycosylation of p70. To further characterize the mIL-3 receptor, 12^I-mIL-3 was bound and DSS crosslinked to B6SUtA j cells. Treatment of crosslinked cells, after solubilization, with N-glycanase revealed that pl40 was decreased by approximately 10 kd, as was p70 (Figure 34a). This result suggested that pl40 was not a dimer of p70 since we would expect a decrease of 20 kd in the pl40. However, it is difficult to accurately quantitate molecular weight changes with proteins of this size. To examine the sugar structure in more detail, 12^I-mIL-3 was bound and DSS crosslinked to B6SUtAj plasma membranes. Crosslinked membranes were then solubilized arid digested with N-glycanase for different times. The results suggested, that two N-linked sugar chains of 8 kd and 3 kd were present on p70 (Figure 34b). Digestion times of up to 24 h revealed no further digestion. 156 a 200-92.5-69-46-B f t * • * 1 N-Glycanase 0 1 3hr 20-92-69-46-30-Figure 34. a) N-glycanase treatment of 12I^-mIL-3 croslinked to intact B6SUtA1 cels. After'2I^-mIL-3 was bound and DS  croslinked to cels, the cels were solubilized in 0.5% SDS, 0.8 mM 2-ME, boiled and diluted with bufer to 20 mM sodium phosphate, pH 8.6, 0.17% SDS, 40 mM 2-ME, 10 mM 1,10 phenanthroline and 1.25% NP40. One unit of N-glycanase was then aded and the solution was incubated at 37°C for 6 h. Lane A represents the starting croslinked material; lane B, material in the N-glycanase bufer without N-glycanase; lane C, material in N-glycanase bufer with 1 unit N-glycanase. b) N-glycanase treatment of l2I^-mIL-3 croslinked to B6SUtAj plasma membranes. 50 pg aliquots of purified membranes were incubated with 3 nM 125I-mIL-3 for 2 h at 4°C, then washed and croslinked with DS. Croslinked membranes were solubilized with 0.5% SDS, 0.8 mM 2-ME, boiled and diluted with N-glycanase bufer. One unit of N-glycanase was then aded and samples were incubated for the times indicated. 4.4 DISCUSSION 157 In this chapter, it was shown that 1 2 5I-mIL-3 was crosslinked by N-hydroxysuccinimidyl esters to two molecular weight species, p70 and pl40, on intact B6SUtAj cells. Although the ratio of pl40/p70 remained constant with time during incubation with ^I-mIL-3 (Figure 27), it decreased with incubation after crosslinking (Figure 30). This apparent discrepancy suggests that the actual process of crosslinking may be inducing a conformational change in the pl40 that makes it more prone to cleavage by a plasma membrane associated protease. This is supported by the finding that p70 is increased relative to pl40 as the crosslinker concentration is increased (Figure 29 and Table 4). More crosslinker per cell would allow for more receptors to be conformationally altered, and thus cleaved to p70. The fact that only p70 was observed when crosslinking studies were carried out with plasma membranes (Figure 32) suggests that cleavage occurs at the cell surface and that the putative associated protease is released from constraints it is under in intact cells. The finding that the addition of sodium azide during the crosslinking step, but not prior to this step, allows for the appearance of pi40 supports the contention that the crosslinking process itself plays a role in making the receptor susceptible to cleavage. Further evidence suggesting that the cleavage of pl40 to p70 is induced by crosslinking comes from earlier cycloheximide studies (CHAPTER III, Figure 18) which suggest that internalized mIL-3 receptors may be recycled to the cell surface and not degraded. As well, in the absence of crosslinking agents, only pl40, and not p70, can be purified from plasma membranes [9]. To gain further insight into the nature of the protease which is apparently quite active even at 4°C, the ability of various agents to alter the pl40/p70 ratio during a 37°C incubation following crosslinking was examined (Figure 30). Boiling for 1 min in SDS sample buffer prior to the 37°C incubation or addition of sodium azide, bacitracin, methylamine during the 37°C incubation period all inhibited p70 formation (Figure 30) Since sodium azide uncouples oxidative phosphorylation, we tested ATP and a non-158 cleavable ATP analog to see if the protease was ATP dependent but no effect was observed (Figure 30). The addition of various protease inhibitors or divalent cation chelators was also without effect. In this regard, addition of sodium azide during crosslinking did not allow for the appearance of pl40 when glutaraldehyde was used as crosslinker. This raises the possibility