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Identification and characterization of human and murine c-fes proteins MacDonald, Ian Andrew 1989

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IDENTIFICATION AND CHARACTERIZATION OF HUMAN AND MURINE c-fes PROTEINS By IAN ANDREW MACDONALD M.D., The University of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1989 0 Ian Andrew MacDonald 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 of 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 of this thesis for f inancia l ga in shall no t b e a l l o w e d w i t h o u 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 of M i c r o b i o l o g y T h e Univers 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 D a t e SeptsmbPr 26. 1 QRQ D E - 6 (2/88) 1 1 ABSTRACT The c - f e s and c - f p s genes a r e , r e s p e c t i v e l y , t h e mammalian and a v i a n p r o t o - o n c o g e n e s w h i c h were a n c e s t r a l t o t h e r e t r o v i r a l £ps/£es o n c o g e n e s . W h i l e t h e p r o t e i n s e n c o d e d by c - f p s / f e s p r o t o - o n c o g e n e s had been i d e n t i f i e d i n a number of s p e c i e s t h e homologous p r o t e i n s had n o t been i s o l a t e d or c h a r a c t e r i z e d i n human or m u r i n e c e l l s . T h i s s t u d y was u n d e r t a k e n i n an a t t e m p t t o i d e n t i f y c - f p s / f e s p r o t e i n s i n human and m u r i n e c e l l s U s i n g a c r o s s - r e a c t i v e r a t a n t i - v - f p s serum two i m m u n o l o g i c a l l y c r o s s - r e a c t i v e p r o t e i n s o f M= 92,000 (p92) and 94,000 (p94) were i m m u n o p r e c i p i t a t e d f r o m a v a r i e t y o f human and m u r i n e c e l l s . F u n c t i o n a l s t u d i e s i n d i c a t e d t h a t b o t h o f t h e s e p r o t e i n s p o s s e s s e d p r o t e i n k i n a s e a c t i v i t y and were a b l e t o a u t o p h o s p h o r y l a t e and t o p h o s p h o r y l a t e exogenous s u b s t r a t e s a t t y r o s i n e r e s i d u e s . S t r u c t u r a l c o m p a r i s o n s s u g g e s t e d t h a t p92 was v e r y c l o s e l y r e l a t e d i n p r i m a r y s e q u e n c e t o t h e v - f e s p r o t e i n P 8 5 ' 3 S " 3 - : E " m w h i l e p94 was more d i s t a n t l y r e l a t e d . On t h e b a s i s o f i m m u n o l o g i c a l c r o s s - r e a c t i v i t y , i n t r i n s i c p r o t e i n - t y r o s i n e k i n a s e a c t i v i t y , and s t r u c t u r a l r e l a t e d n e s s i t was c o n c l u d e d t h a t p92 r e p r e s e n t s t h e human and murine c - f e s gene p r o d u c t ; p94, w h i l e p o s s e s s i n g some s i m i l a r i t i e s , i s p r o b a b l y n o t a c - f e s p r o t e i n b u t i s l i k e l y a r e l a t e d p r o t e i n - t y r o s i n e k i n a s e . i i i Examination of human and murine c e l l s and c e l l l i n e s revealed that the expression of c-fes was confined to those of hemopoietic o r i g i n and e s p e c i a l l y to those c e l l s belonging to myeloid lineages. The expression of c-fes was investigated in human myeloid leukemia c e l l l i n e s during exposure to a chemical inducer of d i f f e r e n t i a t i o n ; complex patterns of expression were observed and these are discussed. The expression of c-fes appears to be d i f f e r e n t i a l l y regulated in responsive and unresponsive c e l l s and i t i s possible that i t plays a role in committment to myeloid lineages. iv TABLE OF CONTENTS Abstract i i L i s t of Tables v i i i L i s t of Figures ix Acknowledgments xi CHAPTER 1 1.0 Introduction 1 1.1 Proto-oncogenes 1 1.2 Isolation and C l a s s i f i c a t i o n of Proto-oncogenes 2 1.3 Functions of Proto-oncogene Products 4 (i. ) Growth factors 6 ( i i . ) Growth factor receptors 8 ( i i i . ) Members of i n t r a c e l l u l a r signal transducing pathways 12 (iv.) Modulators of t r a n s c r i p t i o n a l a c t i v i t y 15 1.4 Mechanisms of Proto-oncogene Activation in Neoplasia 16 (i . ) Insertional mutagenesis by viruses 18 ( i i . ) Chromosomal translocation 19 ( i i i . ) Gene amplification 21 (iv.) Point mutation 22 (v.) Deletion 23 (vi.) Autocrine stimulation 24 1.5 Protein-tyrosine Kinases 26 V 1.6 The £ps/fes Oncogenes 34 1.7 Hemopoiesis 37 1.8 Myelogenous Leukemias 44 1.9 Purpose and Experimental Approach 49 CHAPTER 2 2.0 Materials and Methods 51 2.1 C e l l s , C e l l Lines, and Tissues 51 2.2 Preparation of Immune Reagents 54 2.3 Radiolabelling of C e l l s 55 2.4 Immunoprecipitation 56 2.5 Immune Complex Kinase Reaction 57 2.6 SDS-Polyacrylamide Gel Electrophoresis 59 2.7 Phosphoamino Acid Analysis 61 2.8 Tryptic Peptide Analysis 63 2.9 P a r t i a l P roteolytic Cleavage with V8 Protease 64 2.10 Isolation and Analysis of Total C e l l u l a r RNA 65 2.11 Morphologic Markers of C e l l u l a r D i f f e r e n t i a t i o n 69 2.12 Preparation of Radiolabeled Nucleic Acid Probes 70 CHAPTER 3 3.0 I d e n t i f i c a t i o n and Characterization of Murine and Human c-fes Proteins 73 3.1 Introduction 73 vl 3.2 Results 74 3.2.1 Characterization of Rat anti-fps Sera 74 3.2.2 Examination of Human and Murine Hemopoietic Cel l s for Expression of c-fes Proteins 78 3.2.3 Examination of Human and Murine C e l l Lines for Expression of c-fes Proteins 83 3.2.4 Characterization of Human p92 and p94 87 3.3 Discussion 107 CHAPTER 4 4.0 Expression of c-fes in Human Leukemia C e l l Lines During Chemically Induced D i f f e r e n t i a t i o n 119 4.1 Introduction 119 4.2 Results 121 4.2.1 Examination of HL-525 Cell s for Expression of c-fes 121 4.2.2 Examination of c-fes Expression During Exposure of HL-60 and HL-525 Ce l l s to the Inducer TPA 123 (i. ) Morphology 124 ( i i . ) c-fes transcripts 131 ( i i i . ) Synthesis of p92<=-e"E* 134 (iv.) p92~~Cm"* in v i t r o kinase a c t i v i t y 140 4.3 Discussion 144 CHAPTER 5 5.0 Summary References vii 154 156 v i i i LIST OF TABLES Table 1.1 C l a s s i f i c a t i o n of Proto-oncogenes Page, 5 Table 1.2 Functional C l a s s i f i c a t i o n of Protein-tyrosine Kinases 28 Table 3.1 Expression of p92 and p94 in v i t r o Kinase A c t i v i t y in Human and Mouse C e l l Lines, C e l l s , and Normal Mouse Tissues 85 Table 4.1 Cytochemical Staining Characteristics of HL-60 and HL-525 Ce l l s Before and After Exposure to TPA. 130 ix LIST OF FIGURES Figure 1.1 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 3.11 Figure 3.12 Figure 3.13 Figure 3.14 Page The human hemopoietic system. 39 Id e n t i f i c a t i o n of v-fes and v-fps proteins by rat anti-fps serum. 76 Id e n t i f i c a t i o n of c-fps and v-fes proteins by rat anti-fps serum. 79 Id e n t i f i c a t i o n of p92 and p94 in human and murine hemopoietic c e l l s . 81 Id e n t i f i c a t i o n of p92 in a murine multipotential hemopoietic progenitor c e l l l i n e . 82 I d e n t i f i c a t i o n of p92 and p94 in human and murine hemopoietic c e l l l i n e s . 86 Phosphoamino acid analysis of p92, p94, and enolase. 89 Phosphorylation of enolase in v i t r o by p92, p94, and NCP98. 91 Tryptic phosphopeptide analysis of enolase phosphorylated in v i t r o by p92, p94, and NCP98. 92 Tryptic phosphopeptide analysis of HL-60 p92 and ST-FeSV P85<3-"3-£-a'. 94 Comparative t r y p t i c phosphopeptide analysis of p92 and p94. 96 Phosphoamino acid analysis of t r y p t i c phosphopeptides from p92 and p94, and of in vivo phosphorylated p92. 98 V8 protease digestion of p92, p94, and pQ5w-f. 100 Synthesis of p92 in HL-60 c e l l s . 102 Metabolic l a b e l l i n g of KG-1 and K562 c e l l s . 103 Figure 3.15 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Ide n t i f i c a t i o n of p92, p94, and P210 = — " in lysates of human hemopoietic c e l l s . I d e n t i f i c a t i o n of p32e=-ema HL-525 c e l l s . in Morphologic c h a r a c t e r i s t i c s of HL-60 and HL-525 c e l l s in suspension culture following exposure to TPA. Morphologic d i f f e r e n t i a t i o n of HL-60 and HL-525 c e l l s . Detection of c-fes and acti n mRNA in uninduced and TPA-induced HL-60 c e l l s . Detection of c-fes and ac t i n mRNA in HL-525 c e l l s and HL-525 c e l l s exposed to TPA. Quantitation of c-fes mRNA in HL-60 c e l l s and HL-525 c e l l s exposed to TPA. Synthesis of p92=~ £°* 3 in uninduced and TPA-induced HL-60 c e l l s . Synthesis of p92=~ c" D in uninduced and TPA-induced HL-60 c e l l s . p92=-*»« in v i t r o kinase a c t i v i t y in uninduced and TPA-induced HL-60 c e l l s . p92c-fes in v i t r o kinase a c t i v i t y in HL-525 c e l l s and HL-525 c e l l s exposed to TPA. 106 122 125 128 132 135 136 138 139 141 143 ACKNOWLEDGEMENTS I would l i k e to thank my present supervisory committee, Drs. Hung-Sia Teh, Rob McMaster, and Gerry Weeks, and e s p e c i a l l y my supervisor Dr. J u l i a Levy for allowing me to work with her. My pa r t i c u l a r appreciation is reserved for Dr. Tony Pawson who, while no longer at the University of B r i t i s h Columbia, supervised me c l o s e l y during his tenure here. Also, I am grateful to Drs. Hugh Brock and Rob McMaster for allowing me to work in their laboratories and for assistance in a research area with which they were largely unfamiliar. This work was assisted by multiple individuals in many tangible ways and I am p a r t i c u l a r l y appreciative of the assistance and suggestions given to me by Dr. Gerry Weinmaster, Dr. Linda Button, Dr. Sheldon Naiman, Dr. Tom Frommel, Dr. Peter Greer, Anthea Stammers, Tosca Mah, Ehleen Hinze, S a l l y Freeman, Tracy Kion, and Frances Lee. Financial support from the Medical Research Council of Canada is g r a t e f u l l y acknowledged. This thesis is dedicated to Diane, to Gwynneth, and to Keith; may i t serve as a symbol of my love, of my dedication, of my commitment. 1 CHAPTER 1 1 . 0 INTRODUCTION 1 . 1 p^to-oncogenes. Oncogenes are so named because they were o r i g i n a l l y discovered to be the genetic sequences carried by some oncogenic retroviruses which conferred upon those viruses the property of acute tumorigenicity. These genetic sequences have, in most cases, replaced certain r e t r o v i r a l structural or replicative genes, and thus, in addition to rendering these viruses oncogenic, concomitantly render them replication-deficient; these unique genes are called v i r a l oncogenes(v-onc's). Subsequently i t was discovered that some non-viral tumors contain homologous DNA sequences which can transform NIH 3 T 3 c e l l s ; these transforming genes are called c e l l u l a r oncogenes, or activated oncogenes, in order to distinguish them from v-onc's. Examination of the various species from which the oncogenic retroviruses were original ly isolated reveals that genes homologous with the v-onc's are present in the normal genome. These normal sequences are called proto-oncogenes (c-onc_'s) and i t is widely believed that acutely oncogenic retroviruses transduced c-onc's in the course of v i r a l repl icat ion, giving rise to v-onc's. In support of recombination events giving rise to v-onc's is the 2 observation that short stretches of i d e n t i c a l nucleotides are found in both v i r a l s t r u c t u r a l genes and in corresponding c-onc 1s at the borders of putative recombination junctions (Coussens, et a l . r 1986). Also, integration of certain non-one retroviruses next to c-myc or c-erbB sometimes results in the emergence of novel, r a p i d l y transforming retroviruses which have transduced the adjacent c-onc into the v i r a l genome (Miles, et a l . r 1985; Nusse, 1986). Presumably, c e l l u l a r genes are transduced randomly from the host genome and the r e a d i l y detectable neoplastic phenotype conferred by the presence of a v-onc results in their i s o l a t i o n ; the finding that d i f f e r e n t strains of oncogenic retroviruses, isolated independently from v i r a l l y -induced tumors, carry the same v-onc is consistent with the random transduction of c-onc's. However, more than mere transduction of a c-onc i s required to activate i t s transforming potential and a l l v-onc's show mutations r e l a t i v e to the o r i g i n a l c-onc. Activated oncogenes isolated from non-viral tumors also inevitably carry s t r u c t u r a l a l t e r a t i o n s , point mutations, or are expressed inappropriately r e l a t i v e to the corresponding c-onc; thus, most normal c-onc 1s are unable, in the absence of mutation or aberrent express ion,to transform normal c e l l s or induce tumors (Land, et a l . , 1983; Foster, et a l . f 1985; Bishop, 1987). 1.2 Isolation and C l a s s i f i c a t i o n of Proto-oncogenes 3 Proto-oncogenes have been isolated by one of four methods (Varmus, 1984): 1.) Probing c e l l u l a r DNA with v-onc probes in order to i d e n t i f y related c e l l u l a r sequences. The majority of c-onc's have been i d e n t i f i e d on the basis of being the normal c e l l u l a r counterparts of the r e t r o v i r a l transforming genes. 2.) Probing DNA from v i r a l tumors with unique r e t r o v i r a l sequences, such as long terminal repeats(LTR 1s), in order to i d e n t i f y genes which have been activated by v i r a l integration. The v i r a l LTR 1s serve as markers and allow i d e n t i f i c a t i o n of adjacent v i r a l l y -activated c-onc's; a number of genes which have no known equivalent v-onc have been isolated using v i r a l sequences as molecular markers. 3.) Transfection of a suitable c e l l line with tumor DNA causes transformation i f an activated, dominantly-acting c-onc is present. This technique has proved useful in i s o l a t i n g oncogenes from a variety of spontaneous tumors as well as chemical or radiation-induced neoplasms. 4.) Chromosomal translocations are seen frequently in neoplastic c e l l s and s p e c i f i c translocations which disrupt c-onc expression exist in a number of tumors; cloning of sequences adjacent to chromosomal translocation breakpoints has allowed the i s o l a t i o n of several novel oncogenes. Proto-oncogenes may be c l a s s i f i e d according to structure, c e l l u l a r location, or a c t i v i t y . Since precise functions have not been ascribed to many c-onc's their c l a s s i f i c a t i o n has been made on the basis of c e l l u l a r 4 location or st r u c t u r a l s i m i l a r i t y to more extensively characterized c-onc's. The majority of currently i d e n t i f i e d c-onc's are categorized in Table 1.1 according to the above c r i t e r i a . 1.3 Functions of Proto-oncogene Products The exact functions of the majority of c-onc gene products are not well understood but their importance to normal c e l l u l a r function is underscored by the extreme evolutionary conservation which i s observed for many of these genes. For example, ras genes have been i d e n t i f i e d in the c e l l s of yeasts (DeFeo-Jones, et a l . , 1983), slime moulds (Reymond, et a l . f 1984), insects (Neuman-Silberberg, et a l . , 1984), and mammals (DeFeo, et a l . , 1981), and the sequence of the highly conserved amino terminal domain is at least 84% homologous between any two species (Barbacid, 1987). Functional i n t e g r i t y i s conserved also: mammalian ras genes are able to complement yeast r a s 1 ~ r a s a ~ mutants while normal yeast ras genes, i f mutated, are able to transform NIH 3T3 c e l l s (Kataoka, et a l . P 1985; DeFeo-Jones, et a l . , 1985). The observation that a single v i r a l oncogene, oblivious to normal c e l l u l a r regulatory constraints, is able to induce the rapid, uncontrolled p r o l i f e r a t i o n of c e l l s c h a r a c t e r i s t i c of malignant neoplasms leads to speculation that the corresponding c-onc's play pivota l roles in the processes of c e l l d i v i s i o n and d i f f e r e n t i a t i o n . This TABLE 1.1 C l a s s i f i c a t i o n of Proto-oncogenes Proto-oncogene A c t i v i t y or location of gene product C - S K C prote in-tyrosine kinase c-abl protein-tyrosine kinase c-fps/fes protein-tyrosine kinase c-erbB(EGF receptor) protein-tyros ine kinase c-fms(CSF-l receptor) protein-tyrosine k inase c-ros protein-tyrosine kinase c-f gr prote in-tyrosine kinase c - y e s protein-tyrosine kinase neu protein-tyrosine kinase trK protein-tyrosine k inase lcK. protein-tyrosine kinase k i t protein-tyrosine kinase met protein-tyrosine kinase ret prote in-tyros ine kinase sea prote in-tyros ine kinase c-mos serine/threonine k inase c-mi1/raf serine/threonine k inase pim-i serine/threonine kinase c-Ha-ras GTP-binding protein c-Ki-ras GTP-binding protein N-ras GTP-binding protein c-sls(PDGF B-chain) growth factor i n t - 2 potential growth factor hst potential growth factor c-erbA(thyroid hormone receptor) t r a n s c r i p t i o n a l activator c-1un(p39) t r a n s c r i p t i o n a l activator c-myc DNA binding protein c-myb DNA binding protein c-fos DNA binding protein c - r e l nuclear protein c-ski nuclear protein l n t - l controls segmentation(in Drosophila melanogaster) 6 hypothesis i s advanced by the finding that activated c e l l u l a r oncogenes can also induce transformation and tumorigenicity (Varmus, 1984). What, then, are the normal functions of c-onc's when expressed at appropriate le v e l s , at appropriate times, in appropriate c e l l s ? A number of a c t i v i t i e s have been described for various c-onc gene products, and while precise mechanisms of action in the normal c e l l are not well understood i t is clear that perturbation of these a c t i v i t i e s may contribute to the neoplastic phenotype. The normal functions of c-onc proteins may be categorized as follows: i.) Growth factors; i i . ) Growth factor receptors; i i i . ) Members of i n t r a c e l l u l a r signal transducing pathways; iv.) Modulators of t r a n s c r i p t i o n a l a c t i v i t y . i.) Growth factors While a number of c-onc's encode proteins which function as growth factor receptors only one c-onc has been shown unambiguously to encode a known growth factor. Amino acid sequence analysis of p l a t e l e t derived growth factor(PDGF) revealed homology with the predicted v-sis protein, with the exception of three conservative substitutions (Waterfield, et a l . , 1983). Subsequently i t was shown that c e l l s transformed by v-sis synthesize and secrete a molecule which i s functionally and immunologically indistinguishable from PDGF (Owen, et al.,1984; Johnsson, et a l . , 1985); nucleotide sequence analysis of the human c-sls 7 gene confirmed that t h i s locus encodes the PDGF B-chain (Chiu, et a l . , 1984). PDGF i s a heterodimeric molecule consisting of an A-chain and a B-chain linked by disulphide bonds, and the A-chain, which i s approximately 60% i d e n t i c a l to the B-chain, i s encoded at a locus d i s t i n c t from c - s i s ; the A-chain, unlike the B-chain, is markedly less e f f i c i e n t in transforming f ibroblasts (Betsholtz, et a l . , 1986; Tong e_t a l . , 1987; Beckmann, et a l . , 1988). While individual PDGF A or B chains are functionally inactive, A-A and B-B homodimers are synthesized and secreted by a variety of tumor c e l l l i n e s and have PDGF agonist a c t i v i t y , r a i s i n g the p o s s i b i l t y of an autocrine mechanism in these c e l l s (Heldin, et a l . , 1986; De Larco, et a l . , 1978). A connection exists between the potential protein product of the gene int-2 and a c i d i c (aFGF) and basic (bFGF) fi b r o b l a s t growth factors. The FGF's are members of a family of heparin-binding protein mitogens, while int-2 i s a potential oncogene implicated in the pathogenesis of murine mammary carcinoma by virtue of the frequent p r o v i r a l integration of mouse mammary tumor virus adjacent to the int-2 locus (Peters, et a l . , 1984). Comparison of the predicted primary sequence of int-2 with that of aFGF and bFGF reveals homology with both of these growth factors, suggesting the int-2 product may also function as a growth factor (Dickson, et a l . , 1987). While the int-2 gene product awaits characterization, a novel oncogene isolated from a Kaposi's sarcoma, and possessing sequence homology 8 with i n t - 2 r aFGF, and bFGF, has been shown to encode a protein which is secreted and can stimulate mitogenic a c t i v i t y ( D e l l i Bovi, et a l . f 1987a; D e l l i Bovi, et a l . , 1987b). Thus, i t appears that the Kaposi's sarcoma oncogene, int-2, hst, and the FGF's are members of a gene family which encode a number of secreted growth factors; the normal c e l l u l a r targets of these factors, the existence of single or multiple receptors, and the mechanisms of action of these receptors a l l remain to be elucidated. i i . ) Growth factor receptors Those c-onc's which function as growth factor or hormone receptors comprise the largest functional group of proto-oncogenes at the present time. The sequences and ligand-binding s p e c i f i c i t i e s have been determined for c-erbA, c-erbB, and c-fms. while a number of other genes including neu, c-ros, trk, and c - k i t are believed to encode receptor molecules by virtue of sequence s i m i l a r i t y with known receptors. The c-erbA gene is the normal c e l l u l a r counterpart of the transforming gene, v-erJbA, of avian erythroblastosis virus (AEV), a virus known to cause erythroleukemias in chickens. The finding that regions of v-erbA show homology to the DNA binding and hormone binding domains of steroid hormone receptors suggested that c-erbA, the progenitor of v-erbA, might also specify a receptor protein (Weinberger, et al.,1985; Krust et a l . , 1986). The chicken and human 9 c-erbA genes were sequenced, revealing s t r u c t u r a l s i m i l a r i t y to human glucocorticoid and estrogen receptors, and ligand binding studies with p u r i f i e d hormones showed that c-erbA protein s p e c i f i c a l l y binds thyroid hormones, an e f f e c t which is abrogated by anti-erbA antibodies; steroid hormones which bind related receptors show no a f f i n i t y for c-erbA protein (Sap, et a l . , 1986; Weinberger,et__al., 1986). The homologous v i r a l protein, p7 5'a"'3_"to*-, does not bind thyroid hormones and shows a cytoplasmic d i s t r i b u t i o n in contrast to the exclusively nuclear location of c-erbA protein; these changes in v-erbA ligand binding and subcellular d i s t r i b u t i o n may r e f l e c t disordered regulation of thyroid hormone-sensitive genes (Sap, et a l . , 1986; Boucher, et a l . f 1988). The r e t i n o i c acid receptor gene possesses domain and sequence s i m i l a r i t y with c-erbA, and recently a novel r e t i n o i c acid receptor gene has been i d e n t i f i e d at the hepat i t i s B virus integration s i t e in a hepatocellular carcinoma (Dejean, et a l . , 1986; Petkovich, et al.,1987; Giguere, et a l . , 1987; Benbrook, et a l . , 1988). Thus, c-erbA i s a member of a large gene family which encodes a number of nuclear hormone receptors displaying sequence homology in the highly conserved DNA binding and ligand binding domains. While c-erbA is a nuclear hormone receptor and has been shown to bind DNA the majority of proposed receptors encoded by proto-oncogenes function at the cytoplasmic membrane and are protein-tyrosine kinases; the 10 two best characterized c-onc's encoding receptors are c-fms and c-erbB. The human c-fms gene encodes a 150 kD glycoprotein, gpl50 , = - i : m s , which is located at the c e l l surface and possesses protein-tyrosine kinase a c t i v i t y (Woolford, §_t a l . , 1985; Rettenmier, et a l . f 1986). Examination of human and f e l i n e tissues for expression of c-fms protein reveals that expression is maximal in spleen; curiously, human placenta and choriocarcinoma c e l l l i n e s express high levels of c-fms mRNA and protein (Muller, et a l . , 1983; Sherr,et a l . , 1985; Rettenmier, et a l . , 1986). Fractionation of splenocytes indicates that c-fms expression is limited to a frac t i o n composed of macrophages, granulocytes, and blast c e l l s , and flow cytometry reveals that c e l l s possessing c-fms epitopes are almost e n t i r e l y macrophages (Sherr, §_t a l . , 1985). This r e l a t i v e l y r e s t r i c t e d expression of c-fms protein and i t s relatedness to other receptor proteins suggested a possible hemopoietic growth factor-receptor function for c-fms, and a number of observations support the assignment of c-fms as the gene encoding the receptor for the macrophage growth factor colony stimulating £actor-l (CSF-1): t y r o s i n e - s p e c i f i c phosphorylation of c-fms protein is increased in the presence of CSF-1, anti-fms antibodies pre c i p i t a t e the CSF-1 receptor, and cloned c-fms confers upon f i b r o b l a s t s the a b i l i t y to bind CSF-1 (Sherr, et a l . , 1985; Roussel, et a l . . 1987). 11 The r e t r o v i r a l gene v-erbB f l i k e v-erbA f is also carried by AEV but these two v-onc's are not s t r u c t u r a l l y related. The primary sequence of t r y p t i c peptides obtained from p u r i f i e d human EGF receptor was i d e n t i c a l to regions of the deduced v-erbB protein, and sequencing of human EGF receptor cDNA revealed 95% id e n t i t y with v-erbB in a region encompassing the highly conserved tyrosine kinase domain (Downward, et a l . r 1984a; U l l r i c h , et a l . r 1984). Thus, avian c-erbB, the progenitor of v-erbB, was almost c e r t a i n l y the avian EGF receptor; d i r e c t evidence of thi s i d e n t i t y was established following sequencing of the avian EGF receptor(Lax, et a l . , 1988). The human EGF receptor is a mol wt 175kD glycoprotein which has the a b i l i t y to autophosphorylate and to phosphorylate exogenous substrates, and while t h i s kinase reaction occurs in the absence of ligand, binding of EGF by the receptor results in enhanced substrate phosphorylation both in vivo and in v i t r o (Erneux, et a l . , 1983; Pike, et a l . , 1984; Downward, et a l . , 1984b; Downward, et a l . , 1985). Like the CSF-1 receptor the EGF receptor consists of an e x t r a c e l l u l a r ligand binding domain, a hydrophobic membrane spanning segment, a conserved intracytoplasmic tyrosine kinase domain, and a regulatory domain at the carboxyl terminus; t h i s t y p i c a l domain structure i s also seen in the receptors for PDGF, i n s u l i n , IGF-1, as well as in the potential receptors neu, c - k i t , and c-ros (Martin, 1986; Carpenter, 1987).While the ligand binding s p e c i f i c i t i e s have been determined for the products 12 of c-fms and c-erbB f the i d e n t i t y and function of c r i t i c a l in vivo substrates has remained elusive, and the p o s s i b i l i t y exists that mechanisms other than protein phosphorylation may also be operative in mediating receptor function; u n t i l other members of the EGF and CSF-1 signal-transducing pathways are i d e n t i f i e d the manner in which the s p e c i f i c receptor transmits the mitogenic signal remains speculative. i i i . ) Members of i n t r a c e l l u l a r signal transducing  pathways A number of c-onc products exist which are thought to function as i n t r a c e l l u l a r transducers of the growth and d i f f e r e n t i a t i o n signals i n i t i a t e d when a va r i e t y of ligands interact with their receptors. These proposed i n t r a c e l l u l a r messenger proteins u t i l i z e diverse mechanisms in order to relay t h i s information from the c e l l surface and yet l i t t l e i s known of either the afferent or efferent effectors which interact with these proteins. The ras family of highly conserved genes consists of c-Ha-ras, c-Ki-ras, and N-ras which encode related proteins of 21kD that are l o c a l i z e d on the inner surface of the plasma membrane (Taparowsky, et a l . , 1983; Fujiyama, et a l . f 1986). The ras proteins bind guanosine diphosphate (GDP) and guanosine triphosphate (GTP) and possess a GTPase a c t i v i t y which hydrolyzes GTP to GDP (Scolnick, et a l . , 1979; Tamanoi, et a l . r 1984; McGrath, et a l . , 1984). The biochemical properties of the ras proteins are very similar 13 to those o£ the G proteins, involved in transmembrane signal transduction, and ras proteins show sequence homology with G proteins; these s i m i l a r i t i e s suggest that ras gene products may also be members of signal transducing pathways (Gilman, 1984). The currently favoured model of ras function proposes that ras proteins, following stimulation by an upstream effector molecule, rapi d l y exchange GDP for GTP, inducing a conformational change in the ras protein; t h i s modified ras protein is then able to interact with downstream molecules r e s u l t i n g in transmission of the relevent signal (Barbacid, 1987). In this scheme ras effector function is abrogated by i t s i n t r i n s i c GTPase action which hydrolyzes GTP to GDP with attendant conversion to the inactive conformation; the observation that oncogenic ras proteins are d e f i c i e n t in GTPase a c t i v i t y i s consistent with t h i s model in that mutant proteins,unable to hydrolyze GTP, would remain in a c o n s t i t u t i v e l y activated state (McGrath, et a l . , 1984; Sweet, et a l . , 1984). The i d e n t i t i e s of the hypothesized upstream and downstream effector molecules remain c r y p t i c , although some evidence suggests possible interaction with the phosphatidylinositol pathway (Fleischman, et a l . , 1986; Wakelam, et a l . , 1986). The genes c-raf/mil and c-mos encode soluble cytoplasmic proteins which, while displaying homology with protein-tyrosine kinases, function as serine/threonine protein kinases; the raf/mil and mos proteins are able to autophosphorylate and phosphorylate exogenous substrates at 14 serine and threonine residues (Papkoff, et a l . , 1983; Hunter, et a l . , 1986). The normal roles of c-raf/mil and c-mos are unknown but by analogy with protein kinase C, a serine/threonine kinase, they may function as i n t r a c e l l u l a r signal transducers. The protein-tyrosine kinase pp60=~~_c: i s the product of the c-src gene and is the prototypical i n t r a c e l l u l a r protein believed to modify function through tyrosine phosphorylation of target substrates (Takeya, et a l . , 1983). This 60kD enzyme i s s t r u c t u r a l l y related to cAMP-dependent protein kinase and consists of a non-catalytic domain occupying the amino half of the molecule and a c a t a l y t i c domain located in the carboxyl half; a l l v-onc, c-onc, and growth factor receptor tyrosine kinases possess domains which are homologous with the kinase domain of c-src (Hunter, et a l . , 1985) . There are 16 amino acid residues which are invariant among a l l protein-tyrosine kinases and these are centred around the ATP-binding s i t e near the N-terminus of the kinase domain and around the c a t a l y t i c s i t e near the C-terminus of the kinase domain; there i s l i t t l e , i f any, sequence conservation in the non-catalytic amino terminal domains and these may be important in specifying the unique c h a r a c t e r i s t i c s of each individual kinase (Hunter, et a l . , 1986) . pp60 < = -" r' c i s not an integral membrane protein, lacking both signal peptide and membrane insertion sequences, but is located on the inner surface of the plasma membrane and attached to i n t r a c e l l u l a r membranes 15 (Courtneidge, et a l . , 1980); pp60°-ax'= has the a b i l i t y to autophosphorylate and to phosphorylate exogenous substrates at tyrosine (Hunter, et a l . , 1980). The i n t r a c e l l u l a r location of pp60 c - < s i : < = and i t s prote in-tyros ine kinase a c t i v i t y are consistent with a role as a member of a s i g n a l -transducing pathway, perhaps coupling a transmembrane receptor to another constituent of the same pathway; however, t h i s conjecture must be balanced with the observation that pp60 o - a , , r , = expression is highest in neurons and p l a t e l e t s , both post-mitotic c e l l s and not known to respond to growth factors (Cotton, et a l . , 1983; Golden, e_t a l . , 1986). While the foregoing observations apply to s p e c i f i c a l l y to pp60t=-"a,1!rc;, most also apply to the large family of i n t r a c e l l u l a r protein-tyrosine kinases, such as c-fgr, c-yes, c-abl, c-fps/fes, which display s i g n i f i c a n t s t r u c t u r a l and functional homology with c-src and do not encode any known receptor function (Hunter, et a l . , 1986). iv.) Modulators of t r a n s c r i p t i o n a l a c t i v i t y A group of c-onc's including c-myc, c-myb, c-fos, and c-1un encode proteins which^ are located in the nucleus of the c e l l and which bind DNA in. v i t r o (Klempnauer, et a l . , 1984; Cole, 1986; Verma, et a l . , 1987; Varmus, 1987). A role for these proteins in t r a n s c r i p t i o n a l control has long been hypothesized and evidence exists that both c-fos and c-myc can participate in t r a n s c r i p t i o n a l regulation, but the most compelling support for this notion has come with 16 characterization of the newest member of t h i s group, c-iun (Maki, et a l . r 1987; Bohmann, et a l . r 1987). The deduced primary sequence of c-iun s p e c i f i e s a protein of MK 37,000 whose carboxyl-terminal region is homologous with the DNA-binding domain of the yeast t r a n s c r i p t i o n factor GCN4, and t h i s region of iun can f u n c t i o n a l l y replace homologous GCN4 sequences in a hybrid molecule (Vogt, et a l . , 1987; Struhl, 1987; Angel, et a l . f 1988). Antisera raised against synthetic iun peptides precipitate the mammalian tr a n s c r i p t i o n factor AP-1, and both AP-1 and jun proteins protect the same s i t e s in DNAase I protection assays, leading to speculation that c-1un encodes AP-1 or a very similar factor (Bohmann, et a l . f 1987). However, recent t r y p t i c peptide mapping data show that p39, a protein found complexed with p 5 5 a - e o ° in c e l l s , is i d e n t i c a l with the product of c-jun; thus, c-jun is a member of a family of e v o l u t i o n a r i l y conserved genes encoding t r a n s c r i p t i o n a l regulatory proteins, and the known association of p39' = _ : , u" with c-fos proteins in nuclei provokes speculation that several c-onc products may act s y n e r g i s t i c a l l y in cascades or complexes which modulate t r a n s c r i p t i o n a l a c t i v i t y (Curran, et a l . , 1982; Rauscher, et a l . , 1988 ). 