that sodium azide, methylamine and bacitracin may be preventing the cleavage of pl40 not by acting directly on the protease but by interfering with N-hychmysuccinimidyl ester crosslinking. While the temperature shift-up experiments, following crosslinking, strongly suggested that pi40 was being converted to p70, it was still conceivable that this post-crosslinking incubation simply preferentially destroyed pl40. To test more directly whether pl40 contained p70 within it, the double crosslinking experiments utilizing glutaraldehyde and DSP were carried out and confirmed that a small but significant amount of p70 could be derived from pl40 (Figure 33). Taken all together, these data suggest the model for the mIL-3 receptor shown in Figure 35. Included in this model is a recent finding [8,10] that mIL-3 stimulates the tyrosine specific phosphorylation of pi40. Further work is necessary to determine the physiological relevance of this cleavage reaction. 159 Figure 35. Model of the mIL-3 receptor. Upon mIL-3 binding, the 140 kd glycoprotein receptor becomes phosphorylated on tyrosine residues [10]. Crosslinking alters the conformation of the receptor such that it becomes more susceptible to a membrane associated protease and is cleaved to a 70 kd glycoprotein. 160 4.5 REFERENCES 1. Sorensen P, Farber NM, Krystal G. Identification of the interleukin 3 receptor using an iodinatable, cleavable, photoreactive crosslinking agent. J Biol Chem 261:9096, 1986. 2. Nicola NA, Peterson L. Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking. J Biol Chem 261:12384, 1986. 3. Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2a). J Biol Chem 261:205,1986. 4. May WS, Ihle JN. Affinity isolation of the interleukin-3 surface receptor. Biochem Biophys Res Commun 135:870, 1986. 5. Schreurs J, Miyajima A, Arai K, Sugawara W, Tanaka H, Onta Y. Murine interleukin-3 (IL-3) receptor structure. FASEB 2:Abstract #7873, 1988. 6. Palaszynski EW, Keller J, Ruscetti F. Biochemical characterization of the murine IL-3 receptor with a polyclonal antibody. J Cell Biochem Suppl 12A:110, 1988. 7. Kindler V, Thorns B, de Kossodo S, Allet B, Eliason JF, Thatcher D, Farber N, Vassalli P. Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3. Proc Natl Acad Sci USA 83:1001,1986. 8. Isfort RJ, Stevens D, May WS, Ihle JN. Interleukin-3 binds to a 140-kDa phosphotyrosine-containing cell surface protein. Proc Nad Acad Sci USA 85:7982, 1988. 9. Mui AL-F, Sorensen P, Krystal G. Purification of the Interleukin 3 receptor. Hemopoiesis Vol 120 (in press). 10. Sorensen P, Mui AL-F, Krystal G. Interleukin-3 stimulates the tyrosine phosphorylation of the 140 kilodalton interleukin-3 receptor. J Biol Chem (in press). 161 CHAPTER V SUMARY Hemopoietic cell proliferation, differentiation and survival, in vitro, is critically dependent upon the presence of HGFs. Although their role in vivo is more difficult to define, there is no doubt that HGFs participate in hemopoietic regulation. Of the HGFs thus far characterized in the murine system, only mlL-3 can support the growth of both primitive multipotential stem cells and progenitor cells committed to differendate along all myeloid, and possibly lymphoid, lineages. Thus a detailed study of mIL-3 should yield insight into hemopoietic stem cell regulation and may provide the conceptual framework for an understanding of various hematologic disorders. It was therefore the intent of this thesis to study the biology and biochemistry of mIL-3 and its surface receptor. 5.1 Purification of Interleukin 3 A simple riiree-step protocol for die purification of mIL-3 from spleen cell conditioned medium has been devised. The advantage of this procedure over those previously reported is the use of glutaraldehyde-fixed mIL-3 receptor-bearing cells to specifically absorb, and subsequendy release, mIL-3 obtained from crude conditioned medium. This initial step in the protocol, target-cell absorption, serves not only to partially purify mIL-3, but also to concentrate the factor. Aside from its obvious application to mIL-3 purification, target-cell absorption may be used for other purposes as well. Specifically, any cell-binding protein factor should, in theory, be able to be specifically absorbed onto cells expressing its receptor. Providing a cell line (or pure primary culture) is available which expresses moderate levels of surface 162 receptor (>10,000) and, that the factor can be assayed, the possibility for partial purification by target-cell absorption exists. The use of this procedure in the identification of putative ligands for cloned receptor-like oncogenes is particularly attractive. Since cells with high levels of these receptor oncogenes can be constructed through the use of new expression vectors, selection of an appropriate source containing the putative ligand is all that is required. The mIL-3 purified from mitogen-stimulated spleen cell conditioned medium migrated as two distinct protein forms on SDS/PAGE analysis, 19.5 kd and 16.5 kd. Only the 16.5 kd band remained after N-linked sugars were removed, suggesting that the difference in molecular weight of the bands was a result of heterogeneity in glycosylation. Interestingly, the differences in glycosylation appeared to have no effect on its affinity for the receptor, as both forms bound equally well to target cells and their binding was inhibited by similar concentrations of unglycosylated recombinant mIL-3. Finally, N-deglycosylation studies suggest that native mIL-3 may have a low level of previously unreported O-linked sugar residues as well. 5.2 Interleukin 3 Receptor Down-Regulation The binding of a peptide growth factor to its cell surface receptor initiates a cascade of events which results, several hours later, in DNA synthesis. The fate of the receptor-ligand complex, and its possible intracellular function, has regained new interest following recent reports that several peptide growth factors may be shuttled to the nucleus by their receptors and directly regulate gene expression [1,2]. With this in mind, the consequences of mIL-3 binding were examined. After binding, mIL-3 was found to be rapidly internalized and specifically processed. Two specific cleavage products could be demonstrated before mIL-3 was completely degraded in lysosomes. It is interesting to speculate on the significance of such intracellular processing. Presumably, specific proteases contained within endosome/lysosome vesicles are responsible for the digestions. However, recent evidence suggests that the mIL-3 receptor itself may possess proteolytic activity, and thus, it is reasonable 163 to hypothesize that the receptor might specifically cleave mIL-3. The function of mIL-3 cleavage and the subsequent generation of small (<5 kd) fragments is not known, although such processing may be necessary for signal transduction. Unlike its ligand, the mBL-3 receptor appears to escape proteolytic cleavage and be recycled back to the membrane for re-expression. Furthermore, the level of re-expression appears to depend upon the concentration of extracellular mIL-3. The mechanism by which a target cell senses the mIL-3 concentration is not known, although in light of evidence that the receptor is phosphorylated at tyrosine residues in response to ligand binding, it is conceivable diat phosphorylated intermediates are involved. Moreover, it was found that the level of receptor expression correlated directly with target cell responsiveness to mIL-3, whereby high receptor expression corresponded to an increased sensitivity to mIL-3. By culturing factor-dependent cells in low concentrations of mIL-3, the level of receptor expression could be increased up to 10-fold and the sensitivity to mIL-3, up to 30-fold. The ability of mIL-3 to down-regulate its receptor could not be mimicked by mGM-CSF, thus confirming, in part, the results of Walker et al [3] that mGM-CSF does not down-modulate the mIL-3 receptor. 5.3 Characterization of the Interleukin 3 Receptor Recently, there have been conflicting reports concerning the size and structure of the mIL-3 receptor. In an attempt to clarify and explain these data, a number of chemical crosslinking agents have been used to characterize die structure of the mIL-3 receptor. When * 25l-mIL-3 was crosslinked with N-hydroxysuccinimidyl esters to B6SUtAj cells, SDS/PAGE analysis revealed die presence of two distinct crosslinked species of 140 kd (pl40) and 70 kd (p70). Since Scatchard analysis consistendy revealed only one affinity class, it was postulated tiiat pl40 and p70 were related to each other and shared a similar ligand binding domain. Further evidence to support this contention was given when it was shown that the ratio of pl40/p70 crosslinked to 12^I-mIL-3 could be decreased (i.e. p70 increased, pl40 decreased) by shifting up the temperature immediately after 164 c r o s s l i n k i n g . T h i s sugges ted that a s p e c i f i c c e l l u l a r p ro tease m i g h t b e c o m e ac t i va ted f o l l o w i n g b i n