1.4 Mechanisms of Proto-oncogene Activation in Neoplasia While individual v-onc's are capable independently of inducing tumors i t is often unclear to what extent the etiology of non-viral tumors can be ascribed to activated 17 c-onc's, and no single type of non-viral tumor, with the exception o£ a few carcinogen induced cancers, has been shown to possess a consistently reproducible c-onc mutation (Balmain, et a l . , 1983; Wiseman, et a l . . 1986; Bishop, 1987). However, the frequent detection of activated or inappropriately expressed c-onc's in a variety of tumors suggests that they may subserve e t i o l o g i c roles which correspond to discrete steps in the development of malignant neoplasms. The mechanisms of c-onc ac t i v a t i o n are manifold but a useful generalization has been formulated based upon the subcellular locations of the c-onc gene products: nuclear proteins are activated by t r a n s c r i p t i o n a l deregulation while cytoplasmic proteins are activated by altered structure or function (Weinberg, 1985). This generalization may be extended further in observing that nuclear and cytoplasmic c-onc proteins, when activated, have d i f f e r e n t , yet complementary, effects which contribute to carcinogenesis: nuclear proteins generally res u l t in immortalization of primary c e l l s such that they w i l l r e p l i c a t e i n d e f i n i t e l y in vitro., while activated cytoplasmic proteins r e s u l t in the appearance of a transformed phenotype (Ruley,1983; Land, et. a l . , 1983; Mougneau, et a l . , 1984; Alema, et a l . , 1985). In most cases the expression of a pair of c-onc's, one cytoplasmic and one nuclear, is required for the e f f i c i e n t transformation of primary c e l l s ; the obvious exceptions are 18 the r e t r o v i r a l v-onc's which carry f u l l transforming potential in a single gene. The ac t i v a t i n g mechanisms which res u l t in loss of t r a n s c r i p t i o n a l control or in synthesis of altered gene products may be c l a s s i f i e d as follows: i.) Insertional mutagenesis by viruses; i i . ) Chromosomal translocation; i i i . ) Gene amplification; iv.) Point mutation; v.) Deletion; vi.) Autocrine stimulation. i.) Insertional mutagenesis by viruses Any virus which integrates randomly within host c e l l DNA to form a provirus has the potential to disrupt normal regulation of c-onc t r a n s c r i p t i o n i f i t integrates within c-onc regulatory sequences; th i s mode of c-onc a c t i v a t i o n is known as i n s e r t i o n a l mutagenesis and is observed when a non-transforming retrovirus integrates in proximity to a c-onc. The provirus can a l t e r t r a n s c r i p t i o n by integrating into upstream regulatory sequences, thus bringing the c-onc under control of a v i r a l promoter, or v i r a l Integration may occur either upstream or downstream of the gene and increase t r a n s c r i p t i o n by virtue of the enhancers carried in the v i r a l LTR's; either of these mechanisms may res u l t in the enhanced t r a n s c r i p t i o n of c-myc seen in avian leukosis virus(ALV)-induced B - c e l l lymphomas (Hayward, et a l . , 1981; Payne, et a l . , 1982). DNA viruses may also be able to induce similar aberrations and the integration of human papillomavirus in some c e r v i c a l carcinoma c e l l lines has 19 been shown to occur adjacent to c-myc and is associated with increased steady-state levels of c-myc mRNA (Durst, et a l . , 1987). Another form of inse r t i o n a l mutagenesis by ALV in murine erythroleukemia involves insertion into and disruption of the coding region of c-erbB such that a truncated EGF-receptor lacking an external domain i s produced; i n t e r e s t i n g l y , in t h i s case, integration of ALV results in the production of a mutated cytoplasmic protein while ALV integration into c-myc results in elevated levels of normal myc protein (Nilsen, et a l . , 1985). Thus, ALV integration serves as a paradigm i l l u s t r a t i n g some of the unique features distinguishing a c t i v a t i o n of cytoplasmic and nuclear c-onc proteins. i i . ) Chromosomal translocation Karyotypic abnormalities are a frequent finding in cancer c e l l s and chromosomal translocations are consistently associated with certain human tumors; c e l l s of Burkitt's lymphoma, a B - c e l l neoplasm, demonstrate translocations involving chromosome 8, close to the c-myc locus, and chromosomes carrying the immunoglobulin heavy or l i g h t chain l o c i ; most exchanges occur with the IgH locus on chromosome 14 (Leder, et a l . , 1983). As a consequence of translocation the c-myc locus loses normal upstream controls or comes under the influence of powerful immunoglobulin enhancers, or both, and pe r s i s t e n t l y elevated levels of c-myc mRNA, re l a t i v e to quiescent c e l l s , are detected; i t is not 20 resolved to what extent elevated levels of c-myc mRNA versus loss of negative t r a n s c r i p t i o n a l control constitute a c t i v a t i o n of c-myc (Taub, et a l . , 1984; Keath, et a l . , 1984). Recently a translocation involving c-myc in a plasma c e l l myeloma was described which resulted in production of a chimeric c-myc tr a n s c r i p t in which translocated c e l l u l a r sequences replaced the downstream regulatory region of c-myc; th i s juxtaposition resulted in greatly enhanced c-myc trans c r i p t s t a b i l i t y , perhaps achieving the same net ef f e c t as increasing t r a n s c r i p t i o n a l a c t i v i t y ( H o l l i s , et a l . , 1988) . The c-abl gene encodes a cytoplasmic protein-tyrosine kinase and i t s structure is altered in chronic myelogenous leukemia (CML) by the translocation which produces the Philadelphia chromosome (Phi), found in 90-95% of CML patients. The Phi i s generated by a reciprocal translocation between chromosomes 9 and 22 and results in fusion of c-abl sequences on chromosome 9 with those of a gene known as bcr on chromosome 22; tra n s c r i p t i o n i s i n i t i a t e d within bcr and spli c e s into c-abl exon 2, producing a chimeric mRNA (Shtivelman, et a l . , 1985). This hybrid mRNA is translated into a novel protein, P210 b"—* t' : L, in CML c e l l s and P210t"=:c~*!'bX possesses greatly enhanced tyrosine kinase a c t i v i t y both in vivo and in v i t r o r e l a t i v e to the normal c-abl product, pl45°- i a l 3 : L (Konopka, et a l . , 1984). While a c t i v a t i o n of kinase a c t i v i t y is apparent in P210 t o o > i r~ o t o : L the contribution i t makes to leukemogenesis is 21 less! clear and the hybrid bcr/abl protein, even when highly expressed, does not transform f i b r o b l a s t s (Daley, et a l . , 1987). A subset of acute lymphocytic leukemia(ALL) patients also demonstrate a Phi, but in these patients a smaller transcript d i s t i n c t from that found in CML is produced and encodes a unique protein,Pies*- 1 , 1 1'-'* 1 3 1 (Clark, et a l . , 1988). Hence, chromosomal translocation can activate c-onc's by a l t e r i n g protein function, as i l l u s t r a t e d here by two disparate hematologic malignancies, or a c t i v a t i o n can occur by a l t e r i n g steady-state t r a n s c r i p t l e v e l s , as seen in the case of a number of c-onc's encoding nuclear proteins such as c-myc. i i i . ) Gene amplification Gene amplification is not normally observed in human c e l l s but is seen frequently in cancerous c e l l s and is often heralded by the occurrence of two karyotypic abnormalities, double minute chromosomes and homogeneously staining regions, which disrupt the banding patterns of normal chromosomes. Amplification of s p e c i f i c c-onc's occurs in a wide variety of tumors and may involve genes coding for either cytoplasmic or nuclear proteins, although amplification is more common in genes encoding nuclear proteins; conceptually, i t i s r e l a t i v e l y easy to understand how elevated levels of c-onc product due to gene amplification could a s s i s t in the development or maintenance of the neoplastic state. Amplifications involving c-myc, 22 c-mybr c-erbA, neu, c-erbB, c-Ha-ras, and N-myc have been observed in diverse tumors, including many of hemopoietic o r i g i n (Lee, et a l . , 1984; P e l i c c i , et a l . , 1984; Yukota e_t_ a l . , 1986; van de Vijver, et a l . , 1987; Wong, et a l . , 1987); however, despite the frequent occurrence of amplified c-myc in s o l i d tumors the elevated l e v e l of c-myc mRNA seen in many leukemias is not usually due to amplification or obvious rearrangement at the c-myc locus, implying some other etiology of t r a n s c r i p t i o n a l deregulation (Westin, e_t_ a l . , 1982; Rothberg, et a l . , 1984). iv.) Point mutation Oncogenic a c t i v a t i o n by point mutation occurs in some c-onc's encoding cytoplasmic proteins and is best characterized in transforming a l l e l e s of the ras genes. Activated ras genes are the most commonly i d e n t i f i e d oncogenes isolated from human tumors of a l l types and point mutation seems to be the dominant mechanism resu l t i n g in oncogenic a c t i v a t i o n . Single nucleotide t r a n s i t i o n r e s u l t i n g in mutation at any of codons 12, 13, 59, or 61 is s u f f i c i e n t to render a given ras protein transforming, and a l l the mutations are located in the highly conserved amino terminal effector domain and resu l t in marked reduction in i n t r i n s i c GTPase a c t i v i t y (McGrath, et a l . , 1984; Sweet, e_t_ a l . , 1984; Barbacid, 1987). Mutations at codon 13 of N-ras occur r e l a t i v e l y frequently in acute myeloid leukemia and mutation at codon 13 of Kl-£as_ or of N-r_a_s_ has been 23 demonstrated in preleukemia patients many months prior to development of frank myeloid leukemia (Bos, et a l . , 1985; H i r a i , et a l . , 1987; Li u , et a l . , 1987). This l a t t e r observation, in concert with the i s o l a t i o n of activated Ki-ras a l l e l e s from colonic adenomas prior to their malignant conversion, suggests that ras mutation occurs r e l a t i v e l y early in the neoplastic process and is consistent with an e t i o l o g i c role for the ras oncogenes (Forrester, e_fc. a l . , 1987; Bos, et a l . , 1987). The neu oncogene can also be activated, in addition to gene amplification, by point mutation; in neu the altered codon is situated in the transmembrane spanning segment and is hypothesized to exert i t s e f f e c t by providing a constitutive stimulatory s i g n a l , in the absence of bound ligand, to the effector kinase domain (Bargmann, et a l . , 1986). v.) Deletion Deletion of regulatory domains from proteins encoded by v-onc's is a well recognized mode of acti v a t i o n and results in unrestrained a c t i v i t y of the v i r a l protein. Both v-src and v-fms have deleted those carboxyl terminal sequences which exert a negative regulatory influence upon tyrosine kinase a c t i v i t y , and v-erbB has deleted most of the EGF-binding domain in addition to carboxyl terminal residues ( U l l r i c h , et a l . , 1984; Coussens, et a l . , 1986; Hunter, 1987a). In a sense the chromosomal translocation which generates P210t,<=*:~"'»toX causes a deletion of amino terminal 24 abl sequences and results in enhanced tyrosine kinase a c t i v i t y comparable to that seen with P160 v -" l t' x, which also has a deleted amino terminal (Davis, et a l . , 1985). Some authors believe that truncation of c-onc's, due to r e t r o v i r a l transduction or as a resu l t of the frequent chromosomal translocations which are observed in malignant c e l l s , i s the dominant mode of c-onc ac t i v a t i o n (Duesberg, 1987). This hypothesis proposes that truncation deletes regulatory regions from the c-onc gene and brings i t under the influence of regulatory elements contained within the gene with which i t recombines; this deletion and recombination mechanism i s advanced as being more important than either point mutation or overexpression in the oncogenic a c t i v a t i o n of c-onc's. Most v-onc 1s are indeed truncated versions of c-onc's controlled by r e t r o v i r a l t r a n s c r i p t i o n a l regulatory elements; the human trk oncogene may also represent an example of thi s process: a protein-tyrosine kinase gene has been truncated and is fused with an upstream tropomyosin gene, re s u l t i n g in the production of a novel fusion protein (Martin-Zanca, et a l . , 1986). While elements of t h i s hypothesis are very a t t r a c t i v e they do not completely explain the frequent, and often reproducible association of activated c-onc's with a variety of tumors. However, the p l a u s i b i l i t y of much of t h i s hypothesis suggests that cloning of sequences adjacent to chromosomal translocations may be a very f r u i t f u l approach to ide n t i f y i n g novel oncogenes. 25 vi.) Autocrine stimulation Autocrine stimulation of c e l l s refers to the inappropriate elaboration of growth factors by c e l l s which possess receptors for the same factor; the unregulated expression of growth factor results in persistent, unconstrained stimulation of the c e l l through the cognate receptor (De Larco, et a l . , 1978). The genes encoding factors which function in an autocrine manner may themselves be c-onc's or, a l t e r n a t i v e l y , the a c t i v a t i o n of a non factor-encoding c-onc may r e s u l t secondarily in the stimulation of growth factor genes. A number of human tumors produce peptides with PDGF-agonist a c t i v i t y and, where characterized, these peptides are usually composed of PDGF A-A chain dimers (Heldin, et a l . , 1986; Bronzert, et. a l . , 1987). While some human tumor c e l l s express c - s i s mRNA and secrete PDGF-like molecules, evidence implicating such factors in an autocrine network comes largely from simian sarcoma virus (v-sis) infected c e l l s , which secrete PDGF-li k e factors, and where i n t r a c e l l u l a r autocrine stimulation of PDGF-receptors i s observed and transformation and p r o l i f e r a t i o n are prevented by anti-PDGF sera (Johnsson, e_t a l . , 1985a; Keating, et a l . , 1988). In the case of simian sarcoma virus transformed c e l l s , the s i s gene is introduced by the v i r u s ; in spontaneous human tumors the nature of the genetic disruption which results in uncontrolled expression of endogenous c- s i s genes has not been elucidated. C e l l s infected with other viruses may participate in a second type 26 of autocrine mechanism in which polypeptide growth factors are released as a consequence of inf e c t i o n ; the secreted factors are encoded by c e l l u l a r genes which are expressed following i n f e c t i o n but the nature of the genetic a l t e r a t i o n which re s u l t s in their expression is unknown (Twardzik, e_t a l . , 1982; Langer-Safer, et a l . , 1985; Weinberg, 1985). These liberated factors exert a variety of ef f e c t s and may influence the producing c e l l i t s e l f or surrounding c e l l s . The expression of c-s i s by some human leukemic c e l l s may be an example of the l a t t e r e f f e c t since hemopoietic c e l l s lack PDGF-receptors and are therefore u n l i k e l y targets for the PDGF-related molecules they synthesize; whether th i s c-sis expression is dependent upon act i v a t i o n of another c-onc is unresolved, but any such a c t i v a t i o n i s u n l i k e l y to be due to v i r a l i n f e c t i o n since the majority of human leukemias are believed to be non-viral (Westin, et a l . , 1982; Williams, 1986; Sunami, et a l . r 1987). 1.5 Protein-tyrosine Kinases The protein-tyrosine kinases constitute the largest functional group of c-onc proteins. In addition to those protein-tyrosine kinases s p e c i f i e d by c-onc's and implicated in neoplasia there are a number of c e l l u l a r protein-tyrosine kinases which bear close s t r u c t u r a l and functional homology with c-onc-encoded kinases but which have not yet been found to be activated in spontaneous tumors nor carried by any known re t r o v i r u s . While these c-onc_-related kinases have 27 not been Isolated d i r e c t l y from tumors i t is clear that they do possess latent oncogenicity: the kinase domain of slk can provide transforming a c t i v i t y when used to replace the equivalent domain of v-f gr, and pp56 l c ! > t becomes transforming when the influence of an i n h i b i t o r y phosphotyrosine residue is ablated (Kawakami, et a l . , 1986; Marth, et a l . , 1988a) The protein-tyrosine kinases can be subdivided into two classes, those acting as receptors for various growth factors, and those which are believed to function i n t r a c e l l u l a r l y in signal transduction. The majority of known protein-tyrosine kinases are categorized in Table 1.2, according to function and oncogenicity, although t h i s compilation is not exhaustive. The EGF receptor, encoded by the c-erbB gene, is the best characterized member of the growth factor receptor protein-tyrosine kinases. The EGF receptor i s synthesized as a precursor protein, and a membrane signal peptide is cleaved c o t r a n s l a t i o n a l l y during insertion of the protein into i n t r a c e l l u l a r membranes yi e l d i n g a mature molecule of 1186 residues ( U l l r i c h , et a l . f 1984; Hunter, et a l . , 1985). The amino terminal 621 residues constitute an e x t r a c e l l u l a r ligand binding domain and the carboxyl terminal 542 amino acids comprise an i n t r a c e l l u l a r protein-tyrosine kinase domain; the two domains are separated by a 22 residue hydrophobic membrane spanning segment which is believed to anchor the receptor in the c e l l membrane. The external TABLE 1.2 Functional C l a s s i f i c a t i o n of Protein-tyrosine Kinases Growth Factor Receptors I n t r a c e l l u l a r Prote in-tyrosine Kinases Known Oncogenic Known Oncogenic Potential potential c-fms(CSF-1 receptor) c-src c-erbB(EGF receptor) c-fgr c-erbB-2(neu) c-yes c-ros c-fps/fes c - k i t c-abl  trk  met Oncogenicity Oncogenicity  Not Demonstrated Not Demonstrated PDGF receptor NCP9 4 Insulin receptor hck IGF-1 receptor lyn f yn  ltk arg lck 29 domain is extensively glycosylated and possesses multiple cysteine residues which participate In intrachain d i s u l f i d e bridges; these features help confer upon the receptor i t s ligand binding c h a r a c t e r i s t i c s (Mayes, et a l . , 1984; Soderquist, et a l . , 1984). The kinase domain displays features t y p i c a l of a l l the protein-tyrosine kinases including the canonical sequences present at the ATP binding s i t e and the c a t a l y t i c s i t e ; the carboxyl terminal 15-20kD is not related in sequence to any known protein-tyrosine kinases and probably f u l f i l l s a regulatory role since three of four autophosphorylation s i t e s are located in this domain and deletion of the major phosphate acceptor, tyrosine-1173, from v-erbB molecules i s associated with reduced in v i t r o kinase a c t i v i t y (Downward, et a l . , 1984b; Carpenter, 1987). The EGF receptor possesses constitutive protein-tyrosine kinase a c t i v i t y toward i t s e l f and exogenous substrates, but tyrosine phosphorylation is enhanced both in v i t r o and in vivo following binding of EGF to the receptor (Erneux, §_t a l . , 1983; Downward, et a l . , 1984b). While EGF binding enhances kinase a c t i v i t y , the action of protein kinase C results in conversion of high a f f i n i t y EGF binding s i t e s to low a f f i n i t y and in decreased kinase a c t i v i t y (Friedman, e_t a l . , 1984; Downward, et a l . , 1985). Protein kinase C, a serine/threonine kinase, s e l e c t i v e l y phosphorylates threonine-654, which i s located in proximity to the membrane spanning region, and t h i s is postulated to induce conformational changes, re s u l t i n g in decreased a c t i v i t y 30 (Hunter, et a l . , 1984). Despite knowledge of 1igand-induced changes in enzymatic a c t i v i t y and i t s modulation by the action of protein kinase C the relevant physiological substrates have been d i f f i c u l t to i s o l a t e ; several proteins which are phosphorylated in vivo in an EGF-dependent manner have been isolated but t h e i r funtional significance is unknown (Hunter, et a l . , 1981; Pepinsky, et a l . , 1986). EGF receptors are expressed on most c e l l s and binding of EGF results in stimulation of p r o l i f e r a t i o n , e s p e c i a l l y of e p i t h e l i a l c e l l s . Hemopoietic c e l l s , however, do not express EGF receptors but are able to respond to EGF stimulation i f a c-erbB gene is introduced, implying either that d i s t a l components of the EGF receptor pathway are present in these c e l l s or that the EGF receptor interacts with a common pathway shared by a number of receptors (Pierce, et a l . , 1988). The protein p p e O 0 - " " is the best characterized of a l l the protein-tyrosine kinases; many of the s t r u c t u r a l and functional features of p p 6 0 0 - " 0 are t y p i c a l of a l l protein-tyrosine kinases but e s p e c i a l l y of the non-receptor, i n t r a c e l l u l a r c-onc kinases. These i n t r a c e l l u l a r proteins lack discernable ligand-binding domains and transmembrane segments, although one gene, l t k , encodes an apparent hydrophobic transmembrane region but lacks an e x t r a c e l l u l a r domain (Ben-Neriah, et a l . , 1988). Much of the current knowledge of the c-src protein has come from study of the 31 v i r a l protein, p p 6 0 v - a , K O / and while many s i m i l a r i t i e s exist between the two molecules the differences have been instrumental in deciphering the oncogenic a c t i v a t i o n of not only pp60= _" r r e = but other protein-tyrosine kinases ( C o l l e t t , et a l . r 1978b). ppSO 0 - 8 3" is synthesized on soluble ribosomes but is t i g h t l y membrane associated due to myristylation of glycine-2 following cleavage of the i n i t i a t i n g methionine (Buss, et a l . , 1985; lba, et a l . , 1985). pp60 < =-™~ = is bound to internal c e l l membranes, es p e c i a l l y to the cytoplasmic side of the plasma membrane, and myristylation may be c r i t i c a l in orienting the protein in r e l a t i o n to i t s normal substrates at the c e l l membrane (Willingham, et a l . , 1979; Courtneidge, et a l . , 1980). pp60 c - o r r = displays a domain structure which serves as a model for other non-receptor protein-tyrosine kinases: an amino terminal domain consisting of residues 1-269 while a carboxyl terminal domain i s comprised of residues 270-533. Mild proteolysis of p p 6 0 v - o _ e generates a 30kD C-terminal fragment which retains f u l l kinase a c t i v i t y but the s p e c i f i c a c t i v i t y of t h i s fragment is a c t u a l l y increased r e l a t i v e to the intact molecule, suggesting that the N-terminal domain subserves some regulatory role (Levinson, et a l . , 1981). Thus, the N-terminal domain is important for proper subcellular l o c a l i z a t i o n and probably for regulation of the i n t r i n s i c kinase a c t i v i t y ; several serine residues are included within t h i s domain and, while not c r i t i c a l for kinase a c t i v i t y , do seem to enhance a c t i v i t y when 32 phosphorylated. The amino domain shows l i t t l e homology with known proteins but the kinase domain shares a number of features with most protein kinases. A l l protein-tyrosine kinases have the sequence X-Ala-X-Lys and about 20 residues upstream a conserved sequence of Gly-X-Gly-X-X-Gly (Hunter, et a l . r 1986). These sequence motifs comprise part of the ATP-binding s i t e and the conserved lysine is absolutely required: mutagenesis of lysine-295 in p p 6 0 v - , s " completely abolishes kinase a c t i v i t y and transforming a b i l i t y (Snyder, et a l . . 1985). Interestingly, t h i s point mutation also prevents serine phosphorylation of the kinase; this could mean that binding of ATP by the c a t a l y t i c domain induces a permissive conformation for phosphorylation of serine residues. Toward the carboxyl end of pp60 o - a , j r o are other highly conserved sequences including residues 385-387(Arg-Asp-Leu), residues 404-406(Asp-Phe-Gly), and residues 430-432(Ala-Pro-Glu); mutagenesis of individual residues within these conserved sequences results in complete ablation of kinase a c t i v i t y (Hunter, et a l . , 1986). Tyrosine-416 i s the major autophosphorylation s i t e in p p e O 0 - " " " and the equivalent residue is conserved among a l l tyrosine-protein kinases, although i t i s not phosphorylated in c-erbB (Downward, et a l . f 1984b). Despite being the major s i t e of autophosphorylation mutagenesis of tyrosine-416 has minor eff e c t s on the kinase a c t i v i t y of pp60°~'s*r,= , while loss of tyrosine-527 dramatically increases a c t i v i t y ; the current evidence suggests that hyperphosphorylation of tyrosine-416 33 and hypophosphorylation or loss of tyrosine-527 may be c r i t i c a l to the oncogenic ac t i v a t i o n of pp60°-"" (Courtneidge, 1985; Kmiecik, et a l . , 1987; Piwnica-Worms, §_t a l . f 1987). The c e l l membrane location of pp60 c _ H , = c : and the dramatic e f f e c t i t exerts upon c e l l s in i t s activated form suggest that t h i s protein may act as an i n t r a c e l l u l a r transducer of growth control signals, perhaps acting in concert with s p e c i f i c phosphatases to dynamically a l t e r the a c t i v i t y of substrates via levels of tyrosine phosphorylation. However, s p e c i f i c substrates and the exact roles they f u l f i l l have been d i f f i c u l t to define. Much of the limited knowledge available on pp60 c = -"~ C I substrates has come from study of the v i r a l protein, and even overexpressing the normal protein does not res u l t in elevation of whole c e l l phosphotyrosine or of s p e c i f i c phosphoproteins to the same extent (Coussens, et a l . , 1985). A number of these phosphoproteins are also elevated in c e l l s transformed by other v-onc-encoded kinases, including v-fps, implying common effector pathways or, a l t e r n a t i v e l y , promiscuous, non-specific phosphorylation of c e l l u l a r proteins (Cooper, et a l . , 1981). A number of cytoskeletal proteins, including p36, p81, and v i n c u l i n , and g l y c o l y t i c enzymes contain elevated levels of phosphotyrosine in transformed c e l l s , and yet i t remains unclear whether any of these is a legitimate substrate of pp60=~™rr= or i f a l t e r a t i o n of any of these proteins is d i r e c t l y involved in transformation (Cooper, et a l . , 1983a; Rohrschneider, e£ 34 a l . , 1983; Kamps, et a l . , 1985). A recently described 81-85kD protein may well represent an authentic substrate of PP60 0-""" and preliminary indications are that t h i s protein is a phoshpatidylinositol kinase (Courtneidge, et a l . , 1987; Kaplan, et a l . , 1987). This protein is intriguing for two reasons: i t could explain the phospholipid kinase a c t i v i t y which has been associated with pp60 v _ , B , r , = but, more importantly, i t would link c-src proteins with a known signal transducing pathway (Sugimoto, et a l . r 1984). If this hypothesized connection i s v e r i f i e d i t s t i l l leaves open the question of which afferent molecules stimulate p p 6 0 a - H , E a , something about which nothing i s known. These upstream regulators of pp60' = _~ i ; = a c t i v i t y may be members of mitogen s i g n a l l i n g pathways but in this regard i t i s interesting to note that highest levels of pp60 o-" K , = are found in p l a t e l e t s and neurons, both post-mitotic c e l l types. However, pp60c-src may serve d i f f e r e n t functions in d i f f e r e n t c e l l s , thus not excluding p a r t i c i p a t i o n in a mitogenic role in other c e l l types. 1.6 The fps/fes Oncogenes c-fes is the c e l l u l a r homologue of the v i r a l gene v-fes, carried by the Snyder-Theilen (ST-FeSV) and Gardner-Arnstein (GA-FeSV) strains of f e l i n e sarcoma vi r u s , while c-fps is the c e l l u l a r counterpart of the v-fps gene, carried by the Fujinami avian sarcoma virus and a number of other avian viruses (Bishop, 1982). Comparison of the sequences 35 of v-fps and v-fes genes indicates remarkable s i m i l a r i t y , suggesting that these v i r a l genes were derived from, respectively, the avian and f e l i n e homologues of the same c-onc; v-fes or v-fps probes anneal with i d e n t i c a l r e s t r i c t i o n fragments of human genomic DNA, supporting this view (Hampe, et a l . , 1982; Groffen, et a l . r 1983). Thus, with respect to the human gene, either fps or fes can be used when r e f e r r i n g to the common locus although c-fes w i l l be employed in t h i s d i s s e r t a t i o n purely because of greater evolutionary relatedness. Human c-fes sequences are located on chromosome 15; they are non-contiguous and, by comparison with the chicken c-fps gene, consist of 18 exons over a span of 11 kilobases(kb) of genomic DNA (Franchini, et a l . , 1982; Heisterkamp, et a l . , 1982; Huang, et a l . f 1985; Roebroek, et. a l . , 1985). The topography of the c-fes gene more c l o s e l y resembles that of the c-src gene family rather than that of receptor-encoding genes, such as c-erbB, and c-fes lacks transmembrane and e x t r a c e l l u l a r sequences suggesting i t is probably not, by i t s e l f , a c e l l surface receptor (Roebroek, et a l . r 1985). The fps/fes genes encode proteins which display homology with other protein-tyrosine kinases including a putative ATP-binding s i t e and a tyrosine residue equivalent to the major s i t e of autophosphorylation in src proteins. The functional properties of v-fes and v-fps proteins were demonstrated prior to the nucleotide sequence data which related these genes to v-src, and normal c e l l s 36 from the presumed species of o r i g i n of v-fps or v-fes express homologous c e l l u l a r proteins which are func t i o n a l l y and s t r u c t u r a l l y related to their v i r a l counterparts (Barbacid, et a l . , 1980; Mathey-Prevot, et a l . , 1982). The v-f ps protein, P130'3*"3~£p", autophosphorylates at tyrosine-1073, which i s equivalent to tyrosine-416 in pp60 v _ = , , r c : :, but unlike p p e O 0 - 0 " the loss of the major autophosphorylation s i t e from p i 3 o , * t t « - : C s , a adversely affects i t s transforming and kinase a c t i v i t y (Patschinsky, et a l . , 1982; Weinmaster, e_t_ a l . , 1984). The homologous avian c-fps product, NCP98, phosphorylates t h i s same tyrosine ln v i t r o but, unlike the v i r a l protein, is phosphorylated only at serine in vivo; furthermore i t d i f f e r s from p i s e 3 " " * - * 5 " 1 3 j. n i t s subcellular d i s t r i b u t i o n , being found lar g e l y in the soluble cytoplasmic f r a c t i o n rather than membrane-associated (Mathey-Prevot, e_t_ a l . , 1982; Young, et a l . f 1984). These d i s t i n c t i v e functional features may be among those necessary to re s u l t in oncogenic a c t i v a t i o n of NCP98. Expression of c-fps transcripts in normal avian tissues i s highest in bone marrow c e l l s , and fractionation studies of spleen, bone marrow, and bursa, the avian hemopoietic organs, indicates that NCP98 kinase a c t i v i t y i s associated with granulocytes and macrophages (Shibuya, et a l . , 1982; Samarut, et a l . f 1985). c-fes transcripts are not detectable in whole mouse fetuses of varying gestation but are detectable in a va r i e t y of human malignancies, mainly hemopoietic tumors (Slamon, et a l . , 1984a; Slamon, et a l . . 37 1984b); prior to the results presented in this d i s s e r t a t i o n c-fes proteins were Identified in c e l l l i n e s from a few mammalian species, but no c-fes proteins from human or mouse c e l l s had been i d e n t i f i e d or characterized (Barbacid, e_t a l . , 1980). 1.7 Hemopoiesis Hemopoiesis, or hematopoiesis, i s the l i f e l o n g process which ensures that s u f f i c i e n t numbers and v a r i e t i e s of a l l blood c e l l s are produced throughout l i f e in order to meet the ongoing needs of the organism. Hemopoiesis must not only resupply those blood c e l l s l o s t normally as a re s u l t of f i n i t e lifespans, but must also be poised to respond f l e x i b l y and rapidl y to emergencies such as bleeding or in f e c t i o n . The most basic building block of the hemopoietic system is the pluripotent stem c e l l , c e l l s which are known funct i o n a l l y by their a b i l i t y to repopulate any of the hemopoietic lineages. Because of the i r r e l a t i v e l y low frequency in blood forming organs and their lack of unique antigenic or morphologic features the functional characterization of these c e l l s has r e l i e d on the i r a b i l i t y to form mixed colonies in semisolid media and to repopulate the hemopoietic systems of l e t h a l l y irradiated mice ( T i l l , et a l . r 1961; Bradley, et a l . , 1966; Dexter, et a l . . 1977). Pluripotent stem c e l l s are unique in that they simultaneously possess the capacity for extensive s e l f renewal and the a b i l i t y to d i f f e r e n t i a t e into committed 38 precursors, which then develop along s p e c i f i c lineages (Lord, 1983). The majority of pluripotent stem c e l l s are not a c t i v e l y involved in either d i v i s i o n or d i f f e r e n t i a t i o n but are in a resting Go state from which they can be aroused by various physiological and non-physiological s t i m u l i ; the mechanisms by which these stressors stimulate entry into the active c e l l cycle are unknown. The pluripotent stem c e l l s give r i s e to at least three other types of stem c e l l s , namely those stem c e l l s which are committed to myelopoiesis or to T or B lymphopoiesis. These committed stem c e l l s also possess the capacity for extensive self-renewal but their progeny can only d i f f e r e n t i a t e along the respective myeloid or lymphoid pathways (Abramson, §_t a l . , 1977; Dexter et a l . , 1987). Once committed to a pa r t i c u l a r myeloid lineage the developing c e l l s acquire progressively the functional and morphologic attributes of that lineage concomitant with a decreasing capacity for s e l f renewal. However, c e l l s of the T and B lymphoid lineages are d i f f e r e n t in that they are able to undergo c l o n a l , s e l f -renewing d i v i s i o n s at later stages of development where s i g n i f i c a n t d i f f e r e n t i a t i o n , such as immunoglobulin gene rearrangement, has already taken place (McCulloch, 1983); a br i e f outline of normal hemopoiesis i s diagrammed schematically in Figure 1.1. The CFC-Mix, or CFC-GEMM, i s the committed progenitor c e l l of myelopoiesis and i s able to d i f f e r e n t i a t e along erythroid, granulocyte/macrophage, and megakarocytic lineages; under some circumstances t h i s c e l l 39 STEM CELL CFC-Mix (CFU-GEMM) Pre-T Eos-CFC BFU-E GM-CFC Bas-CFC Meg-CFC Pre-B T lymphocytes Eosinophils Erythrocytes Neutrophils Macrophages/ monocytes Basophils Megakaryocytes B lymphocytes Figure 1.1. The human hemopoietic system. The pluripotent stem c e l l compartment possesses the capacity for extensive s e l f renewal. With increasing d i f f e r e n t i a t i o n , c e l l s become committed to individual lineages and gradually lose their p r o l i f e r a t i v e a b i l i t y . (CFC-Mix = mixed colony forming c e l l ; CFU-GEMM = granulocyte/erythrocyte/monocyte/macrophage colony forming unit; Pre-T = pre-T lymphocyte; Eos-CFC = eosinophil colony forming c e l l ; BFU-E = erythrocyte burst forming unit; GM-CFC = granulocyte/macrophage colony forming c e l l ; Bas-CFC = basophil colony forming c e l l ; Meg-CFC = megakaryocyte colony forming c e l l ; Pre-B = pre-B lymphocyte.) 40 may also generate lymphocytes (Messner, et a l . , 1981; Fauser, et a l . , 1985), but i t lacks the extensive s e l f renewal capacity c h a r a c t e r i s t i c of true pluripotent stem c e l l s and is therefore c l a s s i f i e d as a multipotent progenitor c e l l (Messner, 1986; G r i f f i n et a l . , 1986). The GM-CFC progenitor c e l l , derived from the CFC-GEMM,is able to produce both macrophages and granulocytes depending on the nature of the microenvironment and colony stimulating factors (CSFs) to which i t is exposed; subsequent d i f f e r e n t i a t i o n of GM-CFC's to mature macrophages or granulocytes is a morphologically well characterized, multistage process and w i l l not be reiterated here. The microenvironment in which a stem c e l l or committed progenitor c e l l finds i t s e l f plays an important role in determining the nature of subsequent d i f f e r e n t i a t i o n events. The microenvironment i s composed of stromal c e l l s , e x t r a c e l l u l a r matrix, and both short and long range soluble factors; a l l of these elements play a part in determining the types of c e l l s which develop. Stem c e l l s which seed in the spleen produce predominantly erythrocytes while granulocytes are the predominant c e l l type in bone marrow (Wolf et a l . , 1968); investigations in v i t r o have shown that a feeder layer established from explanted stromal c e l l s is v i t a l to the maintenance and growth of long term bone marrow cultures (Dexter, et a l . , 1977). The e x t r a c e l l u l a r matrix is a glycoprotein mesh secreted by stromal c e l l s and probably serves to allow physical attachment of stem c e l l s 41 at s i t e s o£ hemopoiesis; recently, a novel adhesion protein, haemonectin, has been Isolated which may help account for the granulocyte predominance of bone marrow as i t s p e c i f i c a l l y binds c e l l s of granulocytic o r i g i n and i s located only in the bone marrow (Campbell, et a l . , 1987). Soluble factors, produced l o c a l l y by stromal c e l l s and acting at short range, or synthesized d i s t a n t l y by c e l l s at s i t e s of i n f e c t i o n or inflammation are important contributors to the microenvironment; the best characterized soluble factors which stimulate granulocyte and macrophage myelopoiesis are the granulocyte-macrophage CSFs. CSFs are glycoproteins which are synthesized ubiquitously throughout the body, and myeloid progenitor c e l l s , at least in v i t r o , display an absolute requirement for these factors: the CSFs are necessary in order to maintain p r o l i f e r a t i o n of progenitor c e l l s , orderly d i f f e r e n t i a t i o n to more mature c e l l s , and functional a c t i v i t y of the mature end-stage c e l l (Metcalf, 1985). This requirement for CSFs i s presumably mirrored in vivo, and the d i v e r s i t y of c e l l types which synthesize and secrete these factors suggests that the CSFs act as integral components of the body's natural defences by functionally a c t i v a t i n g granulocytes and macrophages at s i t e s of i n f e c t i o n and by stimulating the production of greater numbers of these c e l l s by the bone marrow and spleen. There are four major granulocyte/macrophage CSFs which have been i d e n t i f i e d and shown to have stimulatory effects on myelopoiesis: 1.) 42 granulocyte-macrophage CSF (GM-CSF); 2.) granulocyte CSF (G-CSF); 3.) macrophage CSF (M-CSF); 4.) multi-CSF (IL-3). There appears to be a hierarchy of CSF actions in that M-CSF and G-CSF stimulate colonies of, respectively, macrophages or granulocytes while GM-CSF results in mixed granulocyte/macrophage colonies and IL-3 produces colonies in which a l l hemopoietic lineages except lymphocytes are represented (Metcalf, 1985). The CSFs are unique glycoproteins possessing l i t t l e sequence homology and appear to be species s p e c i f i c : in general, murine CSFs do not stimulate p r o l i f e r a t i o n of human granulocyte/macrophage precursors (Sachs, 1987). While there i s no sequence homology between the CSFs and known oncogene proteins or tumor growth factors, the important niche which the CSFs occupy in the control of myelopoiesis suggests that their dysregulation might be involved in the genesis of myeloid dyscrasias. There are currently no data to indicate that the pathogenesis of human myeloid leukemias involves the unregulated production of CSFs acting in some form of autocrine loop, and the expression of the GM-CSF gene in transgenic mice does not lead to the development of leukemia (Lang, et a l . , 1987). However, the unregulated expression of a hemopoietic growth factor cDNA in a factor dependent c e l l l i n e results ln conversion to factor-independence and tumorigenicity, suggesting that an autostimulatory loop is c e r t a i n l y a potential mechanism in leukemogenesis (Lang, ejt a l . , 1985). 43 While CSFs are not known to be encoded by c-onc's the product of at least one c-onc functions as a c e l l surface receptor for a CSF: the c-fms gene encodes a protein, gpl50=- f i m-, which is the receptor for CSF-1 (M-CSF). CSF-1 is absolutely required in v i t r o for both the ac t i v a t i o n and maintenance of macrophages and for the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of precursor c e l l s to mature macrophages, and t h i s factor i s s e l e c t i v e l y destroyed by the same c e l l s which i t stimulates (Tushinski, et a l . , 1982). Examination of a number of human c e l l s and c e l l l i n e s revealed that c-fms tra n s c r i p t s and kinase a c t i v i t y are only detectable in monocyte/macrophages or in c e l l s undergoing induced macrophage d i f f e r e n t i a t i o n , with the exception of c e l l s derived from the placental trophoblast (Sariban, et a l . , 1985; Woolford, et a l . , 1985; Rettenmier, et a l . , 1986). The elevated expression of c-fms in placenta probably r e f l e c t s some non-hemopoietic developmental role in thi s tissue as the pregnant uterus secretes high levels of CSF-1 in response to the hormones e s t r a d i o l and progesterone (Pollard, et a l . r 1987). Binding of murine CSF-1 to i t s receptor on macrophages results in enhanced tyrosine kinase a c t i v i t y of the c-fms protein, and anti-fms antisera are able to immunoprecipitate ""I-CSF bound to i t s receptor, suggesting that gpl50° _ : e m" and the CSF-1 receptor are id e n t i c a l (Sherr, et a l . , 1985); formal proof that c-fms encodes the CSF-1 receptor came with the demonstration that a transfected c-fms gene confers upon f i b r o b l a s t s the 44 a b i l i t y t o b i n d and t o be t r a n s f o r m e d by CSF-1 ( R o u s s e l , §_£. a l . , 1987). Thus, a t l e a s t one CSF i s bound by a c-ojic_-encoded r e c e p t o r a l t h o u g h the mechanisms by which the r e c e p t o r mediates the myriad of C S F - l - i n d u c e d r e s p o n s e s are c o m p l e t e l y unknown; the e f f e c t s a r e l i k e l y t o be the r e s u l t of p r o t e i n - t y r o s i n e p h o s p h o r y l a t i o n but s u b s t r a t e s f o r such proposed m o d i f i c a t i o n have not y e t been i d e n t i f i e d . I t i s p o s s i b l e , i f not l i k e l y , t h a t o t h e r c-onc's w i l l encode h e m o p o i e t i c growth f a c t o r r e c e p t o r s s i n c e l i g a n d s have not y e t been c h a r a c t e r i z e d f o r a number of p r o t e i n - t y r o s i n e k i n a s e s which d i s p l a y the t o p o l o g y t y p i c a l of transmembrane r e c e p t o r s . 1.8 Myelogenous Leukemias Leukemias are h e m o p o i e t i c m a l i g n a n c i e s which r e s u l t as a consequence of a r r e s t e d m a t u r a t i o n of a committed c e l l l i n e a g e l e a d i n g t o overgrowth of immature, n o n f u n c t i o n a l c l o n a l p o p u l a t i o n s . Normal h e m o p o i e t i c c e l l s and t h e i r p r e c u r s o r s are crowded out of t h e i r u s u a l m i c r o e n v i r o n m e n t a l n i c h e s i n b l o o d - f o r m i n g organs by r e l e n t l e s s p r o d u c t i o n of the u n d i f f e r e n t i a t e d c l o n e , e v e n t u a l l y l e a d i n g t o c l i n i c a l symptoms a s s o c i a t e d w i t h d e f i c i e n c i e s of the v a r i o u s normal c e l l s . T h i s o v e r p r o d u c t i o n of immature progeny i s not the r e s u l t of a reduced c e l l c y c l e t i m e , i n f a c t l e u k e m i c c e l l s o f t e n have a p r o l o n g e d c e l l c y c l e time compared w i t h t h e i r normal c o u n t e r p a r t s ( B a s e r g a , 1981), but i s due t o 45 p r o l i f e r a t i o n becoming uncoupled from normal regulatory constraints. The myelogenous leukemias are c l a s s i f i e d as either acute (AMD or chronic (CML) according to c l i n i c a l presentation and the predominant c e l l type i d e n t i f i e d in smears of peripheral blood and bone marrow, the c e l l s in CML being mainly defective granulocytes while those in AML are primarily blast c e l l s or p a r t i a l l y d i f f e r e n t i a t e d granulocyte/macrophage precursors. Both AML and CML are clonal hemopathies, that i s , the leukemic c e l l s are believed to a l l be the progeny of a single malignant ancestral c e l l ; evidence of c l o n a l i t y has come from a number of sources including analysis of isoenzymes, karyotypic abnormalities, and analysis of DNA polymorphisms (Fialkow, et a l . , 1977; Fialkow, et a l . , 1981a; Fearon, et__al., 1986). In CML the malignant clone is believed to originate in a pluripotent stem c e l l since the Philadelphia chromosome(Phi), when present, can be detected in c e l l s of multiple lineages (Kalousek, et a l . , 1984), and the clonal nature of th i s disease has been confirmed by examining isoenzymes of glucose-6-phosphate dehydrogenase (G6PD) in CML c e l l s (Fialkow, 1984). Despite evidence that the defect in CML has i t s roots in a pluripotent stem c e l l and that a l l lineages are derived from th i s abnormal precursor, normal d i f f e r e n t i a t i o n programs are followed by these other lineages; t h i s raises the p o s s i b i l i t y that the genetic 46 derangement in the stem c e l l manifests as a sele c t i v e growth advantage unique to the abnormal granulocytic c e l l s . It has been suggested that such a growth advantage might be due to the elevated protein-tyrosine kinase a c t i v i t y of P210 t o"~** t o l in granulocytic c e l l s (Fialkow, 1984), but t h i s must be balanced with the observation that not a l l CML's are Phi-positive and some cases of Phi-positive acute lymphocytic leukemia(ALL) also display increased abl-associated tyrosine kinase a c t i v i t y (Kurzrock, et a l . , 1987; Chan, et a l . , 1987). However, the very frequent association of the Phi with CML strongly suggests that t h i s translocation is involved in the pathogenesis of t h i s disease, although i t may not be the i n i t i a t i n g event since c l o n a l i t y can be demonstrated in CML c e l l s prior to the emergence of the Phi (Fialkow, et a l . , 1981b). Interestingly, those cases of ALL which are Phi-positive, l i k e CML, appear to originate in a pluripotent stem c e l l while Phi-negative ALL *s show no evidence of involvement of myeloid c e l l s , suggesting in these cases that the disease arises in stem c e l l s committed to lymphopoiesis (Tachibana, et a l . , 1987). The AML's comprise a more heterogenous group of diseases than CML, as assessed by morphology of the malignant c e l l s and the presumed c e l l of o r i g i n . The clonal nature of AML has been demonstrated in studies of G6PD isoenzymes and of DNA restriction-fragment polymorphisms, but the c e l l of o r i g i n remains obscure. While i t i s clear that a l l three lineages of myelopoiesis may derive from a 47 malignant progenitor, implying Involvement of a multipotent stem c e l l , i t is not resolved whether lymphopoiesis, suggesting a lesion at the le v e l of a true pluripotent stem c e l l , i s also affected (Fialkow, et a l . , 1981a; G r i f f i n , et a l . , 1986). Studies of c e l l surface markers and other indicators of c e l l lineage have indicated the existence of lineage i n f i d e l i t y in AML, that is , the co-expression of several d i s t i n c t i v e l i neage-specific c h a r a c t e r i s t i c s in the same c e l l (Smith, et a l . f 1983). It i s not clear whether the co-existence of markers of both myeloid and lymphoid lineages in AML blasts represents unique, highly abnormal pathways of d i f f e r e n t i a t i o n or whether AML i s a clonal expansion of transient precursor c e l l s which exist during normal hemopoiesis and which normally co-express several markers (McCulloch, 1983; Greaves et a l . , 1986). A multistep pathogenesis for AML has been suggested in which genetic damage results in the generation of abnormal clones of preleukemic c e l l s and subsequent events allow the emergence of a frankly malignant clone (Fialkow, 1984). Support for t h i s proposal has come from the findings that clonal expansion is already present in the preleukemic syndromes which are frequent forebears of AML (Jacobson, §_t a l . , 1982), and that a c t i v a t i n g mutations are present in the ras genes of some preleukemic patients long before progression to acute leukemia ( H i r a i , et a l . , 1987; L i u , et. a l . , 1987). In thi s model, the aq u i s i t i o n of chromosomal abnormalities or other genetic damage occurs later and 48 confers a selec t i v e advantage upon the leukemic clone; the existence of abnormal karyotypes, which are frequently i d e n t i f i e d in AML's, may herald such unrestrained growth, although consistent involvement of c-onc's in the chromosomal alte r a t i o n s i s not an invariant feature (Yunis, 1983). The leukemic c e l l s in AML are phenotypically less mature than those of CML and the leukemic AML c e l l s appear to be blocked from d i f f e r e n t i a t i n g beyond i d e n t i f i a b l e precursor forms to mature granulocytes or monocytes. However, t h i s blockage i s not absolute and apparently normal granulocytes derived from the malignant clone have been i d e n t i f i e d in AML patients in remission (Fearon, et a l . , 1986), and some leukemic c e l l s are able to d i f f e r e n t i a t e normally, both in vivo and in v i t r o , when stimulated with CSF's (Dexter, et a l . , 1987; Sachs, 1987). Also, characterizing the p r o f i l e s of myeloid surface markers on leukemic c e l l s suggests that some d i f f e r e n t i a t i o n , a l b e i t aberrent, does occur in AML patients in vivo ( G r i f f i n , ejt a l . r 1986); however, th i s d i f f e r e n t i a t i o n i s c l e a r l y i n e f f e c t i v e in div e r t i n g the majority of blast c e l l s into normal developmental programs. The fact that d i f f e r e n t i a t i o n can take place at a l l belies the assumption that a l l leukemic blasts are equal in their p r o l i f e r a t i v e capacity, and i t has been shown that the vast bulk of leukemic c e l l s are p r o l i f e r a t i v e l y inert ( G r i f f i n , et a l . , 1986); the c e l l s responsible for the rele n t l e s s production of blasts are clonogenic AML "stem c e l l s " , morphologically 49 indistinguishable from other blasts but possessing extensive self-renewal capacity (Buick, e_£ a l . , 1979; Chang, e j t_ a l . , 1980). Thus, in populations of blast c e l l s a hierarchy of diminishing r e p l i c a t i v e capacity ex i s t s , but, unlike normal myelopoiesis, i t is not accompanied by a concomitant committment to d i f f e r e n t i a t i o n . Much e f f o r t has been expended investigating the induction of d i f f e r e n t i a t i o n in AML's using both physiologic and non-physiologic inducers in the hope that both therapeutic and mechanistic insights might be r e a l i z e d (Pegoraro, et a l . , 1980; Koef f l e r , •et_a_l., 1984). Much useful knowledge has come from the examination of fresh c e l l s but more information has resulted from the systematic study of the effects of inducers upon myeloid leukemia c e l l lines (Koeffler, 1983), and the use of leukemic c e l l l i n e s has allowed characterization of changes in expression of c-onc's which accompany d i f f e r e n t i a t i o n , thereby allowing glimpses of their normal functions. 1.9 Purpose and Experimental Approach While well characterized at the DNA l e v e l (Groffen, §_t a l . , 1982; Shibuya, et a l . , 1982), d e t a i l s of the expression of c-fps and c-fes proto-oncogenes in human and murine c e l l s have remained largely undeciphered. Previous studies have characterized the c-fps/fes gene products from a number of avian and mammalian sources but did not i d e n t i f y or characterize either the human or murine proteins (Barbacid, et a l . r 1980; Mathey-Prevot, et a l . r 1982). Detectable 50 levels of c-fes transcripts are present in human hematologic malignancies but in few other tumors (Slamon, et a l . , 1984b), and thus I employed immunoprecipitation techniques and a vari e t y of functional and st r u c t u r a l assays in order to t r y and i d e n t i f y the human and murine c-fps/fes homologous proteins in a vari e t y of hemopoietic c e l l s . D i f f e r e n t i a t i o n to a mature phenotype i s an i n t r i n s i c property of normal hemopoietic c e l l s and is a phenomenon which can be studied to some extent in v i t r o by employing c e l l l i n e s and exogenous inducers of d i f f e r e n t i a t i o n . Evidence indicates that levels of protein-tyrosine kinases may vary with the d i f f e r e n t i a t i v e state of hemopoietic c e l l s (Barnekow, et a l . r 1986; Golden, et a l . , 1986; Q u i n t r e l l , §_£ a l . f 1987), and may play a supporting role in leukemogenesis (Dexter, et a l . , 1984). Therefore, I have investigated human myeloid leukemia c e l l l i n e s during exposure to a chemical inducer of macrophage d i f f e r e n t i a t i o n in order to determine i f similar changes in expression of c-fes occur: I evaluated the expression of both c-fes mRNA and p92 a - e' B' a kinase a c t i v i t y before and after exposure of c e l l lines to the inducer tetradecanoyl phorbol acetate (TPA). 51 CHAPTER 2 2.0 Materials and Methods 2.1 C e l l s . C e l l Lines, and Tissues The following human c e l l lines were used: HL-60, derived from a patient with promyelocytic leukemia ( C o l l i n s , et a l . f 1977); HL-525, a subline derived from HL-60, was obtained by subculturing HL-60 c e l l s 102 times at 5-8 day intervals in increasing concentrations of TPA (Mitchell, §_£. a l . , 1986; Homma, et a l . , 1986); KG-1, derived from a patient with acute myelogenous leukemia (Koeffler, et a l . , 1978); KG-la, an undifferentiated subline of KG-1 which has lost the capacity to d i f f e r e n t i a t e (Koeffler, et a l . , 1980); K562, a c e l l l i n e showing erythroid c h a r a c t e r i s t i c s but o r i g i n a l l y obtained from a patient with chronic myelogenous leukemia (CML; Lozzio, et a l . , 1975); HEL, an erythroleukemia c e l l l i n e (Martin, et a l . , 1982); MOLT-4, an immature T-lymphocyte derived c e l l l i n e (Minowada, et a l . , 1972); SU-DHL-4, . an immunoglobulin-positive h i s t i o c y t i c B-lymphoma c e l l l i n e (Epstein et a l . , 1978); WAY-1, an Epstein-Barr virus-transformed B-lymphocyte c e l l l i n e (D. Howard, personal communication); NCI-H82, a s m a l l - c e l l lung carcinoma c e l l l i n e ( L i t t l e , et a l . . 1983); U-937, a monocyte-1ike c e l l l i n e obtained from a patient with generalized h i s t i o c y t i c lymphoma (Sundstrom, et a l . , 1976). 52 The following murine c e l l lines were used: P388AD-4, an adherent macrophage-like c e l l l i n e (Cohen, et a l . , 1981); WEHI-3B, a myelomonocytic leukemia derived c e l l l i n e (Warner, et a l . , 1969); B6SUtA, an IL-3-dependent, non-tumorigenic c e l l l i n e with some properties of a multipotential progenitor c e l l (Greenberger, et a l . , 1983); EL-4, a thymoma c e l l l i n e (Farrar, et a l . , 1980); NS-1, a non-secreting myeloma c e l l l i n e (Kohler, et a l . , 1976); P815, a mastocytoma c e l l l i n e (Dunn, et a l . 1957); MEL, a Friend virus-induced erythroleukemia c e l l l i n e (Friend, et a l . , 1971); P19 and OlAl, embryonal carcinoma c e l l lines (McBurney, et a l . , 1982); YI, an adrenocortical tumor c e l l l i n e (Yasamura, et a l . , 1966); and NIH 3T3, a f i b r o b l a s t c e l l l i n e (Barbacid, et a l . . 1981). A number of virus-transformed c e l l l i n e s were used: E26 virus-transformed avian myeloblasts were grown as previously described (Beug, et a l . , 1984); FSV-transformed Rat II c e l l s were grown according to published methods (Weinmaster, et. a l . , 1983); and ST-FeSV-transformed NIH 3T3 fi b r o b l a s t s were grown as previously described (Barbacid et a l . , 1981). No infectious viruses were produced by any of these c e l l l i n e s as a l l the viruses were r e p l i c a t i o n defective retroviruses. Adherent c e l l l i n e s were maintained ln 100 mm Falcon dishes while suspension cultures were grown in 250 ml tissue culture f l a s k s . C e l l s were grown in RPMI-1640 medium (Gibco) supplemented with HEPES, 10% f e t a l bovine serum 53 ( F B S ) , p e n i c i l l i n and streptomycin at 37°C in a 5% C O a atmosphere, or in Dulbecco's modified Eagle's medium receiving the same supplements. When required, individual c e l l l i n e s were supplemented with 3% horse serum ( c e l l l i n e Yl) or 2% chicken serum (avian c e l l l i n e s ) . A l l c e l l lines were tested p e r i o d i c a l l y for contamination by Mycoplasma species using a commercially available c y t o t o x i c i t y test k i t (Mycotect; Bethesda Research Laboratories), and a l l experiments were performed on Mycoplasma-negative c e l l s . Normal mouse bone marrow c e l l s were obtained from the femurs and t i b i a s of DBA/2 mice by flushing the marrow c a v i t i e s with cold PBS and p e l l e t i n g the c e l l s , while early erythroblasts were obtained from the spleens of acetylphenylhydrazine-treated mice as described previously (MacDonald, et a l . , 1985). Normal mouse spleens, hearts, l i v e r s , and kidneys were obtained from DBA/2 mice, the tissues homogenized, and lysates prepared for use in in  v i t r o kinase reactions. Peripheral blood leukocytes and bone marrow c e l l s from healthy donors or from patients with CML or AML were separated by buoyant density centrifugation over Ficoll-Hypaque and contaminating erythrocytes were removed by l y s i s with TRIS/NH4C1, as described (Territo, e_t a l . , 1977). Exposure of HL-60 or HL-525 cultures to TPA was done as follows: healthy c e l l s , in logarithmic growth phase, were s p l i t with fresh media 24 hr prior to addition of TPA. On 54 the day of induction the c e l l s were counted and c e l l density was adjusted to 8 X 10 s / ml with fresh medium. Ce l l s were placed in 750 ml tissue culture flasks and TPA (10 ug / ml in 95% ethanol; Sigma) was added d i r e c t l y to the culture and gently mixed; f i n a l concentration of TPA was 1.6 X 1 0 - e H and ethanol 0.1%. No morphologic alt e r a t i o n s were ever seen in control cultures receiving only ethanol. L i t t l e , i f any, adherence was seen in cultures of less than 8 hr exposure to TPA and hence these early cultures were harvested by simply pouring the c e l l s into centrifuge tubes and p e l l e t i n g them. In cultures induced for longer than 8 hr, the flasks were f i r s t scraped gently with a p l a s t i c c e l l scraper (Costar) to dislodge any adherent c e l l s prior to p e l l e t i n g . 2.2 Preparation of Immune Reagents Anti-fps serum (J6) was obtained from T. Pawson and was o r i g i n a l l y prepared by i n j e c t i n g 4-week-old Fischer X Wistar rats with 5 X 10 s FSV s t r a i n L5-transformed Rat-1 c e l l s as previously described (Ingman-Baker, et a l . , 1984). Non-immune serum was obtained from the same rats prior to inoculation with FSV-transformed c e l l s . Anti-pEX-2-abl rabbit antiserum (Konopka, et a l . f 1984) was obtained from 0. Witte and goat antiserum to ST-FeSV P87'3"'3-*-" (a n t i -S T a u t ) was obtained from M . Barbacid (Barbacid, et a l . , 1980). Staphylococcus aureus s t r a i n Cowan I (IgGsorb; The Enzyme Center) was prepared by incubation at 4°C with rabbit 55 a n t i - r a t gamma globulin (RARIG; Cappel-Worthington) for 2 hr. The RARIG-coated Staph, aureus was washed three times with PBS and resuspended at a f i n a l concentration of 10% in either l y s i s buffer or kinase l y s i s buffer prior to use in immunoprecipitation. 2.3 Radiolabelling of C e l l s Healthy c e l l s in logarithmic growth phase were counted and 1 X 10"7 c e l l s were pelleted and washed with PBS prior to resuspension in 1.0 ml of RPMI-1640 medium lacking methionine and supplemented with 3-5% FBS. After a period of pre-incubation of 10-30 min in a 35 mm well (2x3 well plate; Linbro), (35S)methionine (lOOuCi/ml, 1000 Ci/mmol; Amersham Corp.) was added to a f i n a l concentration of 250 uCi/ml and the c e l l s were incubated at 37°C for periods of time ranging from 20 min to 8 hr. Adequate incorporation of label was determined to have taken place after a 30 min period of incubation. Following the l a b e l l i n g period the c e l l s were pelleted and washed twice with 2.0 ml ice cold PBS and then lysed on ice in 0.5 ml of l y s i s buffer (1% Nonidet P-40; 0.5% sodium deoxycholate; 10 mM T r i s -HCl(pH7.5); 100 mM NaCl; 1 mM EDTA; 2 mM ATP; 10 ug/ml aprotlnin; ImM phenylmethylsulfonylfluoride (PMSF); 50 ug/ml leupeptin). The crude lysates were cleared by centrifugation at 27,000 X g at 4°C for 30 min and the cleared supernatants were transferred to fresh 1.5 ml 56 Eppendorf tubes and held at 4°C u n t i l immunoprecipitated; the p e l l e t s were discarded. For in vivo phosphate l a b e l l i n g , medium was switched from RPMI-1640 to DMEM several days prior to r a d i o l a b e l l i n g ; c e l l s tolerated changes in media well with no discernable a l t e r a t i o n in growth ki n e t i c s or morphology. 5 X 10 s c e l l s were pelleted, washed once with PBS, and suspended in 1.0 ml of phosphate-free DMEM supplemented with 3-5% FBS, following which 3 2P-orthophosphate (2.0 mGi/ml, c a r r i e r free; ICN Pharmaceuticals Inc.) was added to a f i n a l concentration of 1.0 mCi/ml. C e l l s were labelled for 12 hr at 37°C following which they were harvested and lysates prepared exactly as described for methionine l a b e l l i n g . To determine i f p92 was phosphorylated in vivo in a l a b i l e manner which was susceptible to the action of phosphotyrosine phosphatases, c e l l s were labelled with 3 2P-orthophosphate as described above except that sodium orthovanadate, an in h i b i t o r of phosphotyrosine phosphatases, was added to the culture medium to a f i n a l concentration of 100 uM. Sodium orthovanadate (100 uM) was also included in the l y s i s buffer and a l l the wash buffers used during the immunoprecipitation of proteins from these c e l l s . 2.4 Immunoprecipitation Cleared c e l l lysates were incubated with either immune or non-immune serum for 45 min at which time 10 volumes, 57 r e l a t i v e to the volume of serum, of a 10% suspension of RARIG-coated Staph, aureus was added and incubated for an additional 45 min. The immune complexes were pelleted in a microfuge and washed successively with 1 M NaCl - 10 mM Tris-HCl(pH 8.0) - 0.1% NP-40; with 100 mM NaCl - 1 mM EDTA - 10 mM Tr is-HCl(pH 8.0) - 0.1% NP-40 - 0.1% SDS; and with 10 mM Tr is-HCl(pH 8.0) - 0.1% NP-40; a l l washes were performed at 4°C. Immune complexes were prepared for SDS-polyacrylamide gel electrophoresis as described in section 2.5. For TPA-kinetic studies the same immunoprecipitation procedure as above was followed except that the volumes of ( 3 BS)methionine-labelled lysates from a l l time points were adjusted so that equivalent amounts of t o t a l TCA-precipitable r a d i o a c t i v i t y were being precipitated. Volumes of serum and of RARIG-coated Staph, aureus were adjusted so that equimolar concentrations of each reagent were maintained for a l l immunoprecipitations. 