d i n g and c r o s s l i n k i n g . P u r i f i c a t i o n o f p l 4 0 and the use o f a he te roc ross l i nk ing strategy revea led that p 7 0 c o u l d be d e r i v e d d i r e c t l y f r o m the p r o t e o l y s i s o f p l 4 0 . C o n s i s t e n t w i t h these , and o the r , d a t a , a m o d e l w a s p roposed w h i c h suggested that the m I L - 3 receptor is a 140 k d m e m b r a n e g l y c o p r o t e i n that, u p o n b i n d i n g and c r o s s l i n k i n g o f 1 2 ^ I - m I L - 3 , i s r a p i d l y c l e a v e d to p 7 0 . T h e p 7 0 c l e a v a g e p r o d u c t rep resen ts that p o r t i o n o f the mJJL-3 recep to r w h i c h con ta ins the m I L - 3 b i n d i n g si te and p r e s u m a b l y rema ins m e m b r a n e b o u n d . H o w e v e r , the fate o f the o ther 7 0 k d f ragment that w o u l d a lso be l ibera ted b y a s i ng le c leavage is u n k n o w n . S i n c e p rev ious s tud ies have s h o w n that the m I L - 3 receptor is r e c y c l e d b a c k to the c e l l sur face , i t appears that p ro teo l y t i c c l eavage o f the receptor ( poss ib l y by the receptor i t se l f ) i s an in f requent event , great ly s t imu la ted b y the con fo rma t i ona l changes induced b y c r o s s l i n k i n g . S t i l l , i t is poss ib le that receptor c l eavage re leases a m o b i l e , in t race l lu la r f ragment necessary fo r s i gna l t ransduct ion . It is a lso poss ib l e that th i s pu ta t i ve i n t r a c e l l u l a r r e c e p t o r f r agmen t m i g h t fac i l i t a te r ecep to r d o w n - r e g u l a t i o n . A l t h o u g h it is i n t e r e s t i n g to s p e c u l a t e o n the p h y s i o l o g i c ro le o f a s p e c i f i c m I L - 3 r e c e p t o r c l e a v a g e , f r o m the d a t a p r e s e n t e d w i t h i n , i t i s c o n c e i v a b l e that the p r o d u c t i o n o f p 7 0 f r o m p l 4 0 i s d u e to the a r t i f i c i a l , n o n -p h y s i o l o g i c ac t i va t ion o f a p ro teo ly t i c act iv i ty by c h e m i c a l c r o s s l i n k i n g . 5.4 Concluding Remarks T h e da ta p resen ted w i t h i n th is thes is s h o u l d h e l p fu r ther o u r u n d e r s t a n d i n g o f the m e c h a n i s m o f ac t i on o f I L - 3 . S i n c e data f r o m in vivo and in vitro s tudies suppor t a ro le f o r I L - 3 i n the regu la t ion o f the hemopo ie t i c sys tem, k n o w l e d g e o f the m e c h a n i s m o f ac t ion o f I L - 3 shou ld p rove use fu l i n de te rm in ing the i n v o l v e m e n t o f I L - 3 i n the g e n e s i s o f m a n y h e m a t o l o g i c d i s o r d e r s , i n c l u d i n g n e o p l a s i a . T o th i s e n d , H u m p h r i e s et a l [4] h a v e recen t l y demonst ra ted that t umor i gen i c h e m o p o i e t i c c e l l l i nes c a n be generated b y A b e l s o n v i r u s i n f e c t i o n o f m u r i n e b o n e m a r r o w , and w i t h sens i t i ve N o r t h e r n b l o t a n d S j m a p p i n g techn iques , demons t ra ted autocr ine m I L - 3 p r o d u c t i o n i n eve ry c e l l l i ne generated. It w o u l d be i n t r i gu ing to e x a m i n e the l e v e l o f m I L - 3 receptor exp ress ion o n these t umor igen i c c e l l l i nes and de te rmine i f , i n fact, 165 autocrine mIL-3 production was a critical step in hemopoietic cell transformation. If so, it would be interesting to determine whether the intracellular receptor activation pathway outlined by Keating and Williams [5] was operative within these cells. In conclusion, mIL-3 has been purified and its receptor studied in detail. As the gene encoding die receptor should soon be cloned, a more clear understanding of the mechanism of action of mIL-3 will be forthcoming. 166 5.5 References 1. Rakowitz-Szulczynska EM, Rodeck U, Herlyn M, Koprowski H. Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors. Proc Nad Acad Sci USA 83:3728, 1986. 2. Smith RM, Jarret L. Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process. Proc Nad Acad Sci USA 84:459,1987. 3. Walker F, Nicola N, Metcalf D, Burgess AW. Hierarchical down-modulation of hemopoietic growth factor receptors. Cell 43:269, 1985. 4. Humphries RK, Abraham S, Krystal G, Lansdorp P, Lemoine F, Eaves CJ. Activation of multiple hemopoietic growth factor genes in Abelson virus-transformed myeloid cells. Exp Hematol 16:774, 1988. 5. Keating MT, Williams LT. Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells. Science 239:914,1988. 


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