2.5 Immune Complex Kinase Reaction Samples of 5 X 10"7 to I X 10 s c e l l s were harvested, washed, lysed in kinase l y s i s buffer (1.0% NP-40 - 20 mM Tr is-HCl(pH 7.5) - 150 mM NaCl - 1 mM EDTA -0.5% sodium deoxycholate), and lysates were cleared at 27,000 xg at 4°C for 30 min. Samples of cleared lysate were immunoprecipitated as described above and the r e s u l t i n g immune complexes were washed once in kinase l y s i s buffer and 58 twice in kinase reaction buffer (20 mM Tris-HCT(pH 7.5) - 10 mM MnCl 2), the immune complexes were then incubated with 2-20 uCi of (gamma-32P)ATP (3,000 Ci/mmol; 10 uCi/ul; Amersham Corp.) in 35 ul of kinase reaction buffer at 20°C for 15 min. Kinase reactions were terminated by the addition of 0.5 ml of kinase l y s i s buffer and the immune complexes were washed three times with kinase l y s i s buffer, on ice. Following t h i s , the immune complexes were suspended in 50 ul of SDS-electrophoresis sample buffer (10% glycerol - 5% 2-mercaptoethanol - 2.3% SDS - 0.0625 Tris ) and heated at 37°C for 10 min in order to disrupt the immune complexes. The RARIG-coated Staph, aureus was pelleted by centifuging at f u l l speed in a microfuge for 3 min, and the supernatants, containing the radiolabelled proteins, were prepared for immediate SDS-polyacrylamide gel electrophoresis or were stored overnight at -20°C. Phosphorylation of exogenous substrates was assayed by adding 5 ug of acetic acid-denatured rabbit muscle enolase (Cooper, et a l . , 1984) to the kinase reaction mixture, prior to the addition of (gamma-32P)ATP, and then incubating at 30°C for 15 min. The reaction was terminated by addition of an equal volume of 2X SDS-electrophoresis sample buffer to the reaction mixture followed by heating at 37°C for 10 min to disrupt the immune complex; supernatants were prepared as above and were subjected to electrophoresis or stored overnight at -20°C. 59 In order to compare In v i t r o kinase a c t i v i t y from one time point to the next in the TPA-kinetic studies, the protein content of each lysate was determined by a Coomassie blue-binding assay (Sedmak, et a l . , 1977) and the volume of individual lysates adjusted to ensure that equivalent amounts of t o t a l protein were subjected to immunoprecipitation; adjustments were made, where necessary, to maintain equimolar concentrations of the immune reagents. 2.6 SDS-Polyacrvlamide Gel Electrophoresis Radiolabelled proteins in SDS-elecrtrophoresis sample buffer were heated at 100°C for 3 min before being loaded onto the SDS-polyacrylamide gel system as o r i g i n a l l y described by Laemmli (1970). A 15 cm v e r t i c a l slab gel apparatus was used (Protean II; Bio-Rad Laboratories) and the stacking gel contained 4.5% polyacrylamide while the separating gel contained 7.5% polyacrylamide; in the case of V8 protease analysis a 12.5% separating gel was employed. The proteins were separated e l e c t r o p h o r e t i c a l l y at a constant power of 2 watts, which was increased to 3 watts once the tracking dye in the sample buffer had entered the separating g e l . A buffer system of 0.025 M T r i s - 0.192 M glycine - 0.1% SDS - pH 8.3 was u t i l i z e d and electrophoresis was continued u n t i l the tracking dye reached the bottom of the gel, at which time the current was stopped. After electrophoresis, gels containing ( 3 2 P ) - l a b e l l e d proteins were soaked overnight in 0.04% Coomassie b r i l l i a n t blue-R -60 7.5% acetic acid - 50% methanol in order to f i x proteins and sta i n molecular weight standards of known s i z e . Stained gels were destained for one to three hours in 7.5% acetic acid - 50% methanol and for 10 min in d i s t i l l e d water before being dried onto f i l t e r paper (3MM; Whatman) using a Hoefer slab gel dryer. Labelled proteins were detected according to the method of Laskey and M i l l s (1977) by exposing the dried gels to Kodak XAR-5 f i l m at -80°C in the presence of an i n t e n s i f y i n g screen (Lightning Plus; DuPont). Gels containing ( 3 = >S )methionine-labelled proteins were treated i d e n t i c a l l y except that following destaining gels were impregnated with fluor (En 3Hance; New England Nuclear Corp.) for one hour before drying and autoradiography. In the case of gels in which the 3 2 P - l a b e l l e d proteins were subjected to further analysis, for example t r y p t i c peptide mapping, the gels were removed from the electrophoresis apparatus, wrapped in Saran wrap and exposed wet to XAR-5 fi l m at 4°C in order to locate bands of interest; these bands were then excised from the wet gel . A l l films were developed using a Kodak Mil fi l m processor, and molecular weights of relevant proteins were calculated from a linear regression plot of the log of molecular weight versus the r e l a t i v e mobility for standard proteins of known molecular weight which were run on the same gel and were detected by Coomassie blue staining. In those immune complex kinase reactions which included enolase, the location of phosphorylated enolase in SDS-61 p o l y a e r y l a m i d e g e l s was c o n f i r m e d by the p a r a l l e l electrophoresis of unlabelled enolase and detection by staining with Coomassie blue. 2.7 Phosphoamino Acid Analysis Analysis of phosphoamino acid composition of in vivo or in v i t r o phosphorylated proteins or phosphopeptides was carried out e s s e n t i a l l y according to published methods (Beemon, et a l . , 1978; Cooper, et a l . f 1983b), with only minor modifications. 3 2 P - l a b e l l e d proteins were recovered by overlaying wet or dried gels with the corresponding autoradiogram and i d e n t i f i y i n g and excising the piece of gel containing the protein of interest. The proteins were eluted from the gel s l i c e s overnight in 5 ml of PAA elution buffer (50 mM NFUHCOa - 0.1% SDS - 5% 2-mercaptoethanol) at 37°C. The gel fragments were recovered by centrifugation at 500 xg for 10 min and a second elution was performed on these, u t i l i z i n g similar conditions, for 4 hr. The two eluates were combined and centrifuged at 27,000 xg for 10 min in order to remove any residual gel fragments. To the combined eluates was added 25-30 ug of bovine gamma-globulin, as a c a r r i e r , and proteins were precipitated overnight at 4°C by the addition of 100% TCA to a f i n a l concentration of 20%. The precipitated proteins were pelleted by centrifugation at 25,000 xg for 20 min at 4°C and washed twice with 95% ethanol prior to drying under vacuum. The dried p e l l e t s were resuspended in 0.2 ml of 6 N 6 2 HC1 and b o i l e d f o r 2 min b e f o r e b e i n g s e a l e d i n a g l a s s t u b e and h y d r o l y z e d a t 11 0 ° C f o r 90 min; t h e h y d r o l y z e d p r o t e i n s were t r a n s f e r r e d t o m i c r o f u g e t u b e s and t h e l i q u i d was removed by l y o p h i l i z a t i o n . The d r i e d p e l l e t s were r e s u s p e n d e d i n 3.0 u l of pH 1.9 b u f f e r ( 8 8 % f o r m i c a c i d -a c e t i c a c i d - w a t e r ; 50:156:1794 by volume) and t h e n mixed w i t h 3.0 u l o f a s o l u t i o n o f n o n - r a d i o a c t i v e a u t h e n t i c phosphoamino a c i d s ( p h o s p h o s e r i n e , p h o s p h o t h r e o n i n e , and p h o s p h o t y r o s i n e ; a l l a t 0.1 mg/ml) p r i o r t o l o a d i n g o n t o a 20 cm X 20 cm t h i n - l a y e r c e l l u l o s e (TLC) p l a t e (0.1 mm; E. Merck L a b ) . F o l l o w i n g sample a p p l i c a t i o n t h e p l a t e was dampened w i t h pH 1.9 b u f f e r and s u b j e c t e d t o e l e c t r o p h o r e s i s a t 1,000 v o l t s f o r 180 min t o w a r d s t h e anode. The p l a t e was t h e n d r i e d , r e - w e t t e d w i t h pH 3.5 b u f f e r ( p y r i d i n e - a c e t i c a c i d - w a t e r ; 10:100:1890 by v o l u m e ) , and e l e c t r o p h o r e s e d t o w a r d s t h e anode a t 1,000 v o l t s f o r 80 min i n a d i r e c t i o n w h i c h was p e r p e n d i c u l a r t o t h e o r i g i n a l e l e c t r o p h o r e s i s . S u b s e q u e n t l y , t h e p l a t e was d r i e d and e x p o s e d t o f i l m , w i t h an i n t e n s i f y i n g s c r e e n , a t -70°C; f o l l o w i n g a u t o r a d i o g r a p h y t h e n o n - r a d i o a c t i v e phosphoamino a c i d s were l o c a t e d by s p r a y i n g t h e p l a t e w i t h a n i n h y d r i n s t a i n (0.1 gra n i n h y d r i n - 70 ml e t h a n o l - 21 ml a c e t i c a c i d - 2.9 ml 2, 4, 6-c o l l i d i n e ) and d e v e l o p i n g t h e c o l o u r by h e a t i n g g e n t l y o v e r a h o t p l a t e f o r 10 min. The p r o c e d u r e f o l l o w e d i n o r d e r t o d e t e r m i n e phosphoamino a c i d c o m p o s i t i o n o f t r y p t i c p h o s p h o p e p t i d e s was s i m i l a r e x c e p t t h a t a r e a s o f t h e TLC p l a t e s c o n t a i n i n g 63 phosphopeptides were scraped o f f into microfuge tubes and the peptides eluted by incubation overnight at 37°C in 0.5 ml pH 2.1 buffer. The c e l l u l o s e fragments were removed by c e n t r i f u g a t i o n and the eluted phosphopeptides were dried by l y o p h i l i z a t i o n before being subjected to hydrolysis and electrophoresis as described above. 2.8 Tryptic p e p t i d e A n a l y s i s Labelled proteins were i d e n t i f i e d and extracted from gels, precipitated with TCA, washed, and dried exactly as described above for phosphoamino acid analysis. Proteins were dissolved in 100 ul formic acid and to t h i s was added 25 ul methanol and 40 ul performic acid (prepared fresh by combining 0.9 ml formic acid with 0.1 ml 30% hydrogen peroxide for 1 hr at 20°C). The dissolved proteins were incubated at -5°C for 2 hr following which 3.0 ml of water was added, the solution quick frozen in a dry ice-ethanol bath, and l y o p h i l i z e d to dryness. The oxidized proteins were dissolved in 0.5 ml of 50 mM NH 4C0 3 and digested with 5 ug of L-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin (TPCK-trypsin; Worthington) for 6 hr at 37°C. Following digestion 2.5 ml water was added, the solution quick frozen, and l y o p h i l i z e d ; the l y o p h i l i z a t i o n was repeated once and the peptides were then dissolved in 1.0 ml of pH 2.1 electrophoresis buffer (water - formic acid - acetic acid; 90:2:8 by volume), transferred to a 1.5 ml microfuge tube, quick frozen and l y o p h i l i z e d to dryness. 64 The l y o p h i l i z e d sample was resuspended in 5 ul of pH 2.1 buffer and applied to a 20 cm X 20 cm TLC plate, the plate was dampened with pH 2.1 buffer and electrophoresed towards the cathode for 1 hr at 1,000 v o l t s . After electrophoresis the plate was a i r dried and subjected to ascending chromatography in N-butanol - acetic acid - water - pyridine (75:15:60:50, by volume) in a d i r e c t i o n perpendicular to that of electrophoresis. 3 2 P - l a b e l l e d phosphopeptides were detected by exposing the TLC plate to XAR-5 fil m in the presence of an in t e n s i f y i n g screen at -80°C. In some cases, in order to investigate relatedness of proteins, two d i f f e r e n t proteins were isolated and digested with trypsin separately before being applied together to the same TLC plate; conditions were otherwise i d e n t i c a l with those used for a single protein. 2.9 P a r t i a l P r o t e o l y t i c Cleavage with V8 Protease Limited proteolysis with V8 protease was performed e s s e n t i a l l y as described previously (Cleveland, et a l . , 1977); proteins labelled in in v i t r o kinase reactions were separated by electrophoresis and the relevent proteins were i d e n t i f i e d by autoradiography and excised from the gels. The gel s l i c e s containing the labelled proteins were soaked in V8 protease buffer (0.125 M Tris-HCl(pH 6.8) - 1 mM EDTA - 0.1% SDS) for 30 min at 20°C and were placed in the bottoms of wells of a fresh SDS-polyacrylamide gel system with a 5 cm-long stacking gel and a 12.5% polyacrylamide 65 separating g e l . The samples were overlaid with the same v 8 buffer but containing 20% g l y c e r o l ; the usual SDS-electrophoresis buffer was then added to the apparatus. To each well was added 20 u l of a solution consisting of: 10% g l y c e r o l , 0.0001% bromphenol blue, and various concentrations of Staphylococcal aureus V8 protease, dissolved in V8 protease buffer. Electrophoresis was performed at a constant power of 2W u n t i l the bromphenol blue tracking dye was 0.5 cm from the separating g e l , at which point the power and cooling system were turned off for 30 min; electrophoresis was then resumed at 3W u n t i l the dye front had reached the end of the separating g e l . Gels were fixed, stained, dried and exposed to f i l m exactly as described in section 2.6. 2.10 Isolation and Analysis of Total C e l l u l a r RNA Total c e l l u l a r RNA was isolated according to published methods (Cathala, et a l . . 1983). B r i e f l y , 1 X 10 s c e l l s were pelleted in a benchtop centrifuge for 3 min at 800 xg in a 50 ml p l a s t i c tubes, washed once with 10 ml of PBS at 4°C, and re-pelleted for 3 min at 800 xg in pre-weighed 30 ml Corex tubes. C e l l cultures which had been induced with TPA and which showed any c e l l adherence were scraped with a c e l l scraper prior to harvesting and washing the c e l l s . The c e l l p e l l e t was weighed after the second centrifugation and 7.0 ml/gm(of wet c e l l s ) of guanidine l y s i s buffer (5 M guanidine monothiocyanate - 10 mM EDTA - 50 mM Tris-HCKpH 66 7.5) - 8%(v/v) 2-mercaptoethanol) was added to each tube and the c e l l s were homogenized by vortexing three times for 10 seconds each. The c e l l lysate was transferred onto ice and t o t a l c e l l u l a r RNA was precipitated by the addition of 7 volumes of 4 M L i C l per volume of homogenate, and incubated 15-20 hr at 4°C. The RNA's were pelleted at 11,000 xg for 60 min at 4°C, and the supernatants, containing mainly DNA and denatured proteins, were discarded. To remove DNA and protein contaminants the p e l l e t was resuspended in 3 M L i C l (volume i d e n t i c a l with that of 4 M L i C l ) ; the p e l l e t was disrupted by forcing i t successively through 18, 22, and 25 gauge needles using a s t e r i l e syringe. The RNA's were re-pelleted at 11,000 xg for 60 min at 4°C, the supernatant discarded, and the p e l l e t dissolved in RNA s o l u b i l i z a t i o n buffer (10 mM Tris-HCl(pH 7.5) - 1 mM EDTA - 0.1% SDS). The s o l u b i l i z e d RNA's were extracted according to accepted methods (Maniatis, et a l . , 1982) with phenol, phenol:chloroform, and chloroform. After the f i n a l chloroform extraction the RNA's were precipitated from the aqueous phase by adding 0.05 volumes of 3 M sodium acetate (pH 5.1) and 2 volumes of 95% ethanol and incubating 12 hr at -20°C. The RNA's were pelleted by centrifugation at 12,000 xg for 10 min at 4°C, washed once with 10 ml of 70% ethanol, re-pelleted at 12,000 xg for 10 min and dried under vacuum. RNA p e l l e t s were dissolved in 100 ul of water and the concentration of RNA was determined 67 spectrophotometrically by measuring the ODaeo of duplicate 1:100 d i l u t i o n s of RNA. Total RNA was fractionated according to the method of Lehrach et a l . (1977) as follows: a 0.8-1.0% agarose-formaldehyde gel was poured into a 9.5 X 14.5 cm horizontal electrophoresis tray (Dedicated Design Line; Hoefer S c i e n t i f i c Instruments), a 15 tooth comb was inserted, and the gel was allowed to set at room temperature. RNA samples were prepared for loading as follows: a volume of RNA solution calculated to contain 10 ug of t o t a l c e l l u l a r RNA was placed in a 1.5 ml microfuge tube and to i t was added 12.0 ul deionized formamide, 4.0 ul 37% formalin, 5.0 ul 5X gel running buffer (0.2 M morpholinopropanesulfonic acid -50 mM sodium acetate - 5 mM EDTA), and a volume of water s u f f i c i e n t to bring the t o t a l volume to 25 u l . After mixing, these samples were incubated at 70°C for 10 min and then 2.5 ul of RNA loading buffer (50% glycerol - 1 mM EDTA - 0.4% bromphenol blue - 0.4% xylene cyanol) was added to each tube and mixed; a 3 ug aliquot of authentic RNA molecular size standards (RNA ladder; Bethesda Research Laboratories) was prepared in exactly the same manner as the RNA samples. The agarose gel was submerged in 1,100 ml of IX gel running buffer, the comb removed, RNA samples and size standards loaded into wells, and subjected to electrophoresis at 70 volts for 3 hr. The gel was stained in 500 ml of acridine orange (15 ug/ml in water) for 30 min and was then destained twice in 500 ml of water for a t o t a l 68 of 60 min; the stained gel was exposed to u l t r a v i o l e t l i g h t in order to measure the locations of the size standards and to ascertain that their was no degradation of the 28s or 18s rRNA. Any gel showing evidence of degradation of either 28s or 18s rRNA was assumed to have been subject to the action of ribonucleases and was discarded. RNA was blot-transferred from the gel to a nylon membrane (HYBOND-N; Amersham Corp.) according to the manufacturer's instructions, which are e s s e n t i a l l y modifications of commonly employed protocols (Southern, 1975; Thomas, 1980). Following blot transfer for 12-16 hr the membrane was removed, rinsed in 2X SSC, wrapped in Saran wrap and exposed to u l t r a v i o l e t l i g h t for 5 min in order to bind the RNA to the membrane. The membrane was placed in a heat-sealable bag and pre-hybridization f l u i d (0.6 M NaCl - 0.18 M Na 2HP0 4 - 3 mM EDTA - 1% N-lauroyl sarcosine - 2.5% dextran sulfate - 100 ug/ml heat-denatured herring testes DNA) was added at a r a t i o of 0.1 ml/cm2 area of nylon membrane. Air bubbles were forced out, the bag sealed, and incubated at 65°C for 5 hr. After pre-hybridization the bag was cut open and the f l u i d emptied out; t h i s was replaced by hybridization f l u i d at 0.05 ml/cm2 area of membrane, the bag was re-sealed, and the membrane allowed to hybridize at 65°C for 16-20 hr; the hybridization f l u i d had exactly the same composition as the pre-hybridization mix except that i t contained the labelled 69 probe, described in section 2.12, at a concentration o£ 0.5-1.0 X 10"7 CPM/ ml of f l u i d . After hybridization the membrane was removed from the bag and washed twice in IX SSC (3 M NaCl - 0.3 M trisodium citrate;pH 7.0) - 0.1% SDS - 0.1% sodium pyrophosphate at 20°C for 15 min each and once in 0.1X SSC - 0.1% SDS - 0.1% sodium pyrophosphate at 65°C for 30 min in a shaking water bath. Following the f i n a l wash the membrane was a i r - d r i e d u n t i l damp, wrapped in Saran wrap, and exposed to XAR-5 fi l m in the presence of an in t e n s i f y i n g screen at -70°C for 1-4 days . 2.11 Morphologic Markers of C e l l u l a r D i f f e r e n t i a t i o n Morphology of TPA-exposed or control c e l l s growing in culture was recorded with a Wild inverted microscope with photographic attachment. Growth and v i a b i l i t y counts were done by preparing d i l u t i o n s of c e l l s , mixing with an equal volume of 0.5% eosin-Y in PBS, and counting viable and non-viable c e l l s in a hemocytometer chamber on an inverted microscope. Cytocentrifuge-slide preparations of unstained c e l l s were prepared from TPA-exposed and control cultures at appropriate intervals using a Cytospin I I apparatus (Shandon Instruments). Slides were stained with Wright-Giemsa stai n using an automated processor (Coulter Instruments) or were stained for (alpha)-naphthyl acetate esterase or for chloracetate esterase according to previously published procedures (Yam, et a l . , 1970). Stained s l i d e s were 70 evaluated for d i f f e r e n t i a t i o n with a Zeiss Photomicroscope II, and photographed with ASA 400 f i l m on the same microscope. 2.12 Preparation of R a d i o l a b e l e d Nucleic Acid Probes The DNA used i n i t i a l l y to probe membrane-bound RNA was a 3.5 kbp Bglll-Xhol fragment of Gardner-Arnstein FeSV provirus (Even, et a l . f 1983) which had been cloned into pGem-1 and was obtained from Dr. Peter Greer; use of t h i s probe was discontinued due to high levels of binding not only to c-fes mRNA but also to 28s and 18s rRNA. Subsequently a human cDNA probe, also obtained from Dr. Peter Greer, was used in hybridization; t h i s probe consisted of a 1.8 kbp EcoRI fragment, encoding exon 8 to the 3' end of the human c-fes gene (Roebroek, et a l . , 1985), which had been cloned into the EcoRI s i t e of pUC18. The a c t i n probe was a 1.3 kb PstI fragment of bovine (beta)-actin cDNA cloned into the PstI s i t e of pBR322(Degen, et a l . r 1983) . The preparation of the above fragments from the o r i g i n a l plasmid DNA was similar in each case and w i l l be outlined only b r i e f l y . Competent E. c o l l were transformed with p u r i f i e d plasmid DNA according to standard procedures (Maniatis, §_t a l . , 1982) and grown on LB plates containing an appropriate a n t i b i o t i c . Resistant colonies were i d e n t i f i e d and grown up overnight in 4-5 ml LB broth tubes supplemented with a n t i b i o t i c s , and plasmid DNA was isolated from the b a c t e r i a l 71 p e l l e t using a modified version (Wilimzig, et a l , , 1985) of the b o i l i n g method for small scale extraction of plasmid DNA (Holmes, et a l . , 1981). Following i s o l a t i o n of the respective plasmids the presence of the appropriately sized insert was confirmed by r e s t r i c t i o n enzyme analysis using standard procedures; an estimate of the concentration of insert, and hence the plasmid, was made by comparing i t with DNA fragments of known size and concentration using an ethidium bromide spotting plate. After determination of plasmid concentration 1 ug of plasmid DNA was digested to completion with the appropriated r e s t r i c t i o n enzyme and the fragments were separated e l e c t r o p h o r e t i c a l l y on a 1% low melting-point agarose g e l . The band representing insert DNA was i d e n t i f i e d in each case and the band excised from the gel and stored at -20°C u n t i l use. Insert DNA (16-22 ng) was labelled to high s p e c i f i c a c t i v i t y (1-3 X 10 9 CPM/ug) using o l i g o - l a b e l l i n g by hexanucleotide primers, exactly as described (Feinberg, et a l . , 1984), in an overnight reaction. The radiolabelled probe was p u r i f i e d from unincorporated nucleotides over an ion-exchange mini-column (NACS Prepac; Bethesda Research Laboratories) e n t i r e l y according to the manufacturer's s p e c i f i c a t i o n s . Aliquots of the unpurified reaction mix and of the column-purified probe were assayed by l i q u i d s c i n t i l l a t i o n counting in order to calculate s p e c i f i c a c t i v i t y and y i e l d for every l a b e l l i n g reaction. P u r i f i e d probe DNA was held at 4°C u n t i l needed at which time i t was denatured by heating at 95°C for 10 min 72 followed by rapid cooling and addition to the calculated volume of hybridization f l u i d . 73 CHAPTER 3 3.0 I d e n t i f i c a t i o n and Characterization of Murine and Human  c-fes Proteins. 3.1 Introduction c-fps/fes proteins from a vari e t y of species are highly conserved, based upon nucleotide sequence analysis, immunological c r o s s - r e a c t i v i t y , and functional comparison (Barbacid, et a l . , 1980; Hampe, et a l . , 1982; Mathey-Prevot, et a l . f 1982; Huang, e t _ a l . , 1985). FSV-transformed or ST-FeSV-transformed rat fi b r o b l a s t s have been used in syngeneic animals to induce the production of anti-tumor antibodies or have been employed to immunize heterologous species; in either case, antisera are produced which are able to s p e c i f i c a l l y recognize v i r a l and c e l l u l a r fps/fes proteins (Barbacid, et a l . , 1980; Ingman-Baker, et a l . r 1984). I n i t i a l l y , v-fps or v-fes proteins possessing protein-tyrosine kinase a c t i v i t i e s s imilar to that of ppSC^--" were i d e n t i f i e d (Barbacid, et a l • , 1980; Feldman, et a l . , 1980); subsequently, immunologically-related normal c e l l u l a r proteins were shown to have similar protein-tyrosine kinase a c t i v i t y and these c e l l u l a r proteins were demonstrated to have sequences similar to v i r a l transforming proteins by pro t e o l y t i c mapping and eventually by nucleotide sequence analysis (Mathey-Prevot, et a l . , 1982; Huang, §£ a l . , 1985; Roebroek, et a l . , 1985). Despite the i d e n t i f i c a t i o n of c-fes proteins in a number of divergent 74 species, these proteins were not detected in human or murine c e l l s (Barbacid, et a l . . 1980); however, th i s study was limited in the number of c e l l l i n e s which were examined for the presence of c-fes proteins, and t h i s suggested that i t might be possible to i d e n t i f y murine and human c-fes proteins by searching a more extensive array of c e l l s , tissues, and c e l l l i n e s for expression. Also, none of the murine or human c e l l lines which were tested were of hemopoietic o r i g i n ; studies of the avian c-fps protein, NCP98, showed that i t is p r e f e r e n t i a l l y expressed in granulocytes and macrophages (Samarut, et a l . , 1985), further suggesting that the previous i n a b i l i t y to detect homologous c-fes proteins might be circumvented by s c r u t i n i z i n g hemopoietic c e l l s of various lineages. 3.2 Results 3.2.1 Characterization of Rat antl-fps Sera In the course of producing monoclonal antibodies directed against v-fps proteins, a number of sera were collected from Fischer or Wistar rats in which sarcomas had been induced by the i n j e c t i o n of FSV-transformed f i b r o b l a s t s , as described previously (Ingman-Baker, et a l . , 1984). These tumor-bearing rat sera had been shown to possess r e a c t i v i t y towards the v-fps. protein pi40 , a* , l , a- c , :" a in ln v i t r o kinase reactions and one serum, designated J6, demonstrated pa r t i c u l a r a b i l i t y to immunoprecipitate Pl40 <3« <a-*E" E (T. Pawson, personal communication). In order 75 to determine i£ this serum might be suitable for lmmunopreclpltation of c-fes proteins, i t was evaluated for i t s a b i l i t y to precipitate v-f ps (pi40' 3 a" 3~ f i E" B) and v-f es (P85««a-*-«») proteins in in v i t r o kinase reactions. J6 serum s p e c i f i c a l l y immunoprecipitated an 85kD protein from ST-FeSV-transformed NIH 3T3 fib r o b l a s t s which was phosphorylated in v i t r o (Figure 3.1, lane 2); comparison with the equivalent protein recognized by caprine antl-v-fes serum (ST*.uT; Barbacid, et a l . , 1980) suggested that this protein was pas^"" 3 - *"" (Figure 3.1, lane 3). The a b i l i t y of J6 serum to recognize pi40<aJ»<s»-*E'" was confirmed by in v i t r o kinase a c t i v i t y of immunoprecipitates prepared from lysates of FSV-transformed Rat 2 fibr o b l a s t s (Figure 3.1, lane 6). Interestingly, the STAUT serum was unable to recognize P14Q<a<*<3-in t h i s assay system, implying that i t was less broadly cross-reactive with fps/fes proteins than J6 (Figure 3.1, lane 4), and that another explanation for the previously documented i n a b i l i t y to detect human and murine c-fes proteins (Barbacid, et a l . , 1980) could be that the proteins were not recognized by the STAUT serum, rather than not expressed by the c e l l s . Enolase is a g l y c o l y t i c enzyme which is known to be phosphorylated both in vivo and in v i t r o by a number of v i r a l protein-tyrosine kinases, including p g s o - x a - * - -(Cooper, et a l . , 1983a; Cooper, et a l . , 1984).' Enolase is frequently included in in v i t r o kinase assays in order to assess phosphorylation of exogenous substrates by protein-76 Figure 3.1: I d e n t i f i c a t i o n of v-fes and v-fps proteins by rat anti-fps serum. To assay for in v i t r o kinase a c t i v i t y , ST-FeSV-transformed NIH 3T3 c e l l s (lanes 1-3) or FSV-transformed Rat II f i b r o b l a s t s were lysed and immunoprecipitated with J6 anti-fps serum (lanes 2 and 6), or S T . u t anti-fes serum (lanes 3 and 4), or non-immune rat serum (lanes 1, 5, and 7). Immune complexes were incubated with (gamma-32P)ATP and in v i t r o -phosphorylated proteins were i d e n t i f i e d by electrophoretic separation and autoradiography. The locations and sizes (in kD) of molecular weight standards are indicated to the r i g h t . 7 7 tyrosine kinases and has been shown to be phosphorylated exclusively at tyrosine in v i t r o (cooper, et a l . f 1984). J6 serum was tested for i t s a b i l i t y to s p e c i f i c a l l y p r e c i p i t a t e P 8 5 " 3 = > < 3 - £ « ~ i n a n jn v i t r o kinase reaction which included denatured rabbit muscle enolase, and thi s resulted in the autophosphorylation of P85'=r^,3-£*"" and the phosphorylation of enolase (Figure 3.2, B); phosphoamino acid analysis indicated that enolase was phosphorylated exclusively at tyrosine (Figure 3.2, C) . This res u l t suggests that P65'sm'3-*•» possesses i n t r i n s i c tyrosine kinase a c t i v i t y : while a co-pre c i p i t a t i n g kinase could conceivably r e s u l t in the "autophosphorylation" of p85«»«-*« i t i s more d i f f i c u l t to invoke t h i s same explanation to account for the phosphorylation of enolase since the amino acids surrounding the s i t e of tyrosine phosphorylation in enolase are much d i f f e r e n t from those in the v-fps/fes transforming proteins (Cooper, et a l . , 1984). Chicken myeloblasts transformed by the E26 virus produce high levels of NCP98, the normal avian c-fps product; J6 serum is also able to precipitate NCP98, which i s autophosphorylated in an immune complex kinase assay (Figure 3.2, A). Thus, the tumor-bearing rat serum employed in t h i s study was able to immunoprecipitate a number of v i r a l and c e l l u l a r fps/fes proteins of the expected molecular weights from c e l l l i n e s known to express such proteins, and each had an associated protein-tyrosine kinase a c t i v i t y . It appeared that t h i s serum recognized a wider spectrum of c-fps/fes 78 products than the caprine antiserum and thi s reagent was therefore used in an attempt to i d e n t i f y cross-reacting human and murine c-fes proteins. 3.2.2 Examination of Human and Murine Hemopoietic Ce l l s for  Expression of c-fes Proteins A previous investigation f a i l e d to detect cross-reactive c-fes proteins in murine and human c e l l s but thi s study did not survey any hemopoietic c e l l s ; in chickens hemopoietic c e l l s are known to express high levels of NCP98, and thus I examined fresh murine and human hemopoietic c e l l s to determine i f an equivalent a c t i v i t y i s present. Fresh mouse spleen c e l l s and bone marrow c e l l s were obtained from DBA/2 mice, and human peripheral blood leukocytes and bone marrow c e l l s were obtained from healthy donors; in addition, a murine multipotential hemopoietic progenitor c e l l l i n e , B6SUtA, was also examined (Greenberger, et a l . , 1983). Mouse bone marrow and spleen c e l l s were coll e c t e d and washed prior to preparation of c e l l lysates and hence represent heterogenous populations of hemopoietic c e l l s . The human peripheral blood samples were collected over Ficoll-Hypaque and were consequently enriched for lymphocytes, monocytes, and th e i r precursors; the human bone marrow samples were also c o l l e c t e d over Ficoll-Hypaque but consisted mainly of erythroid and myeloid precursor c e l l s , with few lymphocytes or monocytes. C e l l p e l l e t s were prepared, lysed, and proteins immunoprecipated for use in In v i t r o immune complex 79 A B 1 2 3 4 Figure 3.2: I d e n t i f i c a t i o n of c-fps and v-fes proteins by rat anti-fps serum. E26-transformed avian myeloblasts (lanes 1 and 2) or ST-FeSV-transformed NIH 3T3 c e l l s (lanes 3 and 4 ) were lysed and immunoprecipitated with J6 anti-fps serum (lanes 2 and 4 ) or non-immune serum (lanes 1 and 3). Immune complexes were incubated with (gamma-32P)ATP and, in the case of panel B, with 5 ug of acid-denatured rabbit muscle enolase; In v i t r o phosphorylated proteins were separated by electrophoresis and i d e n t i f i e d by autoradiography. The locations and sizes (in kD) of molecular weight standards for panel A are indicated to the l e f t . The location of enolase was confirmed by electrophoresis of unlabelled enolase and detection by Coomassie blue staining. The 85 kD protein i d e n t i f i e d in panel B was isolated from the gel and subjected to acid hydrolysis, followed by two-dimensional electrophoretic separation of phosphoamino acids, as shown in panel C. Authentic phosphoserine (P-S), phosphothreonine (P-T), and phosphotyrosine (P-Y) were located by staining with ninhydrin and are indicated. 80 kinase assays. A 92kD protein (p92) present in lysates of both human peripheral leukocytes and bone marrow c e l l s was s p e c i f i c a l l y phosphorylated in an ln v i t r o kinase reaction (Figure 3.3, lanes 1 and 3); a protein of similar size was also present and was phosphorylated in lysates prepared from mouse bone marrow c e l l s (Figure 3.3, lane 5). Unfractionated spleen c e l l s consist mainly of lymphocytes and erythrocytes, and to a lesser degree of monocyte/macrophages; analysis of mouse spleen c e l l s reveals a 92kD phosphoprotein which co-migrates with p92 found in bone marrow c e l l s (Figure 3.3, lane 7). However, lysates of t o t a l spleen c e l l s also contain a 94kD protein (p94) which is recognized by the anti-fps serum and which is also phosphorylated in v i t r o (Figure 3.3, lane 7). Examination of c e l l s from the multipotential hemopoietic progenitor c e l l l i n e B6SUtA indicated that they too expressed p92 (Figure 3.4, lane 3); t h i s c e l l l i n e , while immortalized, displays many of the features of normal bone marrow progenitors. In no case did the non-immune serum precipitate any unique protein: p92 and p94 were s p e c i f i c a l l y immunoprecipitated by the anti-fps tumor serum. Fresh human peripheral leukocyte preparations occasionally demonstrated the presence of p94 but t h i s was not a consistent observation and seemed to correlate with the presence of erythrocytes in the c e l l p e l l e t . On the basis of immunological c r o s s - r e a c t i v i t y with the v i r a l proteins pi40«-«-e->- and pss-5"""3-*""0, and the c-f ps Figure 3.3: I d e n t i f i c a t i o n of p92 and p94 in human and murine hemopoietic c e l l s . Lysates of normal human peripheral blood leukocytes (lanes 1 and 2), normal human bone marrow c e l l s (lanes 3 and 4), normal mouse bone marrow c e l l s (lanes 5 and 6), and normal mouse spleen c e l l s (lanes 7 and 8) were immunoprecipitated with J6 antl - f j a s . serum (lanes 1, 3, 5, and 7) or non-immune serum (lanes 2, 4, 6, and 8). The immune complexes were incubated with (gamma-32P)ATP and the In  vitro-phosphorylated proteins were separated by electrophoresis and detected by autoradiography. The locations and sizes (in kD) of molecular weight standards are indicated to the l e f t . 82 MOUSE B O N E M A R R O W B6SUtA 1 2 3 4 F i g u r e 3.4: I d e n t i f i c a t i o n o f p92 i n a murine m u l t i p o t e n t i a l h e m o p o i e t i c p r o g e n i t o r c e l l l i n e . Bone marrow c e l l s f r o m DBA mouse femurs ( l a n e s 1 and 2) or B6SUtA c e l l s were l y s e d and i m m u n o p r e c i p i t a t e d w i t h J6 a n t i - f P S serum ( l a n e s 1 and 3) or non-immune serum ( l a n e s 2 and 4 ) . Immune com p l e x e s Were i n c u b a t e d w i t h (gamma- 3 2P)ATP and t h e i n v i t r o p h o s p h o r y l a t e d p r o t e i n s were s e p a r a t e d by e l e c t r o p h o r e s i s and i d e n t i f i e d by a u t o r a d i o g r a p h y . 83 protein NCP98, i t seems l i k e l y that the proteins i d e n t i f i e d in c e l l lysates of human and murine hemopoietic c e l l s represent c-fes proteins. This assertion i s supported by the finding that the molecular weights of these two proteins, 92kD and 94kD, are similar to the molecular weights of f e l i n e NCP92 and of avian NCP98 (Barbacid, ejfc. a l . , 1980; Mathey-Prevot, et a l . F 1982). Also, a protein-kinase a c t i v i t y i s associated with both p92 and p94, and i t is not inconceivable that t h i s a c t i v i t y is due to autophosphorylation at tyrosine residues, a property associated with authentic fes proteins. Either or both of p92 or p94 may be c-fes proteins: the differences in molecular weight can be accounted for by a number of potential mechanisms including post-translational protein processing, glycosylation, or production of d i f f e r e n t i a l l y s pliced t r a n s c r i p t s . 3.2.3 Examination of Human and Murine C e l l l i nes for  Expression of c-fes Proteins R e l a t i v e l y large amounts of protein are required for more extensive characterization and in order to have a r e a d i l y available supply of c e l l s expressing c-fes proteins a number of mouse and human c e l l lines were screened for expression of p92 and p94 using immune complex kinase assays. The c e l l l i n e s examined ref l e c t e d a variety of d i f f e r e n t tissue sources but were predominantly of hemopoietic o r i g i n , as these appeared to hold the most 84 promise of expressing detectable levels of p92 or p94. A l l of the hemopoietic c e l l lines screened expressed either p92, p94, or co-expressed both these proteins (Table 3.1); a d d i t i o n a l l y , several non-hemopoietic c e l l l i n e s expressed p94, but p92 was not observed in any of them. Representative autoradiographs are shown in Figure 3.5, demonstrating the presence of p92 or p94 or the p92/p94 doublet in individual c e l l l i n e s ; also shown are the results of an immune complex kinase assay of HL-60 c e l l s u t i l i z i n g a d i f f e r e n t anti-fps serum (Figure 3.5, lanes 9 and 10). This antiserum, F l - 1 , was also obtained from tumor-bearing rats but consistently showed lower a f f i n i t y for both p92 and p94 and thus was not routinely employed. Most of the c e l l s expressing p92 were derived from myeloid leukemias, although two B-lymphoma c e l l l i n e s and an erythroleukemia c e l l l i n e also expressed p92 (Table 3.1), consistent with published res u l t s which indicate that c-fes transcripts are present in most hematologic malignancies, e s p e c i a l l y AML and CML, but in few other tumors (Slamon, et a l . . 1984). This study also found low levels of c-fes transcripts in four out of four lung tumors examined, but the one lung carcinoma c e l l l i n e tested here for the presence of p92/p94 was negative. Two other groups also reported that c-fes transcripts were detectable only in normal myeloid c e l l s , AML, and CML, with the exception that c-fes mRNA could be detected in megakaryocytes and in immature erythroid precursors ( F e r r a r i , et a l . f 1985; Emilia, et a l . r 1986). The presence 85 TABLE 3.1 Expression of p92 and p94 in v i t r o Kinase A c t i v i t y in Human  and Mouse C e l l Lines, C e l l s , and Normal Mouse Tissues. Species C e l l type or tissue Expression 3-p92 p9 4 Human HL-60(promyelocytic leukemia) + KG-1(myeloblastic leukemia) + KG-la(KG-l variant c e l l line) K562(erythroleukemia) - + HEL(erythroleukemia) + + MOLT-4(T-cell leukemia) - + SU-DHL-4(B-cell lymphoma) + + WAY-l(B-lymphocyte) + + U937(histiocytic lymphoma) + NCI-H82(small-cell lung cancer) - + Peripheral leukocytes(normal) + + Peripheral leukocytes(AML) + + Bone marrow cells(normal) + Mouse B6SUtA(progenitor c e l l ) + WEHI-3B(myeloblast leukemia) + + P815(mastocytoma) + P388AD(macrophage-like) + EL-4(thymoma) - + MEL(erythroleukemia) - + NS1(myeloma) - + P19, 01A1(embryonal carcinoma) - + NIH 3T3(fibroblast) + + YKadrenal cortex) - + bone marrow + spleen(normal) + + spleen(early erythroblasts) - + heart l i v e r kidneys 1 The presence or absence of detectable p92 or p94, as determined by in v i t r o kinase assays, i s Indicated by a "+" or "-" respectively. 86 7 8 9 10 11 12 13 14 Figure 3.5: I d e n t i f i c a t i o n of p92 and p94 in human and murine hemopoietic c e l l l i n e s . C e l l s from MEL (lanes 1 and 2), HEL (lanes 3 and 4), K562 (lanes 5 and 6), HL-60 (lanes 7, 8, 9, and 10), MOLT-4 (lanes 11 and 12), or KG-1 (lanes 13 and 14) c e l l lines were lysed and immunoprecipitated with J6 anti-fps serum (lanes 2, 4, 6, 7, 11, and 13), Fl-1 anti-fps serum (lane 9), or non-immune serum (lanes 1, 3, 5, 8, 10, 12, and 14). Immune complexes were incubated with (gamma-32P)ATP and ln v i t r o phosphorylated proteins were i d e n t i f i e d by electrophoretic separation and autoradiography. 87 of p94 did not correlate with any pa r t i c u l a r c e l l type or lineage, and i f p92 and p94 are linked in some precursor/product relationship i t i s curious that more of •the c e l l l i n e s did not display both p92 and p94 a c t i v i t y . The human c e l l l i n e HL-60 was chosen as a source of p92, K562 as a source of p94, and HEL as a source of p92/p94 (Figure 3.5). These c e l l l i n e s were chosen for further characterization for a variety of reasons, including a v a i l a b i l i t y of the c e l l lines and ease of culture, r e l a t i v e l y high levels of expression of their respective proteins, and r e l a t i v e l y low levels of non-specific p r e c i p i t a t i o n of unrelated c e l l u l a r proteins from lysates of these c e l l s . Also, c e l l l i n e s , while not necessarily c l o n a l , represent far more homogeneous populations of c e l l s than could have been arrived at by using fresh tissues. 3.2.4 Characterization of Human p92 and p94 The sine qua non of protein-tyrosine kinases, whether encoded by v i r a l or by c e l l u l a r genes, i s their a b i l i t y to phosphorylate substrates at tyrosine residues; the majority of v-onc and c-onc protein-tyrosine kinases have now been shown to autophosphorylate at one or more tyrosine residues within the enzyme i t s e l f . p92 and p94 would be expected to contain phosphotyrosine i f the incorporation of label seen in the immune complex kinase assay is due to an analogous autophosphorylating a c t i v i t y ; accordingly, p92 and p94 proteins, phosphorylated in v i t r o , were extracted from SDS-88 polyacrylamide gels and analyzed for phosphoamino acid content. HL-60 p92 i s phosphorylated at tyrosine, although phosphoserine and phosphothreonine are also present (Figure 3.6, A), and K562 p94 i s phosphorylated exclusively at tyrosine (Figure 3.6, B). While Figure 3.6, A suggests that phosphoserine and phosphotyrosine are present in equimolar amounts, repeat experiments showed that the majority of phosphate label was incorporated as phosphotyrosine (see Figure 3.11, A). The detection of phosphoserine and phosphothreonine in p92 could be due to an i n t r i n s i c serine/threonine kinase a c t i v i t y , such as that described for mos or m i l / r a f f or might be due to contaminating, co-p r e c i p i t a t i n g c e l l u l a r kinases which u t i l i z e p92 as a substrate. Low levels of phosphoserine or phosphothreonine have also been detected in the autophosphorylated products of well characterized protein-tyrosine kinases such as p p 6 0 Q - D E O , pp60v-""r=, and pito-a-v-*** ( C o l l e t t , et a l . , 1978a; Hunter, et a l . f 1980; Feldman, et a l . r 1980); further characterization was undertaken in order to t r y and resolve whether the observed phosphorylation of serine and threonine was due to an i n t r i n s i c a c t i v i t y of p92 or to some co-precipitating kinase. A number of g l y c o l y t i c enzymes, including enolase, are phosphorylated at tyrosine by fps/fes-encoded protein-tyrosine kinases (Cooper, et a l . , 1983a). The phosphorylation of enolase in v i t r o occurs at a single tyrosine residue and t h i s same unique tyrosine is also 89 C B p s e r p - t h r p t y r D p-ser P ~ s e l v p-ser „ p-thr -p-thr p-ty ^ ^ t y r • p t y Figure 3.6: Phosphoamino acid analysis of p92, p94, and enolase. 3 2 P - l a b e l l e d proteins, phosphorylated in in  v i t r o kinase reactions as described, were isolated from SDS-polyacrylamide gels and subjected to acid hydrolysis, followed by two-dimensional electrophoretic separation of phosphoamino acids. The positions of authentic phosphoserine (p-ser), phosphothreonine (p-thr), and phosphotyrosine (p-tyr) were determined by staining with ninhydrin and are indicated. (A) HL-60 p92; (B) K562 p94; (C) enolase phosphorylated in v i t r o by HL-60 p92; ( D ) enolase phosphorylated in v i t r o by K562 p94; (E) enolase phosphorylated in v i t r o by NCP98. 90 modified in vivo (Cooper, et a l . , 1984); i f p92 and p94 represent authentic c-fes proteins i t i s l i k e l y that they too w i l l phosphorylate enolase at tyrosine. The a b i l i t y of these proteins to phosphorylate rabbit muscle enolase in an in v i t r o kinase assay was determined; p92 from HL-60 (Figure 3.7; lane 1) and p94 from K562 (Figure 3.7; lane 5) were able to cause the phosphorylation of enolase in the immune complex kinase assay. Under the same reaction conditions NCP98 from E26-transformed c e l l s also resulted in the phosphorylation of enolase (Figure 3.7, lane 3); phosphoamino acid analysis of enolase from each of these reactions demonstrated predominantly phosphotyrosine (Figure 3.6; C, D, and E). Phosphorylated enolase was extracted from SDS-polyacrylamide gels and subjected to digestion with trypsin; trypsin cleaves after lysine and arginine residues and, depending on the content of amino acid residues in the phosphate-labelled cleavage fragments, unique patterns re s u l t following electrophoretic and chromatographic separation. Digestion of enolase labelled in v i t r o by p92, p94, or NCP98 showed that a single co-migrating t r y p t i c phosphopeptide was generated, indicating that a l l three proteins possessed the same substrate s p e c i f i c i t y for enolase (Figure 3.8; A, B, and C). Phosphoamino acid analysis of thi s peptide revealed, in each case, exclusively phosphotyrosine (data not shown), establishing that p92 and p94 phosphorylate the same tyrosine residue as known fps/fe3 proteins. These results show that the kinase a c t i v i t i e s 91 1 2 3 4 5 F i g u r e 3.7: Phosphorylation of enolase in v i t r o by p92, p94, and NCP98. HL-60 c e l l s (lanes 1 and 2), E26-transformed avian myeloblasts (lane 3), or K562 c e l l s (lanes 4 and 5) were lysed and immunoprecipitated with J6 antl-fps serum (lanes 1, 3, and 5) or non-immune rat serum (lanes 2 and 4). Each immune complex was precipitated and resuspended in a 30 ul reaction mixture containing 5 ug of acid-denatured rabbit muscle enolase prior to the addition of (gamma-32P)ATP. I n v i t r o phosphorylated proteins were i d e n t i f i e d by electrophoretic separation and autoradiography. The location of enolase was confirmed by electrophoresis of unlabelled enolase and detection by staining with Coomassie blue. 92 Figure 3.8: Tryptic phosphopeptide analysis of enolase phosphorylated in v i t r o by p92, p94, and NCP98. Rabbit muscle enolase, phosphorylated in v i t r o by p92, p94 or NCP98 (as in Figure 3.7), was eluted from SDS-polyacrylamide gels and digested with trypsin. The res u l t i n g digests were analyzed by electrophoresis on TLC plates at pH 2.1, followed by chromatography in the second dimension. The locations of the 3 2P-containing phosphopeptides were determined by autoradiography. (A) enolase phosphorylated by p94; (B) enolase phosphorylated by NCP98; (C) enolase phosphorylated by p92. 93 associated with p92 and p94 phosphorylate enolase at tyrosine contained almost exclusively within a single major t r y p t i c phosphopeptide, implying that these proteins are indeed protein-tyrosine kinases. These data also support the assumption that the phosphoserine and phosphothreonine observed in phosphorylated p92 (Figures 3.6, A; 3.11, A) were due to a co-precipitating kinase since enolase phosphorylated in v i t r o by NCP98, a known t y r o s i n e - s p e c i f i c kinase, likewise contained trace amounts of phosphoserine and phosphothreonine; also, a single t r y p t i c peptide containing the major phosphoacceptor tyrosine residue is completely consistent with previously published results for other fps/fes proteins (Cooper, et a l . f 1984). Mapping of t r y p t i c peptides can be used to esta b l i s h s t r u c t u r a l relatedness between proteins, and t h i s technique was employed to investigate how c l o s e l y p92 and p94 might resemble a known fes protein. HL-60 p92, K562 p94, and ST-FeSV/NIH 3T3 pQ5v*»a-£**<» w e r e phosphorylated in v i t r o in individual immune complex kinase reactions and the label l e d proteins were extracted from SDS-polyacrylamide gels and analyzed by t r y p t i c peptide mapping. Trypsin cleavage of p92 generated two major phosphate containing peptides (Figure 3.9; A, 1 and 2). Cleavage of PQSVW-C™ also generated two predominant phosphopeptides, c l o s e l y resembling p92 in t h i s respect (Figure 3.9, B). A mixing experiment was performed to establish i f the two peptides from p92 co-migrated with those of p85' a r a , 3~ £ o s" 3 / which would 94 A B r Figure 3.9: Tryptic phosphopeptide analysis of HL-60 p92 and ST-FeSV P85*m,;'-*mm. Human p92 and v i r a l P85'="»,a-*•", phosphorylated in in v i t r o kinase reactions, were eluted from SDS-polyacrylamide gels and digested with trypsin. The res u l t i n g digests were analyzed by electrophoresis on TLC plates at pH 2.1, followed by chromatography in the second dimension. The locations of the 3 aP-containing phosphopeptides were determined by autoradiography. An equal amount of radioactive label was loaded onto each plate. (A) HL-60 p92; (B) ST-FeSV P 8 5 « m « - C " ; (C) mixture of equal amounts of p92 and p85«- < a -*"". The two major phosphopeptides isolated from HL-60 p92 are numbered 1 and 2; the sample origins are shown with arrows. 95 imply id e n t i t y ; as shown in Figure 3.9, C, phosphopeptides 1 and 2 from p92, while not separating i d e n t i c a l l y to those from P8 5'am'a~c"a, have migrated in a very similar fashion, suggesting a high degree of amino acid sequence s i m i l a r i t y . Trypsin digestion of ln v i t r o phosphorylated K562 p94 released only one major phosphopeptide (3.10; B, peptide 3), and the appearance of the p94 t r y p t i c peptide map was quite d i f f e r e n t from that of p92 (Figure 3.10, A); p94 obtained from the murine erythroleukemia c e l l l i n e MEL resulted in a pattern i d e n t i c a l with that of human p94 (Figure 3.10; C). In order to est a b l i s h d e f i n i t i v e l y that p94 peptide 3 was d i s t i n c t from either of p92 peptides 1 or 2, phosphorylated p92 and p94 were digested with trypsin and the r e s u l t i n g peptides were separated on the same thin-layer chromatography plate in a mixing experiment (Figure 3.10, D): i t i s quite clear that p92 and p94 phosphopeptides migrate independently. The human erythroleukemia c e l l l i n e , HEL, co-expresses p92 and p94, both of which can be phosphorylated in v i t r o and resolved on SDS-polyacrylamide gels as a doublet (see Figure 3.5); t r y p t i c peptide mapping of the p92/p94 doublet generates a pattern i d e n t i c a l to that obtained from mixing p92 and p94 (Figure 3.10, E). This evidence confirms that the divergent patterns observed in the in d i v i d u a l t r y p t i c peptide maps of p92 and p94 were not a res u l t of obtaining these two proteins from d i f f e r e n t c e l l l i n e s or some other experimental a r t i f a c t : i s o l a t i n g and mapping the same proteins from a single c e l l l i n e , HEL, gave 96 • • 1 • 1 • 2 Figure 3.10: Comparitive t r y p t i c phosphopeptide analysis of p92 and p94. HL-60 p92, K562 p94, HEL p92/p94, or MEL p94 were phosphorylated in in v i t r o kinase reactions, eluted from SDS-polyacrylamide gels, and digested with trypsin. The resu l t i n g digests were analyzed by electrophoresis on TLC plates at pH 2.1, followed by chromatography in the second dimension. The locations of the 3 2P-containing phosphopeptides were determined by autoradiography; an equal amount of radioactive label was loaded onto each plate. (A) HL-60 p92; (B) K562 p94; (C) MEL p94; (D) mixture of equal counts per minute of phosphopeptides from HL-60 p92 and K562 p94; (E) HEL p92/p94 doublet. The two phosphopeptides isolated from p92 are numbered 1 and 2 (as in Figure 3.9), and the major phosphopeptide isolated from p94 is numbered 3. 97 i d e n t i c a l r e s u l t s . Peptides 1, 2, and 3 were scraped o££ of the i r respective locations on the thin-layer chromatography plates and analyzed for phosphoamino acid content; a l l three of the peptides, representing the major phosphoacceptor s i t e s for p92 and p94, contained exclusively phosphotyrosine (Figure 3.11; B, C, E). These data further support the r proposal that p92 and p94 are t y r o s i n e - s p e c i f i c kinases: p92 and p94 have been shown to phosphorylate the exogenous substrate enolase at the same tyrosine as other protein-tyrosine kinases, and the results of t r y p t i c peptide mapping show that the enzymes themselves are autophosphorylated almost exclusively at tyrosine residues contained within one or two t r y p t i c peptides. The absence of detectable phosphoserine or phosphothreonine in either of peptide 1 or 2 from p92, and the lack of any other major phosphopeptides which might represent s i t e s of serine or threonine phosphorylation support the contention that the phosphoserine and phosphothreonine seen in Figure 3.6 is due to some contaminating kinase; none the less, the observed phosphorylation of p92 at serine or threonine residues may well represent s i g n i f i c a n t regulatory modifications. The s t r u c t u r a l comparison of p92 and p94 was extended by performing p a r t i a l p r o t e o l y t i c digests of the in v i t r o phosphorylated proteins with Staphylococcus aureus V8 protease and mapping the r e s u l t i n g fragments; V8 protease cleaves after glutamic acid and aspartic acid residues and is well suited to generate peptides for s t r u c t u r a l 98 Figure 3.11: Phosphoamino acid analysis of t r y p t i c phosphopeptides from p92 and p94, and of in vivo phosphorylated p92. The areas on TLC plates (A) and (B) shown in Figure 3.10 containing, phosphopeptides 1 and 2(p92) and phosphopeptide 3(p94), were scraped o f f , the phosphopeptides eluted from the cel l u l o s e fragments, and subjected to acid hydrolysis. In separate experiments, HL-60 p92 was phosphorylated in an in v i t r o reaction or was labelled in vivo with 3 2 p -orthophosphate; the 3 2 P - l a b e l l e d proteins were eluted from SDS-polyacrylamide gels and subjected to acid hydrolysis. The phosphoamino acids were analyzed by two-dimensional electrophoresis and autoradiography; the locations of authentic phosphoserine (p-S), phosphothreonine (p-T), and phosphotyrosine (p-T) were determined by staining with ninhydrin. (A) HL-60 p92, labelled in v i t r o ; (B) HL-60 p92, peptide 1; (C) HL-60 p92, peptide 2; (D) HL-60 p92, labelled in vivo: (E) K562 p94, peptide 3. 99 comparison of proteins (Cleveland, et a l . , 1977). p92, p94, and P85gag-fes were phosphorylated in individual immune complex kinase reactions and subjected to V8 protease mapping; in p a r a l l e l with the results of t r y p t i c peptide mapping, the pattern of proteo l y t i c fragments generated from p92 (Figure 3.12; lanes 3, 4) c l o s e l y resembled that of P85gag-fes (Figure 3.12; lanes 1, 2), and a majority of P85gag-fes phosphopeptides co-migrated with equivalent peptides from p92, as indicated. A number of peptides unique to p92 were also observed and these presumably represent divergent primary structure r e l a t i v e to P85gag-fes, r e s u l t i n g in a somewhat d i f f e r e n t mosaic of peptides. P a r t i a l p r o t e o l y t i c digestion of p94 yielded a pattern d i s t i n c t from that of either p92 or P85gag-fes and did not appear to share any of the seven i d e n t i f i e d co-migrating fragments (Figure 3.12; lanes 5, 6). Thus, these data extend and support the results of t r y p t i c peptide mapping which suggest that p92 is s t r u c t u r a l l y very c l o s e l y related to the fes protein P85gag-fes, and that p94, while demonstrating functional s i m i l a r i t i e s , i s more d i s t a n t l y related at the l e v e l of primary sequence. The f e l i n e (NCP92) and avian (NCP98) fps/fes homologues are synthesized de novo in the same c e l l s in which they are detected by immune complex kinase assays (Barbacid, et a l . f 1980; Mathey-Prevot, et a l . f 1982). HL-60 c e l l s were metabolically labelled and radioactive proteins were immunoprecipitated and separated e l e c t r o p h o r e t i c a l l y ; p92 100 P 8 5 p 9 2 p 9 4 1 2 3 4 5 6 — 205 Figure 3.12: V8 protease digestion of p92, p94, and PSS'—*-*". HL-60 p92, K562 p94, and ST-FeSV P85"-«-were phosphorylated in v i t r o and the labelled proteins were i d e n t i f i e d by electrophoresis and autoradiography. Gel fragments containing the 3 = P - l a b e l l e d proteins were excised and applied to the wells of a new 12.5% SDS-polyacrylamide gel, the proteins were digested in s i t u with V8 protease, and the re s u l t i n g cleavage products i d e n t i f i e d by electrophoresis and autoradiography. The proteins digested and the amounts of V8 protease used were: (lane 1) P85*-*-*—, 50 ng; (lane 2) PB5"-°-*mm, 200 ng; (lane 3) p92, 50 ng; (lane 4) p92, 200 ng; (lane 5) p94, 50 ng; (lane 6) p94, 200 ng. Comigrating fragments are i d e n t i f i e d by arrows to the l e f t , while molecular weight standards (in kD) are on the r i g h t . 101 was s p e c i f i c a l l y precipitated by the J6 anti-fp^. serum from lysates of HL-60 c e l l s labelled with either ( 3 BSMethionine (Figure 3.13, lane 1) or with 3 2P-orthophosphate (Figure 3.13, lane 5), indicating that i t is synthesized in these c e l l s . In vivo labelled p92 co-migrates with p92 labelled in in v i t r o kinase reactions (Figure 3.13; lane 3). p92 was also detectable in the c e l l l i n e KG-1 (Figure 3.14; lane 1), while p94 could not be immunoprecipitated from lysates of K562 c e l l s labelled with ( 3 9SMethionine (Figure 3.14; lane 3) or with 3 ZP-orthophosphate (data not shown). These data are consistent with the conclusion that p92 is immunologically related to known fps/fes proteins; the i n a b i l i t y to precipitate labelled p94 could mean that i t is not synthesized by these c e l l s or that i t i s labelled but not detected. The s t r u c t u r a l comparisons presented here show that p94 i s more d i s t a n t l y related than p92 to P8 5 < = r — * s u g g e s t i n g , as a consequence, that fewer cross-reactive epitopes may be present and hence low levels of labelled p94 remain undetected by J6 anti-fps serum. Al t e r n a t i v e l y , p94 might be bound by the antiserum with the same a f f i n i t y as p92 but not detected due to a s i g n i f i c a n t l y lower rate of synthesis or smaller steady-state pools of protein; t h i s explanation is probably not the correct one since repeated experiments u t i l i z i n g longer l a b e l l i n g periods and greater volumes of lysate a l l f a i l e d to demonstrate synthesis of p94 (data not shown). 102 Figure 3.13: Synthesis of p92 in HL-60 c e l l s . HL-60 c e l l s were metabolically labelled in vivo with ( 3 BS)methionine (lanes 1 and 2) or with 3 2P-orthophosphate (lanes 5 and 6) and lysates were prepared, or lysates were prepared from unlabelled HL-60 c e l l s (lanes 3 and 4 ) . Lysates were immunoprecipitated with J6 anti-fps serum (lanes 1, 3, and 5) or with non-immune serum (lanes 2, 4, and 6); the immune complex from the unlabelled lysate was labelled in an in v i t r o kinase reaction. The r a d i o l a b e l e d proteins in each case were then separated by electrophoresis through 7.5% SDS-polyacrylamide gels and i d e n t i f i e d by autoradiography; the location of p92 is indicated with an arrow. 103 45 -1 2 3 4 Figure 3.14: Metabolic l a b e l l i n g of KG-1 and K562 c e l l s . KG-1 c e l l s (lanes 1 and 2) or K562 c e l l s (lanes 3 and 4) were metabolically labelled in vivo with ( 3 SS)methionine and lysates prepared. These lysates were immunoprecipitated with J6 anti-fps serum (lanes 1 and 3) or with non-immune serum (lanes 2 and 4) and the labelled proteins were i d e n t i f i e d by electrophoretic separation and autoradiography; the locations of molecular weight markers (in kD) are indicated to the l e f t and the location of p92 is shown with an arrow. 104 The 3 2P-orthophosphate-labelled p92 in Figure 3.13 was extracted from the gel and analyzed for i t s phosphoamino acid content; in contrast to in v i t r o labelled p92 (Figure 3.11; A) in which phosphotyrosine is the predominant phosphorylated species, only phosphoserine was detected in p92 from 3 2P-orthophosphate-labelled c e l l s (Figure 3.11; D). A similar pattern of phosphorylation has been described for NCP98, in which in v i t r o autophosphorylation occurs at tyrosine and yet only phosphoserine is detectable when the protein i s labelled in vivo (Mathey-Prevot, et a l . . 1982); however, the equivalent v i r a l transforming protein of PRC II v i r u s , P105 < 3* < 3 - : E S J", is phosphorylated at tyrosine both in  vivo and in v i t r o , suggesting that r e t r o v i r a l transduction may re s u l t in unmasking of previously unavailable tyrosine residues (Beemon, 1981). c-fes proteins may also be susceptible to similar modifications, since human c-fes protein is not phosphorylated at tyrosine in vivo while v-fes protein, l i k e v-fps, i s phosphorylated at tyrosine both in vivo and in v i t r o (Barbacid, et a l . , 1980). Preincubation of 3 2 P - l a b e l l e d c e l l s with sodium orthovanadate, an i n h i b i t o r of phosphotyrosine phosphatases, and inclusion of t h i s reagent in a l l buffers did not re s u l t in the appearance of discernable phosphotyrosine in p92 (data not shown). This suggests that p92, l i k e NCP98, is not phosphorylated at tyrosine in vivo and that the lack of detectable phosphotyrosine is not due to phosphatase cleavage during i s o l a t i o n ; removal of c-fps/fes proteins 105 from their normal c e l l u l a r context, either by c e l l disruption or r e t r o v i r a l expression, f a c i l i t a t e s autophosphorylation of tyrosine, possibly by removing these enzymes from the v i c i n i t y of in h i b i t o r y molecules. The K562 c e l l l i n e was derived from a patient with CML and possesses an amplified and translocated c-abl gene, res u l t i n g in the production of a c-abl protein, P210 , = _~ f c , x, which i s considerably larger than the normal pi45<=-«j=i (Heisterkamp, et a l . f 1983); as a resu l t of i t s aberrant structure, P210<=~"toX possesses considerably greater protein-tyrosine kinase a c t i v i t y than i t s normal human counterpart (Konopka, et a l . , 1984). In order to show that J6 anti-fps serum did not react promiscuously with other protein-tyrosine kinases, and to show that p94 detected in K562 c e l l s did not represent a processed form of P210<=~"toX, K562 lysates were immunoprecipitated with either J6 anti-fps serum or with pEX-2 anti- abl serum and used in immune complex kinase assays. J6 serum recognized only p94 (Figure 3.15; lane 3) and pEX-2 serum recognized only P210<=~ot>x (Figure 3.15; lane 4); for comparison, HL-60 p92 and HEL p92/p94 are shown in Figure 3.15, lanes 1 and 2. These data imply that J6 serum does not react indiscriminately with any protein-tyrosine kinase and that p94 i s not a processed form of P210 C !~" t , x; furthermore, the results suggest that p94, while being s t r u c t u r a l l y d i s t i n c t , shares some common antigenic determinants with fps/fes-encoded proteins which 106 1 2 3 4 P210 c - a b l p94 3 2 P -in-vitro F i g u r e 3.15: I d e n t i f i c a t i o n of p92, p94, and P210C=--,:,:L i n l y s a t e s of human h e m o p o i e t i c c e l l s . L y s a t e s were pr e p a r e d from HL-60 c e l l s ( l a n e 1 ) , HEL c e l l s ( l a n e 2), or K562 c e l l s ( l a n e s 3 and 4) and were i m m u n o p r e c i p i t a t e d w i t h J6 a n t i - f p s serum ( l a n e s 1, 2, and 3) or w i t h pEX-2 a n t i - a b l serum ( l a n e 4 ) . Immune complexes were i n c u b a t e d w i t h (gamma- 3 2P)ATP and i n  v i t r o p h o s p h o r y l a t e d p r o t e i n s were i d e n t i f i e d by e l e c t r o p h o r e t i c s e p a r a t i o n and a u t o r a d i o g r a p h y . The l o c a t i o n s and s i z e s of m o l e c u l a r weight s t a n d a r d s ( i n kD) are i n d i c a t e d , as are the l o c a t i o n s of p92, p94, and P 2 1 0 o - m t > 1 . 107 may not be present in other protein-tyrosine kinases, as exemplified by P210°— k f a : L. 3. 3 Discussion The data presented here extend previously published r e s u l t s , which demonstrated the presence of immunologically cross-reactive fps/fes proteins possessing protein-tyrosine kinase a c t i v i t y in c e l l s from a variety of species (Barbacid, et a l . , 1980; Mathey-Prevot, et a l . , 1982). One of these previous reports alluded to the expression of NCP98 in hemopoietic c e l l s (Mathey-Prevot, et a l . , 1982), but the association of maximal kinase a c t i v i t y with granulocytic c e l l s and macrophages was not made u n t i l somewhat later (Samarut, et a l . , 1985). The other early report in this area demonstrated a 92kD protein-kinase in non-hemopoietic c e l l s obtained from several species, but human and murine c e l l l i n e s , derived from f i b r o b l a s t s , were negative for an equivalent a c t i v i t y (Barbacid, et a l . , 1980). My r e s u l t s demonstrate c l e a r l y that proteins possessing immunological determinants in common with v-fps and v-fes proteins are present in a v a r i e t y of murine and human c e l l s . These proteins, p92 and p94, are of similar molecular mass as the c-fps/fes proteins described previously and also appear to possess i n t r i n s i c protein-tyrosine kinase a c t i v i t y . It i s not established unequivocally from my results that the associated kinase a c t i v i t y is i n t r i n s i c to p92 and p94 , as discussed in section 3.2.1.; however, 108 applying the same burden of proof which other investigators have employed, i t is l i k e l y that the observed "autophosphorylation" and phosphorylation of enolase i s due to an enzymatic a c t i v i t y present within these molecules. Investigators confronted t h i s same conundrum soon after the discovery that pp60 v _ E , r : o i s associated with a protein kinase a c t i v i t y , and the p o s s i b i l i t y was raised that a c e l l u l a r kinase might be activated by v-src, rather than the kinase a c t i v i t y residing in the v i r a l protein (Richert, et a l . , 1979). Ample evidence consistent with the l a t t e r interpretation is available, including the demonstration that p p 6 0 v - " ° , translated in v i t r o from v i r i o n RNA, results in the phosphorylation of tyrosine (Hunter, et a l . . 1980), and also that pp60 v - , , , = : c ! is inseparable from i t s presumed kinase a c t i v i t y despite extensive p u r i f i c a t i o n (Purchio, 1982). The vast weight of evidence also supports the conclusion that protein-tyrosine kinase a c t i v i t y i s an i n t r i n s i c property of fps/fes proteins; t h i s evidence includes sequence homology between fps/fes kinase domains and the src kinase domain (Hampe, et a l . , 1982; Groffen, et. a l . , 1983), mutant viruses encoding conditional kinase a c t i v i t y (Pawson, et a l . f 1980), and functional comparisons with other protein-tyrosine kinases (Feldman, et a l . , 1980; Cooper, et a l . , . 1984). Thus, since p92 and p94 are s p e c i f i c a l l y immunoprecipitated by an anti-fps sera which reacts with known fps/fes proteins, and since they appear to possess an i n t r i n s i c protein-tyrosine kinase a c t i v i t y , I 109 conclude that i t is l i k e l y that either or both are c e l l u l a r fp s/fes proteins. It is apparent that p92 Is synthesized ln the same c e l l s in which i t is i d e n t i f i e d by in v i t r o kinase a c t i v i t y , but p94 synthesis could not be i d e n t i f i e d by metabolic l a b e l l i n g c e l l s with either 3 2P-orthophosphate or with ( 3 BS)methionine. While i t i s possible that the par t i c u l a r c e l l lines examined did not themselves synthesize p94 i t i s more l i k e l y that the antiserum did not detect low lev e l synthesis due to fewer cross-reactive epitopes being present on p94 than on p92; other investigators, using d i f f e r e n t anti-fps sera, have demonstrated synthesis of p94 in hemopoietic c e l l s , suggesting that the l a t t e r p o s s i b i l i t y is the correct one (Feldman, et a l . f 1985; Feldman, et a l . . 1986). Also, the results of mapping p r o t e o l y t i c fragments generated by cleavage of p92 and p94 with trypsin or with V8 protease imply that p92 i s s t r u c t u r a l l y more c l o s e l y related to p8 5<3a"=,-e,B'3, a known fes protein, than p94; the two major phosphopeptides derived from trypsin digestion of p92 v i r t u a l l y co-migrated with the two major peptides generated from p e s ' 5 " " 3 - * " " , indicating that, while not i d e n t i c a l , the primary sequences of these peptides are c l o s e l y related. Presumably t h i s conservation of structure i s mirrored in the conservation of relevant epitopes, allowing low levels of metabolically labelled p92 to be detected by immunoprecipitation. Digestion of p94 with trypsin yielded only one major phosphopeptide which did not appear to co-migrate with either of the peptides from p92 or 110 PQ^oMLV-cmm. published data indicate that t r y p t i c digestion of NCP98 yiel d s a pattern reminiscent of that seen for either p92 or p 8 5 ' - « - f " (Mathey-Prevot, e_t aJL., 1982), while phosphopeptide mapping of avian p94 indicates that i t is d i s t i n c t from NCP98 (Feldman, et a l . . 1986). The observation of Feldman, et al.,(1986 ) that two t r y p t i c phosphopeptides were generated following cleavage of avian p94, as opposed to the single peptide reported here for human p94, suggests that they may be d i f f e r e n t proteins, rather than homologues, or that d i f f e r e n t phosphorylation s i t e s are u t i l i z e d in avian and human p94; while they also i d e n t i f i e d a cross-reactive 94kD protein-tyrosine kinase in human hemopoietic c e l l s i t was not compared with avian p94 by t r y p t i c peptide mapping. Tryptic peptide mapping of ( 3 BS)methionine labelled p92 and p94 would l i k e l y have yielded valuable insights into relatedness of these two proteins but, as shown in Figure 3.14, labelled p94 could not be detected with the available antiserum; also, i n s u f f i c i e n t ( 3 SS)methionine-labelled p92 was recovered from gels to allow exploitation of thi s technique. Structural comparison by mapping V8 protease digests of in v i t r o phosphorylated p92 and p94 was consistent with the data obtained from t r y p t i c peptide mapping and indicated that p92 and pQ5<a-"3-**"> shared several co-migrating fragments while p94 exhibited i t s own unique pattern; since V8 protease cleaves peptide bonds at s i t e s d i s t i n c t from trypsin t h i s i s further evidence that p92 is related to p e S " 3 " " * - * " . A I l l number of V8 protease cleavage fragments of In v i t r o phosphorylated p92, obtained from either HL-60 c e l l s or normal human bone marrow c e l l s , have been reported to co-migrate with those obtained from NCP98 (Feldman, et a l . , 1985) , supporting the i d e n t i f i c a t i o n of p92 as a c-fps/fes protein; while p94 was also i d e n t i f i e d in t h i s study no s t r u c t u r a l comparisons were undertaken. In summary, I have isolated and characterized a 92kD protein, present in both mouse and human c e l l s , which I believe to be the product of the c-fes gene, and I designate t h i s protein pS2c~SmiB. I have also characterized a 94kD protein which, while displaying some st r u c t u r a l and functional s i m i l a r i t i e s , appears to be the product of another gene; I designate this protein p94. E s s e n t i a l l y i d e n t i c a l results to those presented here were obtained independently by other investigators (Feldman, et a l . . 1985; Feldman, et a l . , 1986) , and formal proof that human p92 < = - e"" is the product of the c-fes gene has recently been published (Greer, et a l . , 1988; Smithgall, et a l . , 1988). Very new data derived from the cloning and sequencing of an apparently f u l l length cDNA encoding p94 establishes conclusively that p92 and p94 are unique, but related, proteins; the gene encoding p94 has t e n t a t i v e l y been assigned the name FER (Hao, et a l . , 1989). Modulation of protein function involving a dynamic balance of phosphorylation and dephosphorylation has been proposed as an important regulatory mechanism in the normal c e l l ; these modifications, catalyzed by protein kinases and 112 protein phosphatases respectively, are believed to r e s u l t in a c t i v a t i o n or deactivation of s p e c i f i c substrate proteins (Ingebritsen, et a l . f 1983; Hunter, 1987b). Envisaged within t h i s concept are cascades or pathways of proteins, their afferent a c t i v a t i o n dependent on regulatory kinases and phosphatases, and these proteins in turn acting in an efferent manner to control the a c t i v i t y of other downstream proteins, perhaps again by reversible phosphorylation and dephosphorylation. Examples of the most proximal members of such proposed pathways exist in the form of c e l l surface receptors, as t y p i f i e d by the EGF receptor. It i s known that the tyrosine kinase a c t i v i t y innate to the EGF receptor is influenced p o s i t i v e l y by the binding of ligand and negatively by phosphorylation of the receptor by protein kinase C; however, the downstream molecule, or molecules, modified by the EGF receptor remain to be i d e n t i f i e d . Thus, the EGF receptor displays some of the c h a r a c t e r i s t i c s of members of these hypothetical s i g n a l l i n g cascades, namely negative regulation by protein kinase C and 1igand-dependent increased phosphorylation of c e l l u l a r substrates; the contribution of t y r o s y l - s p e c i f i c phosphatases to the regulation of EGF receptor a c t i v i t y i s less clear but s t i l l completely plausible. The association of pp60=_'n,cc' with an 81-85kD protein believed to be a phosphatidylinositol (PI) kinase has been described (Kaplan, et a l . . 1987; Courtneidge, et a l . , 1987); th i s protein may be a legitimate example of a p h y s i o l o g i c a l l y relevant substrate of 113 pp60 c ,- f f l l c o, and might account for the PI kinase a c t i v i t y reported to be associated with v-src and v-ros (Macara, g i a l . , 1984; Sugimoto, et a l . , 1984). Direct regulation of th i s putative PI kinase by c-src-mediated phosphorylation has yet to be demonstrated; however, i t i s known that the phosphorylation of key tyrosine residues in many protein-tyrosine kinases, including c-src and v-fps r has profound effects upon enzymatic a c t i v i t y (Weinmaster, et a l . , 1984; Kmiecik, et a l . r 1987; Piwnica-Worms, et a l . r 1987). In th i s respect i t i s interesting that p92 c : _ e"" i s phosphorylated at tyrosine in v i t r o and yet phosphotyrosine is not detected in vivo; p92 0- e" m might be released from some i n h i b i t o r y complex during c e l l l y s i s , allowing phosphorylation at tyrosine in v i t r o , or the turnover of phosphate at tyrosine residues in vivo might be at an extremely low l e v e l , not allowing detection. However, this second p o s s i b i l i t y seems less l i k e l y since the addition of sodium orthovanadate, an i n h i b i t o r of phosphotyrosine-phosphatases, did not resu l t in detectable phosphotyrosine. It i s also e n t i r e l y possible that c-fes functions normally In vivo without s i g n i f i c a n t phosphorylation of tyrosine and i t i s only in an aberrant context, such as an in v i t r o reaction or when c-fes is transduced by a retrovirus to y i e l d v-fes, that phosphotyrosine is detected: the phosphoserine detected in pS2°~ema in vivo may well represent an important regulatory modification in the normal c e l l . 114 L i t t l e is known of the ln vivo substrates upon which p92=-£«» acts, or of the proteins which might u t i l i z e p 9 2 a - e " as a substrate; these molecules have been d i f f i c u l t to elucidate for a l l protein-tyrosine kinases. While a number of protein-tyrosine kinases act as c e l l surface receptors i t i s probable that most, lacking external domains and transmembrane sequences, do not act independently as receptors. However, protein-tyrosine kinases lacking the t y p i c a l features might s t i l l function in receptor complexes by supplying the intracytoplasmic s i g n a l l i n g apparatus to a transmembrane receptor protein. A protein-tyrosine kinase operating in t h i s fashion could be envisaged as associating exclusively with a s p e c i f i c 1igand-binding protein or, a l t e r n a t i v e l y , acting as a common transducer for a number of d i f f e r e n t 1igand-binding proteins. The l a t t e r model is c e r t a i n l y consistent with observations that d i f f e r e n t growth factors, acting through d i f f e r e n t receptors, often r e s u l t in similar biochemical changes within c e l l s (Kruijer, et a l . , 1984; Muller, et a l . , 1984; Gee, et a l . . 1986), while the former model c l o s e l y p a r a l l e l s the structure of the receptors for i n s u l i n and IGF-1. The i n s u l i n and IGF-1 receptors consist of an external ligand-binding protein complexed through disulphlde bonds with an i n t e r n a l l y situated protein-tyrosine kinase ( U l l r i c h , et a l . , 1985; U l l r i c h , et a l . , 1986); these receptors d i f f e r s l i g h t l y from the model presented here in that the internal chain supplies the transmembrane segment in addition to the kinase domain. 115 However, the recently described association of p56 i a , c with the T-lymphocyte antigen CD4 supports the f i r s t model: p56 l e V c is e n t i r e l y internal while CD4 spans the membrane; however, the ligand which binds CD4 i s unknown (Rudd, e_t a l . , 1988; V e i l l e t t e , et a l . , 1988). The gene l t k , encoding a transmembrane protein-tyrosine kinase devoid of an ex t r a c e l l u l a r domain, has been described; t h i s too may be an example of a c-onc acting as part of a multi-unit receptor where the ligand binding region is on a separate molecule (Ben-Neriah, et a l . , 1988). It i s e n t i r e l y possible that p92«=-*-« i s also a constituent of a multi-unit receptor, and a recent report suggests that the c-fes protein may be an essential component for responsiveness to GM-CSF (Smithgall, et a l . , 1988). It is noteworthy that the gene immediately upstream of human c-fes, designated fur, has a predicted protein sequence displaying many features found in known receptors, including a cysteine-rich e x t r a c e l l u l a r domain, a transmembrane segment, and a short i n t r a c e l l u l a r segment (Roebroek, et a l . , 1986a; Roebroek, et a l . , 1986b); the short intracytoplasmic region of fur might associate with p92 a _ 1 !"", or with some other effector molecule, in response to binding of ligand. Both c-fes and fur are in close proximity cytogenetically to the gene encoding gpl50, a myeloid membrane antigen expressed in the same types of hemopoietic c e l l s as p 9 2 ° _ c - " (Look, et a l . , 1986); i t is possible that a l l three of these genes are expressed coordinately to form a receptor complex for some hemopoietic 116 g r o w t h f a c t o r . However, f u r i s e x p r e s s e d i n a much g r e a t e r v a r i e t y o f t i s s u e s t h a n e i t h e r c - f e s or g p l 5 0 ( S c h a l k e n , e t a l . r 1987), s u g g e s t i n g e i t h e r t h a t i t s l o c a t i o n a d j a c e n t t o c - f e s i s o n l y c o i n c i d e n t a l and t h e y a r e n o t e x p r e s s e d t o g e t h e r , or t h a t f u r s u b s e r v e s d i f f e r e n t f u n c t i o n s , p e r h a p s i n a s s o c i a t i o n w i t h d i f f e r e n t e f f e c t o r p r o t e i n s , when e x p r e s s e d i n n o n - h e m o p o l e t i c t i s s u e s ; s u p p o r t f o r t h e l a t t e r h y p o t h e s i s comes from c-fms, e n c o d i n g t h e CSF-1 r e c e p t o r o f m a c r ophages, w h i c h i s a l s o h i g h l y e x p r e s s e d i n p l a c e n t a and p l a y s a p a r t i n c o n t r o l l i n g p l a c e n t a l d e v e l o p m e n t ( P o l l a r d , e t a l . r 1 9 8 7 ) . W h i l e i t i s t e m p t i n g t o a s c r i b e a h e m o p o i e t i c g r o w t h f a c t o r - r e c e p t o r r o l e t o p 9 2 a _ : C ' " D , b a s e d on e x p r e s s i o n r e s t r i c t e d t o h e m o p o i e t i c c e l l s as r e p o r t e d h e r e and e l s e w h e r e (Feldman, e t a l . , 1985; S m i t h g a l l , e_£ a l . r 1 9 8 8 ) , t h i s c o n j e c t u r e must be b a l a n c e d by s e v e r a l c a v e a t s . F i r s t l y , p92=~*mm i s e x p r e s s e d n o r m a l l y i n p o s t -m i t o t i c l e u k o c y t e s ; t h e s e c e l l s do n o t r e s p o n d t o g r o w th f a c t o r s by e n g a g i n g i n a new r o u n d o f c e l l d i v i s i o n . A s i m i l a r p a t t e r n o f e x p r e s s i o n has been d e s c r i b e d f o r c - s r c , i n w h i c h h i g h e s t l e v e l s o f p p 6 0 a _ " = o a r e f o u n d i n p l a t e l e t s and n e u r o n s , and i t has been p r o p o s e d t h a t i t s f u n c t i o n i s t o m a i n t a i n t h e d i f f e r e n t i a t e d s t a t e ( C o t t o n , e t a l . . 1983; G o l d e n , e t a l . r 1 9 8 6 ) . S e c o n d l y , p 9 2 ° - e - ° a c t i v i t y , w h i l e most o f t e n d e t e c t e d i n m y e l o i d c e l l l i n e s , i s a l s o p r e s e n t i n c e l l s o f e r y t h r o i d , e o s i n o p h i l i c , and B - l y m p h o c y t i c o r i g i n ; t h e o n l y known g r o w t h f a c t o r t o a f f e c t c e l l s i n a l l t h e s e l i n e a g e s I s I L - 3 and i t s r e c e p t o r a p p e a r s t o be 117 d i s t i n c t from p92a-fi-*a (Palaszynski, et a l . , 1 9 8 4 ; Nicola, et a l . f 1986). Thirdly, p92°- e , B- a c t i v i t y is present in f i b r o b l a s t s , as reported here for NIH 3T3 and elsewhere (Barbacid, et a l . , 1980); however, these observations are not inconsistent with c-fes expression r e s t r i c t e d primarily to hemopoietic c e l l s since f i b r o b l a s t s can be derived from pluripotent hemopoietic stem c e l l s (Lord, 1983). F i n a l l y , c-fes t r a n s c r i p t s can be detected in some lung neoplasms (Slamon, et a l . , 1984b), and low levels of p92'=-e""* a c t i v i t y are found in normal mouse lung(Feldman, et a l . , 1985), although the single lung carcinoma-derived c e l l l i n e examined here was negative. Whole lung tissue is composed of several d i f f e r e n t c e l l s , including large numbers of alveolar macrophages, and the precise contribution of any one c e l l type to the overa l l To92°~e"a a c t i v i t y is unknown; however, fur transcripts have also been detected in human lung cancers, suggesting that the concomitant presence of c-fes t r a n s c r i p t s may be more than casual (Schalken, et a l . , 1987). While the preceeding discussion has focussed on the p o s s i b i l i t y that p92°-*- D may function as a c e l l surface receptor, either alone or in concert with other proteins, i t is equally l i k e l y that i t functions as an i n t r a c e l l u l a r member of a signal transducing pathway. Subcellular fractionation studies have not been performed for p92 a - f !" < 8, but the d i s t r i b u t i o n of NCP98 was found to be largely, but not e n t i r e l y , cytoplasmic (Young, et a l . , 1984); th i s might 118 imply that c-fps f and by inference c-fes f does not usually function at the c e l l membrane, although the equivalent v i r a l protein, pi2Q<smta~Cs'a, is membrane-associated (Feldman, g i a l . , 1983). p92 e =- C c" s may be a member of an expanding family of I n t r a c e l l u l a r protein-tyrosine kinases, including hck, lck, and l t k , which do not f u l f i l l the c r i t e r i a of t y p i c a l transmembrane receptors and yet are believed to play a role in signal transduction (Marth, et a l . , 1985; Ziegler, §i a l . , 1987; Ben-Neriah, et a l . r 1988). Several of these genes, including c-fes, are expressed in post-mitotic c e l l s , and detectable hck transcripts increase in myeloid c e l l s following induction of macrophage d i f f e r e n t i a t i o n ( Q u i n t r e l l , et a l . , 1987). These genes are similar to c-src in this respect and may serve in maintaining the d i f f e r e n t i a t e d phenotype; i f thi s is so their gene products may hold some promise as lineage-specific markers of d i f f e r e n t i a t i o n . Many of the considerations and speculations presented above for p 9 2 a - c " also apply to p94; while the data here and elsewhere (Feldman, et a l . . 1985; Feldman, et a l . . 1986) estab l i s h that p94 is not a c-fes protein, i t does appear to be a legitimate member of the protein-tyrosine kinase family. p94 is expressed in a more ubiquitous fashion than p92<=-*-» a n f j [ thus may participate in some process c r i t i c a l to a l l c e l l s ; unfortunately, much less is known about p94 in a l l respects than the fps/fes proteins, thus e f f e c t i v e l y l i m i t i n g speculation on potential functions and regulation 119 o£ p94 to that which can be made about protein-tyrosine kinases in general. CHAPTER 4 4.0 Expression of c-fes in Human Leukemia C e l l Lines During  Chemically Induced D i f f e r e n t i a t i o n . 4.1 Introduction AML may be conceptualized as a disease of disordered maturation of myeloid c e l l s , due to an unknown defect, r e s u l t i n g in the clonal overgrowth of blast c e l l s which appear blocked from d i f f e r e n t i a t i n g normally. However, this arrested maturation i s not absolute and analysis of X-chromosome-linked DNA polymorphisms has indicated, in some patients, that morphologically normal granulocytes do develop from the malignant clone in vivo (Fearon, et a l . , 1986). Also, many primary myeloid leukemias w i l l undergo d i f f e r e n t i a t i o n in v i t r o following stimulation with physiologic inducers of d i f f e r e n t i a t i o n , such as p u r i f i e d CSF's or vitamin D3, or with non-physiologic inducers, such as TPA or DMSO (Pegoraro, et a l . , 1980; Koeffler, et a l . , 1984; Sachs, 1987). However, leukemic c e l l explants, l i k e a l l primary tissue cultures, have a f i n i t e l i f e s p a n in. v i t r o , thus precluding systematic studies of d i f f e r e n t i a t i o n over a prolonged period of time; in order to circumvent t h i s l i m i t a t i o n in v i t r o , investigators have employed myeloid leukemia c e l l l i n e s . A number of myeloid leukemia c e l l lines e x i s t , representing developmental blocks at various i d e n t i f i a b l e stages, and the finding that many of these c e l l lines d i f f e r e n t i a t e in a fashion similar to primary 121 leukemias ln v i t r o suggests that they may be v a l i d models for studying leukemic d i f f e r e n t i a t i o n (Koeffler, et a l . f 1981; Koeffler, 1983; McCarthy, et a l . , 1983). Considerable evidence has emerged l i n k i n g the expression of c-onc's with the control of c e l l d i v i s i o n and d i f f e r e n t i a t i o n ; studying c-onc expression during d i f f e r e n t i a t i o n of leukemic c e l l s i s valuable because i t affords mechanistic insights into normal and leukemic myeloid d i f f e r e n t i a t i o n , and because the p o s s i b i l i t y of therapeutic progress e x i s t s . c-onc 1s of a l l functional classes are expressed in myeloid leukemias but the significance of t h i s expression to the emergence, maintenance, and expansion of the malignant clone is unknown. However, the finding that mutated ras genes are present in preleukemic c e l l s prior to the emergence of acute leukemia i s suggestive of an e t i o l o g i c r o l e , as opposed to some views which propose that point mutations of c-onc 1s are not a major mechanism in malignant neoplasia (Duesberg, 1987). Furthermore, many v-onc's cause acute leukemia in animals, implying that unregulated or mutant oncogene expression may be an important mechanism in leukemogenesis. Examining c-fes expression during myeloid d i f f e r e n t i a t i o n is hampered by the requirement of r e l a t i v e l y large numbers of c e l l s in order to obtain s u f f i c i e n t RNA and protein for study; in order to circumvent th i s problem I elected to examine d i f f e r e n t i a t i o n of the human myeloid 122 leukemia c e l l l i n e HL-60 following exposure to the inducer TPA. Additionly, HL-60 was chosen because a TPA-resistant subline, designated HL-525, became available, allowing evaluation of differences in c-fes expression between a differentiation-responsive c e l l l i n e and i t s unresponsive counterpart. 4 .2 Results 4.2.1 Examination of HL-525 Ce l l s for Expression of c-fes HL-60 c e l l s have been shown to synthesize active p92 e -*"" but whether or not HL-525 possessed a similar a c t i v i t y was unknown. Results published for KG-1 and i t s d i f f e r e n t i a t i o n - r e s i s t a n t subline, KG-la, indicated that KG-1 possesses p 9 2 e _ e " ° a c t i v i t y while KG-la does not (Feldman, et a l . f 1985). This raised the intriguing p o s s i b i l i t y that expression of p92°-e"a protein-tyrosine kinase a c t i v i t y in myeloid leukemias correlates with a b i l i t y to respond to inducers, while absence of this same a c t i v i t y correlates with resistance; accordingly, HL-525 c e l l s were tested for the presence of functional p92 < = _ j :"~ in an in  v i t r o kinase assay. The results indicate c l e a r l y that p92=-e-« c a n t,e immunoprecipitated from HL-525 c e l l s and that i t i s capable of autophosphorylation and phosphorylation of the exogenous substrate enolase (Figure 4.1, lane 3), similar to that seen in parental HL-60 c e l l s (Figure 4.1, lane 1); these results would seem to be quite d i f f e r e n t from those obtained for KG-1 and i t s subline E N O L A S E * * ^ E N O L A S E 1 2 3 4 Figure 4.1: I d e n t i f i c a t i o n of p 9 2 ° - e " in HL-525 c e l l s . To assay for p92=-*"" a c t i v i t y , lysates of HL-60 c e l l s (lanes 1 and 2) and HL-525 c e l l s (lanes 3 and 4) were immunoprecipitated with J6 anti-f_£2. serum (lanes 1 and 3) or with non-immune rat serum (lanes 2 and 4) and the immune complexes were incubated with (gamma-32P)ATP in reaction mixtures which contained 5 ug of acid-denatured rabbit muscle enolase. The in v i t r o -phosphorylated proteins were analyzed by electrophoretic separation and autoradiography; the location of enolase was confirmed by electrophoresis of unlabelled enolase. The locations of p92 and enolase are indicated by arrows. 124 KG-la. However, other investigators subsequently determined that p92'=~Xmo i s detectable in KG-la c e l l s when cultures of low passage-number, r e l a t i v e to the o r i g i n a l i s o l a t e , were tested as opposed to high passage-number cultures; thus, the loss of pS2°~£aa from KG-la c e l l s appears to be an a r t i f a c t related to length of time in culture (T. Pawson, personal communication). Therefore, there does not seem to be a c o r r e l a t i o n between the a b i l i t y to detect p 9 2 = - : C " kinase a c t i v i t y and the a b i l i t y to d i f f e r e n t i a t e in v i t r o in response to inducers. 4 .2 .2 Examination of c-fes Expression During Exposure of  HL-60 and HL-525 Ce l l s to the Inducer TPA. HL - 6 0 c e l l s , growing in suspension culture, have the a b i l i t y to d i f f e r e n t i a t e to more mature c e l l s in response to a v a r i e t y of physiologic and non-physiologic inducers. C e l l s resembling granulocytes, monocytes, macrophages, and eosinophils can a l l be derived from HL - 6 0 c e l l cultures following addition of the appropriate inducer; however, i t should be emphasized that a l l of these "diferentiated" c e l l s are d e f i c i e n t in some feature possessed by their normal counterparts ( C o l l i n s , 1987). The phorbol ester TPA, a tumor promoter, exerts a somewhat paradoxical e f f e c t on HL - 6 0 c e l l s in that i t stimulates them to undergo morphologic transformation to c e l l s resembling mature macrophages and displaying some of the same functional attributes of these c e l l s (Huberman, et a l . , 1979; Rovera, 125 et al.. f ,1979a; Rovera, et a l . , 1979b; Koeffler, e_£ a l . , 1981). The TPA-resistant HL-60 subline, HL-525, is refractory to the macrophage-inducing effects of TPA, and numerous morphological and biochemical differences between HL-60 and HL-525 following exposure to TPA have been documented (Solanki, et a l . , 1981; Huberman, et a l . , 1982; Fisher, et a l . . 1984; Anderson, et a l . , 1985). i.) Morphology In order to ensure that the expected morphologic differences between HL-60 and HL-525 developed following addition of TPA, cultures were observed microscopically for ov e r a l l morphology and samples were withdrawn at timed intervals for preparation of cytocentrifuge s l i d e s and diagnostic staining. HL-60 and HL-525 c e l l s in culture are indistinguishable prior to the addition of TPA (Figure 4.2, A and C); following induction with TPA for 48 hr cultures of HL-60 showed marked phenotypic change, including development of pseudopodia, enlarged macrophage-like c e l l s , marked adherence to the p l a s t i c f l a s k s , and clumping of c e l l s (Figure 4.2, B). In contrast, HL-525 c e l l s exposed to TPA for an equivalent period of time showed no discernable morphologic changes, and the most s t r i k i n g feature of these cultures was extreme crowding of c e l l s , i ndicative of ongoing mitosis (Figure 4.2, D). Sequential c e l l counts performed on induced and uninduced cultures of HL-60 and HL-525 c e l l s confirmed that there was cessation of c e l l 126 Figure 4.2: Morphologic c h a r a c t e r i s t i c s of HL-60 and HL-525 c e l l s in suspension culture following exposure to TPA. HL-60 c e l l s (A) and HL-525 c e l l s (B) were grown in tissue culture flasks in suspension culture. TPA was added to each culture to reach a f i n a l concentration of 10 ng/ml; HL-60 c e l l s (C) and HL-525 c e l l s (D) were photographed in s i t u 48 hr following addition of TPA to the culture flasks. Magnification, x40. 127 128 d i v i s i o n in HL-60 after addition of TPA while the growth kin e t i c s of HL-525 were unaffected; these observations were completely consistent with published results for these c e l l l i n e s (Huberman, et a l . , 1979; Huberman, et a l . , 1982). Wright-Giemsa staining of s l i d e s prepared from uninduced HL-60 c e l l s showed uniform b l a s t - l i k e c e l l s (Figure 4.3, A), while HL-60 c e l l s induced for 48 hr showed the presence of large macrophage-like c e l l s , monocyte-like c e l l s , and the persistence of some blast c e l l s s imilar to those seen in the uninduced culture (Figure 4.3, B); these observations are similar to published reports (Rovera, et a l . , 1979a; Rovera, et a l . , 1979b). HL-525 c e l l s prior to treatment with TPA appeared very similar to uninduced HL-60 c e l l s (Figure 4.3, C); following 48 hr of induction a few morphologic changes were apparent, but these were quite d i f f e r e n t from those observed for HL-60 and consisted mainly of v a r i a t i o n in c e l l s i z e , some mild c e l l enlargement, and vacuolization. Some monocytic c e l l s but no macrophage-like c e l l s were i d e n t i f i e d , and similar changes have been noted by other investigators (E. Huberman, personal communication; Homma, et a l . f 1986). Diagnostic staining for cloracetate esterase, a marker of granulocytes, and (alpha)-naphthyl esterase, a marker of monocyte/macrophages, confirmed that induced HL-60 c e l l s displayed the staining c h a r a c t e r i s t i c s of monocyte/macrophages while HL-525 did not (Table 4.1); these data are also consistent with published observations (Huberman, et a l . r 1982; M i t c h e l l , et a l . f 1986) . Thus, by 129 Figure 4.3: Morphologic d i f f e r e n t i a t i o n of HL-60 and HL-525 c e l l s . HL-60 c e l l s (A) and HL-525 c e l l s (C) growing in suspension culture were cytocentrifuged onto glass s l i d e s and stained with Wright-Giemsa s t a i n . HL-60 c e l l s (B) and HL-525 c e l l s (D) exposed t o 10 ng/ml TPA f o r 48 hr were c y t o c e n t r i f u g e d o n t o g l a s s s l i d e s and stained with Wright-Giemsa; adherent HL-60 c e l l s were dislodged gently with a c e l l scraper before cytocentrifugation. Magnification, x400. 131 TABLE 4.1 Cvtochemical Staining Characteristics of HL-60 and HL-525  Ce l l s Before and After Exposure to TPA. Stain C e l l type % of c e l l s staining p o s i t i v e 1 n i l TPA 48 hr TPA (alpha)-naphthyl acetate esterase HL-60 HL-525 0% 5% 80% 20% Chloracetate esterase HL-60 HL-525 100% 90% 20% 80% 1 Cytocentrifuge s l i d e mounts of control c e l l s or of c e l l s exposed to TPA for 48 hr were prepared and stained for (alpha)-naphthyl acetate esterase and chloracetate esterase according to the methods of Yam, et a l . (1970); c e l l s were evaluated microscopically in order to determine numbers of c e l l s displaying positive staining c h a r a c t e r i s t i c s . 132 a number of morphologic c r i t e r i a , HL-60 c e l l s responded to TPA by assuming a macrophage-like morphology while the TPA-resistant subline HL-525 did not. i i . ) c-fes transcripts Published data indicate that the expression of some c-onc's f including protein-tyrosine kinases, i s altered when d i f f e r e n t i a t i o n is induced in HL-60 c e l l s with TPA (Mitchell, et a l . , 1985; Barnekow, et a l . , 1986; Gee, e_t a l . , 1986). Although reports indicate that c-fes transcripts and in v i t r o kinase a c t i v i t y increase in HL-60 c e l l s after stimulation of granulocyte d i f f e r e n t i a t i o n by dimethyl sulfoxide, TPA-induced d i f f e r e n t i a t i o n was not examined ( F e r r a r i , et a l . , 1985; Smithgall, et a l . , 1988). In order to determine whether or not changes in the expression of c-fes accompany the morphologic alt e r a t i o n s of TPA-induced d i f f e r e n t i a t i o n , levels of steady-state c-fes mRNA in uninduced and induced HL-60 c e l l s were examined. Uninduced HL-60 c e l l s synthesize a 2.5 kb RNA which hybridizes to a human c-fes probe in Northern blots (Figure 4.4, A; 0 hr); a t r a n s c r i p t of i d e n t i c a l size was detected when RNA blots were probed instead with a 3.5 kbp Bglll-Xhol fragment encoding Gardner-Arnstein FeSV v-fes sequences (data not shown). The c-fes t r a n s c r i p t i d e n t i f i e d in Figure 4.4 was calculated to be 2.5 kb in length using authentic RNA molecular size standards as references, and this size is in good agreement with a 2.6 kb c-fes t r a n s c r i p t which was 133 A Oh 1h 2h 4h 8h 24h B 4 . 4 0 -2 . 3 7 -1 .35 -< actin Oh 1h 2h 4h 8h 24h Figure 4.4: Detection of c-fes and act i n mRNA in uninduced and TPA-induced HL-60 c e l l s . Total RNA was extracted from uninduced (0 hr) c e l l s and from c e l l s induced with TPA for 1, 2, 4, 8, or 24 hr. 10 ug of t o t a l RNA from each sample was electrophoresed, subjected to blot hybridization with a human c-fes probe, and transcripts were detected by autoradiography (A). After autoradiography, membranes were stripped of c-fes probe and hybridized to a bovine a c t i n probe u t i l i z i n g conditions i d e n t i c a l to those for c-fes (B). Sizes of authentic RNA molecular size standards are given to the l e f t , in kb. 134 i d e n t i f i e d in mRNA isolated from human hemopoietic malignancies (Slamon, et a l . r 1984b). Although the concentration of c-fes transcripts appears to be decreasing slowly with length of induction, a t r i v i a l explanation for the observed decline might be that the amount of RNA loaded in each lane was unequal, accounting for th i s perceived decline. In order to control for th i s p o s s i b i l i t y the membrane was stripped of c-fes probe and re-probed for (beta)-actin mRNA, which has been shown to remain constant in TPA-induced HL-60 c e l l s (Sariban, et a l . , 1985). c-fes t r a n s c r i p t s , were therefore normalized to (beta)-actin tr a n s c r i p t s by densitometric scanning of the autoradiograms and establishing a r a t i o of c-fes expression to that of (beta)-actin for each lane. These rat i o s were calculated as a percentage, where the value obtained for uninduced c e l l s was defined as 100%, and plotted as a function of time following induction with TPA (Figure 4.6, HL-60); When d i s p a r i t i e s in loading are accounted for in t h i s manner i t is clear that levels of c-fes mRNA decrease rapi d l y following exposure of HL-60 c e l l s to TPA: the number of c-fes transcripts dropped to approximately 50% of pre-induction levels in the f i r s t 1 hr and then declined more slowly to a lev e l approximately 10% of pre-induction by 24 hr (Figure 4.6, HL-60). Although th i s p a r t i c u l a r induction was terminated at 24 hr, continuing the induction to 48 hr in p a r a l l e l experiments (data not shown) did not result in any s i g n i f i c a n t change in levels of c-fes 135 tr a n s c r i p t s , which remained constant at approximately 10% of pre-lnduction values, as shown in Figure 4.6. Cultures of HL-525 c e l l s were also exposed to TPA and Northern blots prepared in order to compare the response of this d i f f e r e n t i a t i o n - r e s i s t a n t subline with that of the parental l i n e . A c-fes t r a n s c r i p t of i d e n t i c a l size to that of HL-60 was detected in uninduced HL-525 c e l l s (Figure 4.5, A; 0 hr), but the response of HL-525 c e l l s to TPA-stimulation was more complex: the addition of TPA resulted in a rapid decline in c-fes mRNA during the f i r s t 8 hr, which e s s e n t i a l l y p a r a l l e l e d the decrease observed in HL-60 c e l l s (Figure 4.6, HL-525). But, between 8 and 24 hr post-induction, c-fes mRNA levels began to increase so that by 24 hr the number of c-fes transcripts had been restored to 100% of uninduced l e v e l s ; over the remaining 24 hr trans c r i p t levels declined somewhat to about 80% of pre-induction (Figure 4.6, HL-525). i i i . ) Synthesis of p92<=~:e'**' Northern blot experiments indicated that levels of steady-state c-fes mRNA declined r a p i d l y following addition of TPA, suggesting some form of t r a n s c r i p t i o n a l regulation. In order to ascertain whether de novo synthesis of £$2°-*""° was altered as well, r a d i o l a b e l e d p92 a - c"' D was isolated from uninduced and induced HL-60 c e l l s which had been metabolically labelled with ( 3 SS)-methionine (Figure 4.7). Exposure to TPA resulted in a decrease in p92 0- : E" , 0 synthesis 136 A Oh 2h 8h 24h 48h B 4.40-2 . 3 7 ^^^^gtm^^^m^^^^^^^^^ 1.35-Oh 2h 8h 24h 48h Figure 4.5: Detection of c-fes and acti n mRNA in HL-525 c e l l s and HL-525 c e l l s exposed to TPA. Total RNA was extracted from uninduced (0 hr) c e l l s and from c e l l s induced with TPA for 2, 8, 24, or 48 hr. 10 ug of to t a l RNA from each sample was electrophoresed, subjected to blot hybridization with a human c-fes probe, and transcripts were detected by autoradiography (A). After autoradiography, membranes were stripped of c-fes probe and hybridized to a bovine ac t i n probe u t i l i z i n g conditions i d e n t i c a l to those for c-fes (B). Sizes of authentic RNA molecular size standards are given to the l e f t , in kb. 0 12 24 36 48 Hours of induction with TPA Figure 4.6: Quantitation of c-fes mRNA in HL-60 c e l l s and HL-525 c e l l s exposed to TPA. The autoradiograms in Figures 4.4 and 4.5 were scanned using soft laser densitometry and a r a t i o of in t e n s i t y of c-fes versus i n t e n s i t y of a c t i n was calculated for each time point. The r a t i o s are plotted as percentages, where the ratios obtained for 0 hr in each c e l l l i n e were assigned a value of 100%. 138 within 2 hr following induction, and production of p 9 2 ° - e " a continued to decrease over the next 2 hr, at which point synthesis s t a b i l i z e d at this reduced l e v e l . The decline in p92=- c-» synthesis lagged behind the decline observed in c-fes mRNA since repeat experiments, concentrating on the i n i t i a l period following induction, showed that no detectable drop in synthesis occurred u n t i l approximately 90 min post-induction (Figure 4.8). By 4 hr the synthesis of p92=- e«= s t a b i l i z e d at the new, reduced equilibrium l e v e l and synthesis remained detectable at 24 hr after induction with TPA (Figure 4.7). Repeat experiments showed that p92=-fi=» synthesis was maintained at a low, but constant, l e v e l from 8 hr to 50 hr post-induction (data not shown). At no point following TPA-induction did de novo synthesis of pg2=>-c-« ever become undetectable, a low le v e l of persistent synthesis always being present, consistent with the results of RNA analysis which indicated that despite a reduction ln the size of the tr a n s c r i p t pool c-fes mRNA remained detectable from 8 hr to 48 hr post-induction (Figures 4.4, 4.6) . It was not possible to investigate the synthesis of p92°- e"" a in r a d i o l a b e l e d HL-525 c e l l s following exposure to TPA due to lack of s u f f i c i e n t J6 anti-fps serum. In an attempt to secure additional antisera, tumors were induced in Fischer X Wistar rats by inje c t i n g v-fps transformed fi b r o b l a s t s and sera were tested exactly as described for the production of J6 antiserum (Ingman-Baker, et a l . , 1984); Figure 4.7: Synthesis of p92=- e— in uninduced and TPA-induced HL-60 c e l l s . HL-60 c e l l s were induced with TPA for 0, 2, 4, 8, and 24 hr, r a d i o l a b e l e d with (35S)methionine for 30 min and c e l l lysates were prepared and immunoprecipitated. Radiolabeled proteins were separated e l e c t r o p h o r e t i c a l l y and i d e n t i f i e d by autoradiography. The times indicated show the t o t a l length of exposure to TPA and include the l a b e l l i n g period. Lysates from each time point were immunoprecipitated with immune (I) or non-immune serum (N), as indicated. The locations and sizes (in kD) of molecular weight standards are indicated to the l e f t . 140 N J N I N I N 0' 45' 90' 180' Figure 4.8: Synthesis of p 9 2 ° - e " in uninduced and TPA-induced HL-60 c e l l s . HL-60 c e l l s were induced with TPA for 0, 45, 90, and 180 min, r a d i o l a b e l e d with (35SM e t h i o n i n e for 30 min and c e l l lysates were prepared and immunoprecipitated. Radiolabeled proteins were separated e l e c t r o p h o r e t i c a l l y and i d e n t i f i e d by autoradiography. The times indicated show the t o t a l length of exposure to TPA and include the l a b e l l i n g period. Lysates from each time point were immunoprecipitated with immune (I) or non-immune (N) serum, as indicated. The position of p 9 2 ° - e " " is indicated with an arrow. 141 however, no antiserum able to immunoprecipitate p 9 2 a - £ " as e f f i c i e n t l y as J6 antiserum was i d e n t i f i e d (data not shown). As an alternative approach, I attempted to raise monoclonal antibodies by inj e c t i n g mice with a recombinant TRP-E/v-fps protein (Sadowski, et a l . . 1986), but the presence of a n t i -fps antibodies could not be demonstrated by immune complex kinase assay and thus hybridomas were not produced (data not shown). i v . D92°-*—• in v i t r o kinase a c t i v i t y p92=-e«» i s the human c-fes protein and possesses protein-tyrosine kinase a c t i v i t y ; in order to determine i f th i s enzymatic a c t i v i t y was altered during TPA-induced d i f f e r e n t i a t i o n , pS2°~Cma from uninduced and induced HL-60 c e l l s was tested for i t s a b i l i t y to autophosphorylate and to phosphorylate enolase in an in v i t r o kinase reaction. In contrast to the data which indicated a decrease in steady-state c-fes mRNA and reduced p92 t = _ i :" synthesis, the in  v i t r o kinase a c t i v i t y of p92G~£mm in HL-60 c e l l lysates remained invariant up to 48 hr post-induction (Figure 4.9). No detectable decrease in enzymatic a c t i v i t y , as assessed by either autophosphorylation or enolase phosphorylation, was seen between 8 hr and 24 hr post-induction despite very low levels of both c-fes tr a n s c r i p t s and p 9 2 ° _ e " " synthesis during the corresponding period of time; both enolase and p92=-*«= were phosphorylated to a similar extent from one time point to the next (Figure 4.9). The In v i t r o kinase 142 H L - 6 0 Oh 12h 18h 2 4 h 3 0 h 3 6 h 4 8 h Figure 4.9: p92a~emm in v i t r o kinase a c t i v i t y in uninduced and TPA-induced HL-60 c e l l s . HL-60 c e l l s were induced with TPA for the indicated lengths of time, c e l l lysates were prepared, and immunoprecipitated with immune (I) or non-immune (N) serum. Each immune complex was resuspended in a 30 ul reaction mixture containing 5 ug of acid-denatured rabbit muscle enolase. Phosphorylated proteins were analyzed by electrophoresis and detected by autoradiography. The location of enolase was confirmed by electrophoresis of unlabelled enolase and detection by Coomassie blue staining. 143 a c t i v i t y of p 9 2 a - e * ° from HL-525 c e l l s exposed to TPA was also determined (Figure 4.10). The data for HL-525 c e l l s i s very si m i l a r to that obtained for HL-60 c e l l s , showing neither a decrease nor an increase in in v i t r o kinase a c t i v i t y . In contrast to HL-60 c e l l s , where f u l l kinase a c t i v i t y i s maintained despite reduced mRNA and pS2cs~£ma synthesis, there is no evidence to indicate that p92=-c,Bra kinase a c t i v i t y i s increased in HL-525 as a res u l t of r e l a t i v e l y higher levels of c-fes t r a n s c r i p t s ; data regarding synthesis of new p92= _ C o" might have been helpful in t h i s regard but could not be obtained, as mentioned, due to lack of immune reagents. The data presented in Figure 4.10 suggests an increase in autophosphorylation and enolase phosphorylation with length of exposure to TPA, but thi s increase can be accounted for by overloading of protein onto gels since duplicate experiments, where more equal loading was obtained, f a i l e d to demonstrate a similar increase (data not shown). In order to obtain additional quantitative information on steady-state levels of p92= - e , B a protein, as opposed to i t s kinase a c t i v i t y , I attempted to i d e n t i f y p92c-fes in fresh lysates prepared from uninduced and induced HL-60 and HL-525 c e l l s using a rabbit anti-v-fps serum (Sadowski, e_t a_l., 1986) in a Western b l o t t i n g procedure. Despite i t s a b i l i t y to i d e n t i f y HL-60 p92'=-±mm in in v i t r o kinase assays (Greer, et a l . , 1988), no unique 92 kD protein could be id e n t i f i e d in Western blots of HL-60 or HL-525 lysates using 144 H L - 5 2 5 J N J N J N I N Oh 12h 24h 48h Figure 4.10: p92<s~emm in v i t r o kinase a c t i v i t y in HL-525 c e l l s and HL-525 c e l l s exposed to TPA. HL-525 c e l l s were exposed to TPA for the indicated lengths of time, c e l l lysates were prepared, and immunoprecipitated with immune (I) or non-immune (N) serum. Each immune complex was resuspended in a 30 ul kinase reaction mixture containing 5 ug of acid-denatured rabbit muscle enolase. Phosphorylated proteins were analyzed by electrophoresis and detected by autoradiography. The location of enolase was confirmed by electophoresis of unlabelled enolase and detection by Coomassie blue staining. 145 th i s antiserum (data not shown). similar i n a b i l i t y to recognize protein-tyrosine kinases in an immunoblot procedure, despite demonstrated a b i l i t y to immunoprecipitate the same proteins from c e l l lysates, has been reported for other rabbit antisera (Feldman, et a l . f 1986). 4.3 Discussion The data presented here establish that c-fes expression in HL-60 c e l l s i s altered during the course of TPA-induced d i f f e r e n t i a t i o n to macrophage-like c e l l s . Steady-state levels of c-fes mRNA decrease very r a p i d l y after induction with TPA, followed c l o s e l y by a decline in p92c-fes synthesis; this suggests that the production of new c-fes protein i s t r a n s c r i p t i o n a l l y regulated, although evidence exists that t r a n s l a t i o n a l regulation may also play an important role in c o n t r o l l i n g synthesis of protein-tyrosine kinases (Marth, et a l . , 1988); the data presented here do not address whether c-fes expression might also be regulated at the t r a n s l a t i o n a l l e v e l . Only a single 2.5 kb species of c-fes t r a n s c r i p t i s i d e n t i f i e d , suggesting that the reduction in steady-state c-fes mRNA is not due to a s h i f t in the balance of subpopulations of mRNA's to one which i s more unstable; presumably, i f subpopulations of c-fes mRNA of d i f f e r i n g s t a b i l i t i e s e x i s t , these would be manifested as trans c r i p t s of d i f f e r e n t sizes as described, for example, for c-myc (Swartwout, et a l . , 1987). The observed decline in c-fes mRNA might conceivably be due to enhanced 146 ribonuclease a c t i v i t y accompanying macrophage d i f f e r e n t i a t i o n , but th i s seems unl i k e l y since there i s no decrease in the ove r a l l population of polyadenylated mRNA associated with TPA-induced d i f f e r e n t i a t i o n of HL-60 (Harley, 1987), and since RNA obtained from induced c e l l s showed no more evidence of degradation than did RNA from uninduced c e l l s . Thus, i t i s most l i k e l y that the reduced amount of steady-state mRNA is due to reduced t r a n s c r i p t i o n from the c-fes gene. Likewise, the decreased synthesis of p92«=-c = => i s probably secondary to a smaller pool of c-fes transcripts being available for tra n s l a t i o n ; increased p r o t e o l y t i c degradation of newly synthesized p92° -*"" seems un l i k e l y to account for the decrease in labelled protein because there i s no detectable reduction at any time in steady-state p92 a _ c~", as assessed by ln v i t r o kinase a c t i v i t y . In fact, the unaltered p92 e = _ e"" protein-tyrosine kinase a c t i v i t y seen during d i f f e r e n t i a t i o n suggests that the normal degradative pathways responsible for p92'=-£ma turnover may be acting at a reduced l e v e l . Neither c-fes tr a n s c r i p t s nor synthesis of new c-fes protein ever disappear completely, implying that there is an ongoing need for p92° _ ± :"" synthesis during d i f f e r e n t i a t i o n , a l b e i t in reduced amounts; th i s low lev e l of synthesis might be enough to resupply the steady-state pool of p92 0- c~", which would be degraded at a reduced rate. An alternative explanation for the apparently constant nature of the p92 a- e-" kinase a c t i v i t y might be that as synthesis of new protein i s 147 decreasing there Is a concomitant increase in the s p e c i f i c a c t i v i t y of the pre-existing pool of p9 2° _ i : , a < , i , / perhaps as a r e s u l t of a change in phosphorylation status. Evidence exists that the s p e c i f i c a c t i v i t y of c-src can be modulated by phosphorylation of s p e c i f i c tyrosine residues (Piwnica-Worms, et a l . f 1987; Kmiecik, et a l . f 1987; Cartwright, §_£ a l . , 1987), and the phosphorylation state of c-src in myeloid c e l l s induced to d i f f e r e n t i a t e with TPA has been reported to change (Gee, et a l . , 1986). However, i t would seem unl i k e l y , in the face of decreased synthesis and normally functioning degradative pathways, that the pre-e x i s t i n g pool of p92c:-*:•,,, could continuously increase i t s s p e c i f i c a c t i v i t y s u f f i c i e n t l y without demonstrable reduction in a c t i v i t y at some time. Clearly, much of the above is speculative; however, observations on the expression c-src during TPA-induced d i f f e r e n t i a t i o n of HL-60 c e l l s generate many of the same questions. Following exposure to TPA the in v i t r o kinase a c t i v i t y of pp60 = -~ = c= increases and yet i t is not clear i f t h i s increase is due to a l t e r a t i o n s in s p e c i f i c a c t i v i t y or due to t r a n s l a t i o n of new protein (Gee, et a l . , 1986; Barnekow, et a l . , 1986) In fact, regulation at both levels may contribute to the observed increase in kinase a c t i v i t y , and although p p e O 0 - " 0 a c t i v i t y increases, as opposed to p92 0- c* B 0' which decreases, i t is somewhat analogous in that augmented pp60c=-'s,r° a c t i v i t y appears not to require any increase in t r a n s c r i p t i o n from c-src just as p92' a- i : - D a c t i v i t y appears 148 to remain constant despite decreased t r a n s c r i p t i o n of c-fes. However, i t should be emphasized that the relationship between in v i t r o kinase a c t i v i t y and normal in vivo kinase a c t i v i t y has not been established for any c-pjic_-encoded protein-tyrosine kinase; therefore, the apparently constant nature of p92t=-fi0"B a c t i v i t y in v i t r o may belie s i g n i f i c a n t l y altered function in vivo. For example, p92 = - l :"" , i n i t i a l rates might be s i g n i f i c a n t l y reduced in vivo and yet this would not be i d e n t i f i e d in the standard in v i t r o kinase assay, employed here, which allows phosphorylation reactions to proceed to completion; however, since the in v i t r o kinase reactions allow complete substrate phosphorylation they do at least act as a good indicator of c-fes steady-state l e v e l s . It i s interesting to note that p p S O 0 - " 0 a c t i v i t y increases during the d i f f e r e n t i a t i o n to m i t o t i c a l l y i n e r t , macrophage-like c e l l s , consistent with r e s u l t s indicating that i t is highly expressed in a number of terminally d i f f e r e n t i a t e d , post-mitotic tissues. A number of other genes encoding protein-tyrosine kinases which are expressed predominantly or exclusively in hemopoietic c e l l s have been described, including lck, hck, and l t k , and the expression of at least one of these, hck, also increases during TPA-induced d i f f e r e n t i a t i o n of HL-60 ( Q u i n t r e l l , et a l . f 1987); thus, the expression of c-fes may be regulated somewhat d i f f e r e n t l y from other protein-tyrosine kinases during TPA-induced d i f f e r e n t i a t i o n of HL-60 c e l l s . 149 What insights, then, can be gleaned about the function of p92°-*m,B from alt e r a t i o n s in i t s expression during induced d i f f e r e n t i a t i o n of a myeloid leukemia c e l l line? The most l i k e l y role for p92"=~*~a, based on strucutural and functional comparisons with other protein-tyrosine kinases, is as a member of some i n t r a c e l l u l a r signal-transducing pathway. If such a pathway were rendered unnecessary by d i f f e r e n t i a t i o n to a more mature c e l l then t h i s could explain the decrease in c-fes transcripts and p92°~±mB synthesis, but the continued presence of high levels of functional protein mitigates against such an interpretation. But, the presence of p92 < = - e*" in myeloid c e l l s of a l l stages of maturity and mitotic capacity, as well as in HL-60 prior to and after d i f f e r e n t i a t i o n , suggests that the expression of c-fes may be necessary to maintain committment to the myeloid lineage. While c-fes mRNA can be detected in some non-myeloid neoplasms (Slamon, et a l . , 1984b) and some normal erythroid precursors (Emilia, et a l . , 1986), these may be examples of normally regulated c-fes expression which might occur t r a n s i e n t l y early in hematopoiesis and disappear as these c e l l s become committed to their respective lineages; the persistence of c-fes kinase a c t i v i t y in some B- c e l l and erythroid neoplasms may therefore reperesent a form of lineage i n f i d e l i t y . Compatible with t h i s proposal of a lineage determination role are the multiple observations that c-fps/fes proteins in vivo are expressed almost exclusively in hemopoietic tissues (Mathey-Prevot, §_t 150 a l . f 1982; MacDonald et a l . r 1985; Feldman et a l . , 1985), and that within hemopoietic tissues c-fps/fes expression i s confined, in mature c e l l s , to those of myeloid lineages (Samarut, et a l . , 1985; Feldman, et a l . , 1985; MacDonald §_t a l . f 1985; Emilia, et a l . , 1986); the few non-hemopoietic tissues which express s i g n i f i c a n t levels of c-fps/fes proteins are known to be r i c h in fixed tissue macrophages and t h i s may account p a r t i a l l y or completely for the a b i l i t y to detect these proteins. If p92 a -*"" functions as one of the subunits of a multimeric receptor complex, as hypothesized in Chapter 3, do the observed changes in the expression of c-fes during induced d i f f e r e n t i a t i o n support such a hypothesis? While at f i r s t glance the change in HL-60 from an immature, p r o l i f e r a t i v e c e l l to a more d i f f e r e n t i a t e d , non-raitotic c e l l without any accompanying change in p 9 2 ° _ e " " a c t i v i t y implies that i t does not function as a growth factor receptor, i t is s t i l l possible to reconcile the observed results with the hypothesis. In thi s regard the CSF's serve as a useful paradigm, their presence being required by dependent c e l l s at a l l stages of maturity for d i v i s i o n , d i f f e r e n t i a t i o n , and proper effector function (Sachs, 1987); by analogy, p92° - 1 !"" could f u l f i l l disparate rol e s , l i k e the CSF's, by binding the same ligand at d i f f e r e n t stages of d i f f e r e n t i a t i o n with vastly d i f f e r e n t consequences. Thus, in undifferentiated c e l l s t h i s hypothetical receptor complex may be necessary for stimulation of mitosis while in 151 d i f f e r e n t i a t e d c e l l s i t might function to maintain v i a b i l i t y or promote an effector function; a precedent for t h i s proposal exists within the family of protein-tyrosine kinases in c-fms, the CSF-l-receptor, which mediates the multiple functions of CSF-1, including stimulation of d i v i s i o n and committment to the macrophage lineage among precursor c e l l s , maintenance of c e l l v i a b i l i t y , and stimulation of macrophage effector functions. It is known that the CSF-l-receptor binds CSF-1 with high a f f i n i t y and the myriad effects mediated by t h i s interaction may involve sele c t i v e substrate u t i l i z a t i o n in a d i f f e r e n t i a t i o n -dependent manner; a similar scheme could be envisaged for p92<=-*«<=»/ thus explaining the persistence of the enzyme. Also, i f p92 < = - : E B B does function as a receptor then i t would not be l i k e l y to bind TPA, which functions through i t s own s p e c i f i c receptor; in the absence of 1igand-mediated receptor turnover the observed kinase a c t i v i t y might not be unexpected. It w i l l be interesting to examine the expression of the fur and gpl50 genes during induced d i f f e r e n t i a t i o n to see i f their regulation resembles that of c-fes, as might be expected i f their gene products are associated in a receptor complex. The changes in expression of c-fes in HL-525 c e l l s add at least a l i t t l e c l a r i f i c a t i o n to some of the preceding conjecture. HL-525 c e l l s , while remaining morphologically immature, possessed in v i t r o kinase a c t i v i t i e s which, l i k e HL-60, remained invariant during exposure to TPA. This is 152 c e r t a i n l y consistent with a myeloid determination role for p92<=-*« / although i t in no way excludes p a r t i c i p a t i o n in a receptor complex. Interestingly, the change in c-fes t r a n s c r i p t s seen in HL-525 exposed to TPA was not uniform, i n i t i a l l y d eclining almost i d e n t i c a l l y to HL-60, but quite r a p i d l y returning to pre-exposure l e v e l s . The divergent regulation of c-fes implies that the mRNA changes seen in HL-60 c e l l s are d i f f e r e n t i a t i o n related and are not the result of some unique d i r e c t action of TPA upon c-fes regulation; u t i l i z a t i o n of d i f f e r e n t inducers which also r e s u l t in macrophage-like d i f f e r e n t i a t i o n would l i k e l y resolve t h i s issue in a more di r e c t manner. The nature of the differences between HL-60 and HL-525 which res u l t in altered regulation of c-fes mRNA are unknown. The changes seen in the c-fes t r a n s c r i p t pool could be explained by induction of some enzyme system which deactivates TPA, thus allowing c-fes t r a n s c r i p t levels to increase; however, human c e l l s are not known to possess the esterase which results in the hydrolysis of TPA in rodent c e l l s , and HL-525 c e l l s have not been shown to degrade or modify TPA (E. Huberman, personal communication). The nature of the defect which renders HL-525 unresponsive to TPA may be of considerable interest as the same defect might be responsible for di f f e r e n t i a t i o n - r e s i s t a n t AML c e l l s which are isolated from patients (Pegoraro, et a l . , 1980). Unfortunately, due to technical constraints, I was unable to determine i f de novo synthesis of p92 a _ e*" exhibited k i n e t i c s similar to those of 153 the mRNA; however, i£ synthesis Is t r a n s c r i p t i o n a l l y regulated, as seems to be the case In HL-60 c e l l s , then return to pre-induction levels of p92 C ! - e"' B synthesis would be expected. Since neither an increase nor a decrease in p92°-*«»» in v i t r o kinase a c t i v i t y is observed, i t would be reasonable to conclude that the degradative pathways are also functioning at normal capacity, maintaining a dynamic balance in steady-state levels of p92°- £"". Many, i f not a l l , of the effects of TPA on c e l l s are believed to be the re s u l t of the d i r e c t stimulation of protein kinase C, a membrane-associated serine/threonine kinase, and ac t i v a t i o n of protein kinase C res u l t s in both stimulatory and i n h i b i t o r y e f f e c t s upon various substrate proteins (Nishizuka, 1988). TPA exposure re s u l t s in reduced phosphorylation of c e l l u l a r proteins in HL-525 c e l l s compared with HL-60 (Anderson, et a l . r 1985), although t h i s is not believed to be due to a reduction in protein kinase C level s or to decreased binding of TPA by protein kinase C (Solanki, et a l . , 1981). The observed differences between HL-60 and HL-525 c e l l s could be due to disruption in the complex regulation of protein kinase C: in addition to modifying the c-fes tr a n s c r i p t pool i t could also conceivably inactivate the breakdown of p92e=_f;«!"3, possibly by phosphorylation of relevant regulatory proteins. However, p92C=-E""" i t s e l f i s not known to be a substrate for protein kinase C, and there i s no evidence that TPA, through protein kinase C, a l t e r s the s p e c i f i c a c t i v i t y of p 9 2 0 - c " D , 154 either in HL-60 or HL-525 c e l l s . But, protein kinase C is known to modulate the a c t i v i t y of other protein-tyrosine kinases by s p e c i f i c serine and threonine phosphorylation (Carpenter, 1987; Livneh, et a l . f 1988), and subtle d i s t i n c t i o n s , which are not detected here, may exi s t in the a c t i v i t i e s of HL-60 and HL-525 p92° - i : ,»" following exposure to TPA. Results have shown that protein kinase C translocates from the soluble to the membrane f r a c t i o n of HL-60 c e l l s within minutes of exposure to TPA; th i s same subcellular translocation does not occur in HL-525 c e l l s and may be one of the c r u c i a l determinants of TPA-unresponsiveness (Homma, et a l . , 1986). A translocation mechanism might be used by the c e l l to expand the repertoire of available substrates for protein kinase C or for other kinases, p o t e n t i a l l y including p9 2 =~ ; the f a i l u r e of such a proposed translocation in HL-525 c e l l s would have major functional ramifications, and yet no indication would be expected from the in v i t r o kinase assay, which does not dist i n g u i s h between membrane-associated and soluble forms of protein-tyrosine kinases. Subcellular protein l o c a l i z a t i o n is c l e a r l y an important correlate of correct function, and aberrent l o c a l i z a t i o n , such as exists when comparing NCP98 with i t s transforming counterpart P H O * * * - * * - , may be an important determinant of oncogenicity (Young, et a l . , 1984); differences in i n t r a c e l l u l a r d i s t r i b u t i o n of p92'=~e'a'a may contribute to the malignant phenotype, yet these alterations would not be detected in the assay system used here. 155 CHAPTER 5-5.0 SUMMARY Thes e i n v e s t i g a t i o n s were u n d e r t a k e n i n o r d e r t o i d e n t i f y and c h a r a c t e r i z e t h e human and m u r i n e c - f e s p r o t e i n s . The i d e n t i f i c a t i o n o f t h e s e p r o t e i n s was i m p o r t a n t b e c a u s e some d a t a f r o m e x p e r i m e n t a l a n i m a l s i n d i c a t e d t h a t homologous c - f p s / f e s p r o t e i n s were c o n f i n e d t o h e m p o i e t i c c e l l s , and d a t a on c - f e s t r a n s c r i p t s i n human tumors t e n d e d t o s u p p o r t t h i s o b s e r v a t i o n . Thus, t h e p o s s i b i l i t y e x i s t e d t h a t d i f f e r e n c e s i n t h e e x p r e s s i o n o f t h e s e p r o t e i n s c o n t r i b u t e s i g n i f i c a n t l y t o t h e n e o p l a s t i c p h e n o t y p e and m i g h t be d e t e c t a b l e . U s i n g a v a r i e t y o f i m m u n o p r e c i p i t a t i o n t e c h n i q u e s two p r o t e i n s o f Mr 92,000 and 94,000 were i d e n t i f i e d and shown t o be p r o t e i n - t y r o s i n e k i n a s e s . S t r u c t u r a l and f u n c t i o n a l c o m p a r i s o n s s u g g e s t e d t h a t t h e 92 kD p r o t e i n i s v e r y c l o s e l y r e l a t e d t o known c - f p s / f e s p r o t e i n s and hence was named p 9 2 a - e " ~ . On t h e o t h e r hand, t h e 94 kD p r o t e i n a p p e a r e d t o be a l e g i t i m a t e p r o t e i n - t y r o s i n e k i n a s e , b u t f u r t h e r c h a r a c t e r i z a t i o n r e s u l t e d i n t h e c o n c l u s i o n t h a t i t does n o t r e p r e s e n t a c - f p s / f e s p r o t e i n . The r e s u l t s o f o t h e r i n v e s t i g a t o r s s h o w i n g t h a t c - f p s / f e s p r o t e i n s a r e p r e s e n t p r i m a r i l y i n h e m o p o i e t i c t i s s u e s was c o n f i r m e d f o r human and m urine c e l l s ; however no o b v i o u s q u a l i t a t i v e o r q u a n t i t a t i v e d i f f e r e n c e s i n p 9 2 = _ £ , a " were d e l i n e a t e d between n o r m a l and l e u k e m i c s o u r c e s . 156 Changes in the expression of c-fes in a differentiation-responsive and unresponsive myeloid leukemia c e l l l i n e were investigated following exposure to the inducer TPA. The most dramatic changes occurred in the levels of c-fes t r a n s c r i p t s , and each c e l l l i n e displayed i t s own d i s t i n c t i v e response. However, despite remarkable alt e r a t i o n s in mRNA lev e l s , p9 2"="" c""n in v i t r o kinase a c t i v i t y remained constant following exposure to TPA. Although changes in s p e c i f i c a c t i v i t y could not be ruled out, the unwavering a b i l i t y to detect f u l l l evels of c-fes protein led to the conclusion that t h i s protein is f u l f i l l i n g some important role In the myeloid leukemia c e l l s examined. The normal role of p92 = _ e"", l i k e most protein-tyrosine kinases, remains enigmatic. However, the ti g h t association between the expression of t h i s enzyme and hemopoietic c e l l s , e s p e c i a l l y of the myeloid lineages, alludes to some important, regulatory or lineage determining r o l e . 157 REFERENCES Abramson, S., M i l l e r , R.G., P h i l l i p s , R.A. (1977). The i d e n t i f i c a t i o n in adult bone marrow of pluripotent and r e s t r i c t e d stem c e l l s of the myeloid and lymphoid systems. J. Exp. Med. 145: 1567-1579. Alema, S., Tato, F., Boettiger, D. (1985). myc and src oncogenes have complementary effects of c e l l p r o l i f e r a t i o n and expression of s p e c i f i c e x t r a c e l l u l a r matrix components in d e f i n i t i v e chondroblasts. Mol. C e l l . B i o l . 5: 538-544. Anderson, N.L., Gemmell, M.A., Coussens, P.M., Murao, S., Huberman, E. (1985). S p e c i f i c protein phosphorylation in human promyelocytic HL-60 leukemia c e l l s susceptible or resis t a n t to induction of c e l l d i f f e r e n t i a t i o n by phorbol-12-myristate-13-acetate. Cane. Res. 45: 4955-4962. Angel, P., A l l e g r e t t o , E.A., Okino, S.T., Hattori, K., Boyle, W.J., Hunter, T., Karin, M. (1988). Oncogene 1un encodes a sequence-specific trans-activator similar to AP-1. Nature 332: 166-171. Balmain, A., Pragnell, I.B. (1983). Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-r_aLS_ oncogene. Nature 303: 72-74. Barbacid, M., Beemon, K., Devare, S.G. (1980). Origin and functional properties of the major gene product of the Snyder-Theilen s t r a i n of f e l i n e sarcoma vi r u s . Proc. Natl. Acad. USA 77: 5158-5162. Barbacid, M., Donner, L., Ruscetti, S.K., Sherr, C.J. (1981). Transformation-defective mutants of Snyder-Theilen f e l i n e sarcoma virus lack t y r o s i n e - s p e c i f i c protein kinase a c t i v i t y . J. V i r o l . 39: 246-254. Barbacid, M. (1987). ras Genes. Ann. Rev. Biochem. 56: 779-827. Bargmann, C.I., Hung, M.-C, Weinberg, R.A. (1986). Multiple independent activations of the neu oncogene by a point mutation a l t e r i n g the transmembrane domain of pl85. C e l l 45: 649-657. Barnekow, A., Gessler, M. (1986). Activation of the pp60=_ "*1CD kinase during d i f f e r e n t i a t i o n of monomyelocytic c e l l s in v i t r o . EMBO J. 5: 701-705 Baserga, R. (1981). The c e l l cycle. N. Engl. J. Med. 304: 453-459. Beckmann, M.P., Betsholtz, C , Heldin, C.-H., Westermark, B., Di Marco, E., Di Fiore, P.P., Robbins, K.C., Aaronson, S.A. (1988). Comparison of b i o l o g i c a l properties and 158 transforming potential of human PDGF-A and PDGF-B chains. Science 241: 1346-1349. Beemon, K., Hunter, T. (1978). Characterization of Rous sarcoma virus src gene products synthesized in v i t r o . J. V i r o l . 28: 551-566. Beemon, K. (1981). Transforming proteins of some f e l i n e and avian sarcoma viruses are related s t r u c t u r a l l y and fu n c t i o n a l l y . C e l l 24: 145-153. Benbrook, D., Lernhardt, E., Pfahl, M. (1988). A new r e t i n o i c acid receptor i d e n t i f i e d from a hepatocellular carcinoma. Nature 333: 669-672. Ben-Neriah, Y., Bauskin, A.R. (1988). Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an e x t r a c e l l u l a r domain. Nature 333: 672-676. Beug, H., Leutz, A, Kahn, P., Graf, T. (1984). Ts mutants of E26 leukemia virus allow transformed myeloblasts, but not erythroblasts or f i b r o b l a s t s , to d i f f e r e n t i a t e at the nonpermissive temperature. C e l l 39: 579-588. Bishop, J.M. (1982). Functions and origins of r e t r o v i r a l transforming genes. In Molecular Biology of Tumor Viruses, RNA Tumor Viruses, ed. R. Weiss, N. Teich, H. Varmus, J. C o f f i n . pp. 999-1108. Cold Spring Harbor Press: Cold Spring Harbor, N.Y. Bishop, J.M. (1987). The molecular genetics of cancer. Science 235: 305-311. Bohmann, D., Bos, T.J., Admon, A., Nishimura T., Vogt, P.K., Tjian, R. (1987). Human proto-oncogene c-1un encodes a DNA binding protein with s t r u c t u r a l and functional properties of tr a n s c r i p t i o n factor AP-1. Science 238: 1386-1392. Bos, J.L., Toksoz, D., Marshall, C.J., Verlaan-de Vries, M., Veeneman, G.H., van der Eb, A.J., van Boom, J.H., Janssen, J.W.G., Steenvoorden, A.CM. (1985). Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. Nature 315: 726-730 Bos, J.L., Fearon, E.R., Hamilton, S.R., Verlaan-de Vries, M., van Boom, J.H., van der Eb, A.J., Vogelstein, B. (1987). Prevalence of ras gene mutations in human col o r e c t a l cancers. Nature 327: 293-297. Bradley, T.R., Metcalf, D. (1966). The growth of mouse bone marrow c e l l s in v i t r o . Austr. J. Exp. B i o l . Med. S c i . 44: 287-300. Bronzert, D.A., Pantazis, P., Antoniades, H.N., Kasid, A., Davidson, N., Dickson, R.B., Lippman, M.E. (1987). 159 Synthesis and secretion of platelet-derived growth factor by human breast cancer c e l l l i n e s . Proc. Natl. Acad. S c i . USA 84: 5763-5767. Buick, R.N., Minden, M.D., McCulloch, E.A. (1979). Se l f -renewal in culture of p r o l i f e r a t i v e blast progenitor c e l l s in acute myeloblastic leukemia. Blood 54: 95-104. Buss, J.E., Sefton, B.M. (1985). Myristic acid, a rare fatty acid, i s the l i p i d attached to the transforming protein of Rous sarcoma virus and i t s c e l l u l a r homolog. J. V i r o l . 53: 7-12. Campbell, A.D., Long, M.W., Wicha, M.S. (1987). Haemonectin, a bone marrow adhesion protein s p e c i f i c for c e l l s of granulocyte lineage. Nature 329: 744-746. Carpenter, C. (1987). Receptors for epidermal growth factor and other polypeptide mitogens. Ann. Rev. Biochem. 56: 881-914 . Cartwright, C.A., Eckhart, W., Simon, S., Kaplan, P. (1987). C e l l transformation by pp60<=_**'e'= mutated in the carboxy-terminal regulatory domain. C e l l 49: 83-91. Cathala, G., Savouret, J.-F., Mendez, B., West, B.L., Karin, M., Mar t i a l , J.A., Baxter, J.D. (1983). A method for i s o l a t i o n of intact, t r a n s l a t i o n a l l y active ribonucleic acid. DNA 2: 329-335. Chan, L.C., Karhi, K.K., Rayter, S.I., Heisterkamp, N., E r i d a n i , S., Powles, R., Lawler, S.D., Groffen, J., Fouikes, J.G., Greaves, M.F., Wiedemann, L.M. (1987). A novel c-abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 325: 635-637. Chang, L.J.A., T i l l , J.E., McCulloch, E.A. (1980). The c e l l u l a r basis of s e l f renewal in culture by human acute myeloblastic leukemia blast c e l l progenitors. J. C e l l . Physiol. 102: 217-222. Chiu, I.-M., Reddy, E.P., Givol, D., Robbins, K.C., Tronick, S.R., Aaronson, S.A. (1984). Nucleotide sequence analysis i d e n t i f i e s the human c-s i s proto-oncogene as a s t r u c t u r a l gene for platelet-derived growth factor. C e l l 37: 123-129. Clark, S.C., McLaughlin, J., Timmons, M., Pendergast, A.M., Ben-Neriah, Y., Dow, L.W., C r i s t , W., Rovera, G., Smith, S.D., Witte, O.N. (1988). Expression of a d i s t i n c t i v e BCR-ABL oncogene in Phi-positive acute lymphocytic leukemia(ALL). Science 239: 775-777. Cleveland, D.W., Stuart, G.F., Kirschner, M.W., Laemmli, K. (1977). Peptide mapping by limited gel proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. 160 J. B i o l . Chem. 252: 1102-1106. Cohen, D.A., Kaplan, A.M. (1981). Adherent Ia* murine tumor lin e s with c h a r a c t e r i s t i c s of dendrit i c c e l l s . J. Exp. Med. 22: 1881-1897. Cole, M.D. (1986). The myc oncogene: i t s role in transformation and d i f f e r e n t i a t i o n . Ann. Rev. Genet. 20: 361-384. C o l l e t t , M. S., Erikson, R.L. (1978a). Protein kinase a c t i v i t y associated with the avian sarcoma virus src gene product. Proc. Natl. Acad. S c i . USA 75: 2021-2024. C o l l e t t , M.S., Brugge, J.S., Erikson, R.L. (1978b). Characterization of a normal avian c e l l protein related to the avian sarcoma virus transforming gene product. C e l l 15: 1363-1369. C o l l i n s , S.J. (1987). The HL-60 promyelocytic leukemia c e l l l i n e : p r o l i f e r a t i o n , d i f f e r e n t i a t i o n , and c e l l u l a r oncogene expression. Blood 70: 1233-1244. Cooper, J.A., Hunter, T. (1981). Four d i f f e r e n t classes of retroviruses induce phosphorylation of tyrosines present in simi l a r c e l l u l a r proteins. Mol. C e l l . B i o l . 1: 394-407. Cooper, J.A., Reiss, N.A., Schwartz, R.J., Hunter, T. (1983a). Three g l y c o l y t i c enzymes are phosphorylated at tyrosine in c e l l s transformed by Rous sarcoma vi r u s . Nature 302: 218-223. Cooper, J.A., Sefton, B.M., Hunter, T. (1983b). Detection and q u a n t i f i c a t i o n of phosphotyrosine in proteins. Meth. Ezymol. 99: 387-402. Cooper, J.A., Esch, F.S., Taylor, S.S., Hunter, T. (1984). Phosphorylation s i t e s in enolase and lactate dehydrogenase u t i l i z e d by tyrosine protein kinases in vivo and in v i t r o . J. B i o l . Chem. 259: 7835-7841. Cotton, P.C., Brugge, J.S. (1983). Neural tissues express high levels of the c e l l u l a r src gene product p p 6 0 Q - " 0 . Mol. C e l l . B i o l . 3: 1157-1162. Courtneidge, S.A., Levinson, A.D., Bishop, J.M. (1980). The protein encoded by the transforming gene of avian sarcoma virus (pp60 E 1 c o) and a homologous protein in normal c e l l s (pp60 E" r c , t o _"~ , =) are associated with the plasma membrane. Proc. Natl. Acad. S c i . USA 77: 3783-3787. Courtneidge, S.A. (1985). Activation of the pp60'=-<B1!:<= kinase by middle T antigen binding or by dephosphorylation. EMBO J. 4: 1471-1477. Courtneidge, S.A., Heber, A. (1987). An 81 kd protein 161 complexed with middle T antigen and p p e O " - " 0 : a possible phosphatidylinositol kinase. C e l l 50: 1031-1037. Coussens, P.M., Cooper, J.A., Hunter, T., Shalloway, D. (1985) . Re s t r i c t i o n of the in v i t r o and in vivo tyrosine protein kinase a c t i v i t i e s of pp60<=-"x:<= r e l a t i v e to pp60 v _ Mol. C e l l . B i o l . 5: 2753-2763. Coussens, L., Van Beveren, C , Smith, D., Chen, E., M i t c h e l l , R.L., Isacke, CM., Verma, I.M., U l l r i c h , A. (1986) . Structural a l t e r a t i o n of v i r a l homologue of receptor proto-oncogene fms at carboxyl terminus. Nature 320: 277-280. Curran, T., Teich, N.M. (1982). I d e n t i f i c a t i o n of a 39,000-dalton protein in c e l l s transformed by the FBJ murine osteosarcoma v i r u s . Virology 116: 221-235. Daley, D.Q., McLaughlin, J., Witte, O.N., Baltimore, D. (1987) . The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 f i b r o b l a s t s . Science 237: 532-535. Davis, R.L., Konopka, J.B., Witte, O.N. (1985). Activation of the c-abl oncogene by v i r a l transduction or chromosomal translocation generates altered c-abl proteins with similar in v i t r o kinase properties. Mol. C e l l . B i o l . 5: 204-213. DeFeo, D., Gonda, M.A., Young, H.A., Chang, E.H., Lowy, D.R., Scolnick, E.M., E l l i s R.W. (1981). Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus. Proc. Natl. Acad. S c i . USA 78: 3328-3332. DeFeo-Jones, D., Scolnick, E.M., Roller, R., Dhar, R. (1983). ras-Related gene sequences i d e n t i f i e d and isolated from Saccharomyces cerevisiae. Nature 306: 707-709. DeFeo-Jones, D., T a t c h e l l , K., Robinson, L.C., S i g a l , I.S., Vass, W.C, Lowy, D.R., Scolnick, E.M. (1985). Mammalian and yeast ras gene products: b i o l o g i c a l function in their heterologous systems. Science 228: 179-184. Degen, J.L., Neubauer, M.G., Friezner Degen, S.J., Seyfrled, C.E., Morris, D.R. (1983). Regulation of protein synthesis in mitogen-activated bovine lymphocytes. J. B i o l . Chem. 258: 12153-12162. De Larco, J.E., Todaro, G.J. (1978). Growth factors from murine sarcoma virus-transformed c e l l s . Proc. Natl. Acad. Sc i . USA 75: 4001-4005. D e l l i Bovi, P., B a s i l i c o , C (1987a). Isolation of a rearranged human transforming gene following transfection of Kaposi's sarcoma DNA. Proc. Natl. Acad. S c i . USA 84: 5660-162 5664 . D e l l i Bove, P., Curatola, A.M., Kern, F.G., Greco, A., Ittmann, M., B a s i l i c o , C. (1987b). An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that i s a member of the FGF family. C e l l 50: 729-737. Dexter, T.M., Allen , T.D., Lajtha, L.G. (1977). Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of hemopoietic stem c e l l s in v i t r o . J . C e l l . Physiol. 91: 335-344. Dexter, T.M., Spooncer, E., Schofield, R., Lord, B.I., Simmons, P. (1986). Haemopoietic stem c e l l s and the problem of self-renewal. Blood C e l l s 10: 315-339. Dexter, T.M., Spooncer, E. (1987). Growth and d i f f e r e n t i a t i o n in the hemopoietic system. Ann. Rev. C e l l B i o l . 3: 423-421. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., U l l r i c h , A., Schlessinger, J., Waterfield, M.D. (1984a). Close s i m i l a r i t y of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 307: 521-527. Downward, J . , Parker, P., Waterfield, M.D. (1984b). Autophosphorylation s i t e s on the epidermal growth factor receptor. Nature 311: 483-485. Downward, J., Waterfield, M.D., Parker, P. (1985). Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. J. B i o l . Chem. 260: 14538-14546. Duesberg, P.H. (1987). Cancer genes: rare recombinants instead of activated oncogenes(a review). Proc. Natl. Acad. S c i . USA 84: 2117-2124. Dunn, T.B., Potter, M. (1957). A transplantable mast c e l l neoplasm in the mouse. J. Natl. Cancer Inst. 18: 587-595. Durst, M., Croce, CM., Gissmann, L., Schwarz, E., Huebner, K. (1987). Papillomavirus sequences integrate near c e l l u l a r oncogenes in some c e r v i c a l carcinomas. Proc. Natl. Acad. S c i . USA 84: 1070-1074. Emilia, G., Donelli, A., F e r r a r i , S., T o r e l l i , U., S e l l e r i , L., Zucchini, P., Moretti, L., V e n t u r e l l i , D., C e c c h e r e l l i , G., T o r e l l i , G. (1986). C e l l u l a r levels of mRNA from c-myc, c-mybf and c-f£s_ onc-genes in normal myeloid and erythroid precursors of human bone marrow: an in s i t u hybridization study. Br. J. Hematol. 62: 287-292. Epstein, A.L., Levy, R., Kim, H., Henle, W., Henle, G., Kaplan, H.S. (1978). Biology of the human malignant 163 lymphomas. IV. Functional characterization o£ ten diffuse h i s t i o c y t i c lymphoma c e l l l i n e s . Cancer 42: 2379-2391. Erneux, C , Cohen, S., Garbers, D.L. (1983). The kinetics of tyrosine phosphorylation by the p u r i f i e d epidermal growth factor receptor kinase of A-431 c e l l s . J. B i o l . Chem. 258: 4137-4142. Even, J., Anderson, S.J., Hampe, A., Galibert, F., Lowy, D., Khoury, G., Sherr, C.J. (1983). Mutant f e l i n e sarcoma proviruses containing the v i r a l oncogene (v-fes) and either f e l i n e or murine control elements. J. V i r o l . 45: 1004-1016. Farrar, J.J., F u l l e r - F a r r a r , T., Simon, P.L., H i l f i k e r , M.L., Stadler, B.M., Farrar, W.L. ( 1 9 8 0 ) . Thymoma production of T c e l l growth factor (interleukin 2). J. Immunol. 125: 2555-2558. Fauser, A.A., Kanz, L., Lohr, G.W. (1985). I d e n t i f i c a t i o n of B c e l l s in multilineage hematopoietic colonies derived from c e l l s of patients with lymphocytic lymphoma. Proc. Natl. Acad. S c i . USA 82: 883-885. Fearon, E.R., Burke, P.J., S c h i f f e r , C.A., Zehnbauer, B.A., Vogelstein, B. (1986). D i f f e r e n t i a t i o n of leukemia c e l l s to polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia. N. Engl. J. Med. 315: 15-24. Feinberg, A.P., Vogelstein, B. (1983). A technique for radiolabeling DNA restriction-endonuclease fragments to high s p e c i f i c a c t i v i t y . Anal. Biochem. 132: 6-13. Feldman, R.A., Hanafusa, T., Hanafusa, H. (1980). Characterization of protein kinase a c t i v i t y associated with the transforming gene product of Fujinami sarcoma vi r u s . C e l l 22: 757-765. Feldman, R.A., Wang, E., Hanafusa, H. (1983). Cytoplasmic l o c a l i z a t i o n of the transforming protein of Fujinami sarcoma vi r u s : s a l t - s e n s i t i v e association with subcellular components. J. V i r o l . 45: 782-791. Feldman, R.A., Gabrilove, J.L., Tarn, J.P., Moore, M.A.S., Hanafusa, H. (1985). S p e c i f i c expression of the human c e l l u l a r fps/fes-encoded protein NCP92 in normal and leukemic myeloid c e l l s . Proc. Natl. Acad. S c i . USA 82: 2379-2383. Feldman, R.A., Tarn, J.P., Hanafusa, H. (1986). Antipeptide antiserum i d e n t i f i e s a widely di s t r i b u t e d c e l l u l a r tyrosine kinase related to but d i s t i n c t from the c-fps/fes-encoded protein. Mol. C e l l . B i o l . 6: 1065-1073. F e r r a r i , S., T o r e l l i , U., S e l l e r i , L., Donelli, A., Ve n t u r e l l i , D., Moretti, L., T o r e l l i , G. (1985). Expression 164 of human c-fes onc-gene occurs at detectable levels in myeloid but not in lymphoid c e l l populations. Br. J. Hematol. 59: 21-25. Fialkow, P.J., Jacobson, R.J., Papayannopoulou, T. (1977). Chronic myelocytic leukemia: Clonal o r i g i n in a stem c e l l common to the granulocyte, erythrocyte, p l a t e l e t and monocyte/macrophage. Am. J. Med. 63: 125-130. Fialkow, P.J., Singer, J.W., Adamson, J.W., Vaidya, K., Dow, L.W., Ochs, J., Moohr, J.W. (1981a). Acute nonlymphocytic leukemia: heterogeneity of stem c e l l o r i g i n . Blood 57: 1068-1073. Fialkow, P.J., Martin, P.J., Najfeld, V., Penfold, G.K., Jacobson, R.J., Hansen, J.A. (1981b). Evidence for a multistep pathogenesis of chronic myelogenous leukemia. Blood 58: 158-163. Fialkow, P.J. (1984) . Clonal evolution of human myeloid leukemias. In J.M. Bishop, J.D. Rowley, and M. Greaves(ed.). Genes and cancer, pp. 215-226. Alan R. L i s s , Inc., New York, New York. Fisher, P.B., Schachter, D., Abbott, R.E., Callaham, M.F., Huberman, E. (1984). Membrane l i p i d dynamics in human promyelocytic leukemia c e l l s sensitive and resi s t a n t to 12-O-tetradecanoylphorbol-13-acetate induction of d i f f e r e n t i a t i o n . Cane. Res. 44: 5550-5554. Fleischman, L.F., Chahwala, S.B., Cantley, L. (1986). Ras-transformed c e l l s : altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites. Science 231: 407-410. Forrester, K., Almoguera, C , Han, K., G r i z z l e , W.E., Perucho, M. (1987). Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 327: 298-303. Foster, D.A., Shibuya, M., Hanafusa, H. (1985). Activation of the transformation potential of the c e l l u l a r fps gene. C e l l 42: 105-115. Franchini, G., Gelmann, E.P., Dalla Favera, R., Gallo, R.C, Wong-Staal, F. (1982). Human gene(c-fes) related to the one sequences of Snyder-Theilen f e l i n e sarcoma v i r u s . Mol. C e l l . B i o l . 2: 1014-1019. Friedman, B., Frackleton, A.R., Ross, A.H., Connors, J.M., F u j i k i , H., Sugimura, T., Rosner, M.R. (1984). Tumor promoters block t y r o s i n e - s p e c l f i c phosphorylation of the epidermal growth factor receptor. Proc. Natl. Acad. S c i . USA 81: 3034-3038. Friend, C , Scher, W., Holland, J., Sato, T. (1971). 165 Hemoglobin synthesis in murine virus-induced leukemic c e l l s In v i t r o : stimulation of erythroid d i f f e r e n t i a t i o n by dimethyl sulfoxide. Proc Natl. Acad S c i . USA 68: 378-382. Fujiyama, A., Tamanoi, F. (1986). Processing and f a t t y acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. S c i . USA 83: 1266-1270. Gee, C.E., G r i f f i n , J . , Sastre, L., M i l l e r , L.J., Springer, T.A., Piwnica-Worms, H., Roberts, T.M. (1986). D i f f e r e n t i a t i o n of myeloid c e l l s i s accompanied by increased levels of pp60°-" r r , = protein and kinase a c t i v i t y . Proc. Natl. Acad. S c i . USA 83: 5131-5135. Gilman, A.G. (1984). G Proteins and dual control of adenylate cyclase. C e l l 36: 577-579. Golden, A., Nemeth, S., Brugge, J.S. (1986). Blood p l a t e l e t s express high levels of the p p e O ^ - ^ ^ - s p e c i f i c tyrosine kinase a c t i v i t y . Proc. Natl. Acad. S c i . USA 83: 852-856. Greaves, M.F., Chan, L.C., Furley, A.J.W., Watt, S.M., Molgaard, H.V. (1986). Lineage promiscuity in hemopoietic d i f f e r e n t i a t i o n and leukemia. Blood 67: 1-11. Greenberger, J.S., Sakakeeny, M.-A., Humphries, R.K., Eaves, C.J., Eckner, R.J. (1983). Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/ basophil) hematopoietic progenitor c e l l l i n e s . Proc. Acad. S c i . USA 80: 2931-2935. Greer, P.A., Meckling-Hansen, K., Pawson, T. (1988). The human c-fps/fes gene product expressed e c t o p i c a l l y in rat fib r o b l a s t s is nontransforming and has restrained protein-tyrosine kinase a c t i v i t y . Mol. C e l l . B i o l . 8: 578-587. G r i f f i n , J.D., Lowenberg, B. (1986). Clonogenic c e l l s in acute myeloblastic leukemia. Blood 68: 1185-1195. Groffen, J ., Heisterkamp, N., Grosveld, F., Van de Ven, W., Stephenson, J.R. (1982). Isolation of human oncogene sequences(v-fes homolog) from a cosmid l i b r a r y . Science 216: 1136-1138. Groffen, J., Heisterkamp, N., Shibuya, M., Hanafusa, H., Stephenson, J.R. (1983). Transforming genes of avian(v-fps) and mammalian(v-fes) retroviruses correspond to a common c e l l u l a r locus. Virology 125: 480-486. Hampe, A., Laprevotte, I., Galibert, F., Fedele, L.A., Sherr, C.J. (1982). Nucleotide sequences of fe l i n e r e t r o v i r a l oncogenes (v-fe_s_) provide evidence for a family of ty r o s i n e - s p e c i f i c protein kinase genes. C e l l 30: 775-785. Hao, Q.-L., Heisterkamp, N., Groffen, J. (1989). Isolation 166 and sequence analysis of a novel human tyrosine kinase gene. Mol. C e l l . B i o l . 9: 1587-1593. Harley, C.B. (1987). Hybridization of oligo(dT) to RNA on n i t r o c e l l u l o s e . Gene Anal. Techn. 4: 17-22. Hayward, W.S., Neel, B.G., Astr i n , S.M. (1981). Activation of a c e l l u l a r one gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 290: 475-480. Heisterkamp, N., Groffen, J., Stephenson, J.R., Spurr, N.K., Goodfellow, P.N., Solomon, E., C a r r i t t , B., Bodmer, W.F. (1982). Chromosomal l o c a l i z a t i o n of human c e l l u l a r homologues of two v i r a l oncogenes. Nature 299: 747-749. Heisterkamp, N., Stephenson, J.R., Groffen, J., Hansen, P.F., de Klein, A., Bartram, C.R., Grosveld, G. (1983). Lo c a l i z a t i o n of the c-abl oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature 306: 239-242. Heldin, C-H., Johnsson, A., Wennergren, S., Wernstedt, C , Betsholtz, C , Westermark, B. (1986). A human osteosarcoma c e l l l i n e secretes a growth factor s t r u c t u r a l l y related to a homodimer of PDGF A-chains. Nature 319: 511-514. H i r a i , H., Kobayashi, Y., Mano, H., Hagiwara, K., Maru, Y., Omine, M., Mizoguchi, H., Nishida, J ., Takaku, F. (1987). A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature 327: 430-432. H o l l i s , G.F., Gazdar, A.F., Bertness, V., Kirsch, I.R. (1988). Complex translocation disrupts c-myc regulation in a human plasma c e l l myeloma. Mol. C e l l . B i o l . 8: 124-129. Homma, Y., Henning-Chubb, C.B., Huberman, E. (1986). Translocation of protein kinase C in human leukemia c e l l s susceptible or resist a n t to d i f f e r e n t i a t i o n induced by phorbol 12-myristate 13-acetate. Proc. Natl. Acad. S c i . USA 83: 7316-7319. Huang, C.-C, Hammond, C , Bishop, J.M. (1985). Nucleotide sequence and topography of chicken c-fps. Genesis of a r e t r o v i r a l oncogene encoding a t y r o s i n e - s p e c i f i c protein kinase. J. Mol. B i o l . 181: 175-186. Huberman, E., Callaham, M.F. (1979). Induction of terminal d i f f e r e n t i a t i o n in human promyelocyte leukemia c e l l s by tumor-promoting agents. Proc. Natl. Acad. S c i . USA 76: 1293-1297. Huberman, E., Braslawsky, G.R., Callaham, M., Fugiki, H. (1982). Induction of d i f f e r e n t i a t i o n of human promyelocytic leukemia(HL-60) c e l l s by t e l e o c i d i n and phorbol-12-myristate-13-acetate. Carcinogenesis 3: 111-114. 167 Hunter, T., sefton, B.M. (1980) . Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. S c i . USA 77: 1311-1315. Hunter, T., Cooper, J.A. (1981). Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor c e l l s . C e l l 24: 741-752. Hunter, T., Ling, N., Cooper, J.A. (1984). Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature 311: 480-483. Hunter, T., Cooper, J.A. (1985) . Protein-tyrosine kinases. Ann. Rev. Biochem. 54: 897-930. Hunter, T., Cooper, J.A. (1986). V i r a l oncogenes and tyrosine phosphorylation. In P.D. Boyer and E.G. Krebs(ed.). The Enzymes; XVII; Control by phosphorylation, Part A, pp. 191-246. Academic Press, Inc., Orlando, Flor ida. Hunter, T. (1987a). A t a i l of two src's: mutatis mutandi. C e l l 49: 1-4. Hunter, T. (1987b). A thousand and one protein kinases. C e l l 50: 823-829. Iba, H., Cross, F.R., Garber, E.A., Hanafusa, H. (1985). Low le v e l of c e l l u l a r protein phosphorylation by nontransf orming overproduced p&0<=-ai:C!. Mol. C e l l . B i o l . 5: 1058-1066. Ingebritsen, T.S., Cohen, P. (1983). Protein phosphatases: properties and role in c e l l u l a r regulation. Science 221: 331-338. Ingman-Baker, J., Hinze, E., Levy, J.G., Pawson, T. (1984). Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between d i f f e r e n t fps-encoded proteins. J. V i r o l . 50: 572-578. Jacobson, R.J., Raskind, W., Sacher, R.A., Shashaty, G., Singer, J.W., Fialkow, P.J. (1982). Refractory anemia(RA), a myelodysplastic syndrome: clonal development with progressive loss of normal committed progenitors. Blood 60(Suppl. 1): 129a. Johnsson, A., Betsholtz, C , Heldin, C.-H., Westermark, B. (1985a). Antibodies against platelet-derived growth factor i n h i b i t acute transformation by simian sarcoma virus. Nature 317: 438-440. Johnsson, A., Betsholtz, C , von der Helm, K., Heldin, C -H., Westermark, B. (1985b). Platelet-derived growth factor 168 agonist a c t i v i t y of a secreted form of the v-sis oncogene product. Proc. Natl. Acad. S c i . USA 82: 1721-1725. Kalousek, D.K., Eaves, C.J., Eaves, A.C. (1984). In-vitro cytogenetic studies of haemopoietic malignancies. Cancer Surveys 3: 439-463. Kamps, M.P., Buss, J.E., Sefton, B.M. (1985). Mutation of NHa-terminal glycine of p60"*:'= prevents both myristoylation and morphological transformation. Proc. Natl. Acad. S c i . USA 82: 4625-4628. Kaplan, D.R., Whitman, M., Schaffhausen, B., Pall a s , D.C, White, M., Cantley, L., Roberts, T.M. (1987). Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase a c t i v i t y . C e l l 50: 1021-1029. Kataoka, T., Powers, S., Cameron, S., Fasano, 0., Goldfarb, M., Broach, J., Wigler, M. (1985). Functional homology of mammalian and yeast RAS genes. C e l l 40: 19-26. Kawakaml, T., Pennington, C.Y., Robbins, K.C (1986). Isolation and oncogenic potential of a novel human s_r_£L-like gene. Mol. C e l l . B i o l . 6: 4195-4201. Keath, E.J., Kelekar, A., Cole, M.D. (1984). Transcriptional a c t i v a t i o n of the translocated c-myc oncogene in mouse plasmacytomas: similar RNA levels in tumor and p r o l i f e r a t i n g normal c e l l s . C e l l 37: 521-528. Keating, M.T., Williams, L.T. (1988). Autocrine stimulation of i n t r a c e l l u l a r PDGF receptors in v-sis-transformed c e l l s . Science, 239: 914-916. Klempnauer, K.-H., Symonds, G., Evan, G.I., Bishop, J.M. (1984). Subcellular l o c a l i z a t i o n of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by the chicken c-myb gene. C e l l 37: 537-547. Kmiecik, T.E., Shalloway, D. (1987). Activation and suppression of p p e o ^ - " * 1 0 transforming a b i l i t y by mutation of i t s primary s i t e s of tyrosine phosphorylation. C e l l 49: 65-73 . Koeffle r , H.P., Golde, D.W. (1978). Acute myelogenous leukemia: a human c e l l l i n e responsive to colony-stimulating a c t i v i t y . Science 200: 1153-1154. Koeffler, H.P., B i l l i n g , R., Lusis, A.J., Sparkes, R., Golde, D.W. (1980). An undifferentiated variant derived from the human acute myelogenous leukemia c e l l l i n e (KG-1). Blood 56: 265-273. Koeffler, H.P., B a r - E l i , M., T e r r i t o , M.C (1981). Phorbol 169 ester e f f e c t on d i f f e r e n t i a t i o n of human myeloid leukemia c e l l l i n e s blocked at d i f f e r e n t stages of maturation, cane. Res. 41: 919-926. Koe f f l e r , H.P. (1983). Induction of d i f f e r e n t i a t i o n of human acute myelogenous leukemia c e l l s : therapeutic implications. Blood 62: 709-721. Koeffler, H.P., Amatruda, T., Ikekawa, N., Kobayashi, Y., DeLuca, H.F. (1984). Induction of macrophage d i f f e r e n t i a t i o n of human normal and leukemic myeloid stem c e l l s by 1,25-dihydroxyvitamin D 3 and i t s fluorinated analogues. Cane. Res. 44: 5624-5628. Kohler, G., Howe, S.C., M i l s t e i n , C. (1976). Fusion between immunoglobulin-secreting and nonsecreting myeloma c e l l l i n e s . Eur. J. Immunol. 6: 292-295. Konopka, J.B., Watanabe, S.M., Witte, O.N. (1984). An a l t e r a t i o n of the human c-abl protein in K562 leukemia c e l l s unmasks associated tyrosine kinase a c t i v i t y . C e l l 37: 1035-1042. K r u i j e r , W., Cooper, J.A., Hunter, T., Verma, I.M. (1984). Platelet-derived growth factor induces rapid but transient expression of the c-fos gene and protein. Nature 312: 711-716 . Kurzock, R., S h t a l r i d , M., Romero, P., Kloetzer, W.S., Talpas, M., T r u j i l l o , J.M., B l i c k , M., Beran, M., Gutterman, J.U. (1987). A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia. Nature 325: 631-635. Laemmli, U.K. (1970). Cleavage of s t r u c t u r a l proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Land, H., Parada,L.F., Weinberg, R.A. (1983). Tumorigenic conversion of primary embryo fi b r o b l a s t s requires at least two cooperating oncogenes. Nature 304: 596-602. Lang, R.A., Metcalf, D., Gough, N.M., Dunn, A.R., Gonda, T.J. (1985). Expression of a hemopoietic growth factor cDNA in a factor-dependent c e l l l i n e results in autonomous growth and tumorigenicity. C e l l 43: 531-542. Lang, R.A., Metcalf, D., Cuthbertson, R.A., Lyons, I., Stanley, E., Kelso, A., Kannourakis, G., Williamson, D.J., Klintworth, G.K., Gonda, T.J., Dunn, A.R. (1987). Transgenic mice expressing a hemopoietic growth factor gene(GM-CSF) develop accumulations of macrophages, blindness, and a f a t a l syndrome of tissue damage. C e l l 51: 675-686. Langer-Safer, P.R., Lehrman, S.R., Skalka, A.M. (1985). 170 v-src i n h i b i t s d i f f e r e n t i a t i o n via an e x t r a c e l l u l a r intermediate(s). Mol. C e l l . B i o l . 5: 2847-2850. Laskey, R.A., M i l l s , A.D. (1977). Enhanced autoradiographic detection of 32P and 1251 using in t e n s i f y i n g screens and hypersensitized f i l m . FEBS Lett. 82: 314-316. Lax, I., Johnson, A., Howk, R., Sap, J., Be l l o t , F., Winkler, M., U l l r i c h , A., Vennstrom, B., Schlessinger, J., Givol., D. (1988). Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse c e l l s , and d i f f e r e n t i a l binding of EGF and transforming growth factor alpha. Mol. C e l l . B i o l . 8: 1970-1978. Leder, P.,Battey, J., Lenoir, G., Moulding, C , Murphy, W., Potter, H., Stewart, T., Taub, R. (1983). Translocations among antibody genes in human cancer. Science 222: 765-771. Lee, W.-H., Murphree, A.L., Benedict, W.F. (1984). Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 309: 458-460. Lehrach, H., Diamond, D., Wozney, J.M., Boedtker, H. (1977). RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a c r i t i c a l reexamination. Biochemistry 16: 4743-4751. Levinson, A.D., Courtneidge, S.A., Bishop, J.M. (1981). Structural and functional domains of the Rous sarcoma virus transforming protein (pp60" =c:) . Proc. Natl. Acad. S c i . USA 78: 1624-1628. L i t t l e , CD., Nan, M.M., Carney, D.N., Gazdar, A.F., Minna, J.D. (1983). Amplification and expression of the c-myc oncogene in human lung cancer c e l l l i n e s . Nature 306: 194-196 . Liu, E., H j e l l e , B., Morgan, R., Hecht, F., Bishop, J.M. (1987). Mutations of the Kirsten-r_as_ proto-oncogene in human preleukaemia. Nature 330: 186-188. Livneh, E., Dull, T.J., Berent, E., Prywes, R., U l l r i c h , A., Schlessinger, J. (1988). Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor. Mol. C e l l . B i o l . 8: 2302-2308. Look, A.T., Peiper, S.C, Rebentisch, M.B., Ashmun, R.A., Roussel, M.F.,. Lemons, R.S., Le Beau, M.M., Rubin, CM., Sherr, C J . (1986). Molecular cloning, expression, and chromosomal l o c a l i z a t i o n of the gene encoding a human myeloid membrane antigen(gp 150). J. C l i n . Invest. 78: 914-921. Lord, B.I. (1983). Haemopoietic stem c e l l s . In C.S. Potten(ed.). Stem c e l l s : their i d e n t i f i c a t i o n and 171 characterisation, pp. 118-154. Chu r c h i l l Livingstone, New York, New York. Lozzio, C.B., Lozzio, B.B. (1975). Human chronic myelogenous leukemia c e l l - l i n e with positive Philadelphia chromosome. Blood 45: 321-334. Macara, I.G., Marinetti, G.V., Balduzzi, P.C. (1984). Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase a c t i v i t y : Possible role in tumorigenesis. Proc. Natl. Acad. S c i . USA 81: 2728-2732. MacDonald, I.A., Levy, J., Pawson, T. (1985). Expression of the mammalian c-fes protein in hematopoietic c e l l s and i d e n t i f i c a t i o n of a d i s t i n c t fes-related protein. Mol. C e l l . B i o l . 5: 2543-2551. Maki, Y., Bos, T.J., Davis, C , Starbuck, M., Vogt, P.K. (1987). Avian sarcoma virus 17 ca r r i e s the iun oncogene. Proc. Natl. Acad. S c i . USA 84: 2848-2852. Maniatis, T., F r i t s c h , E.F., Sambrook, J. (1982). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Marth, J.D., Peet, R., Krebs, E.G., Perlmutter, R.M. (1985). A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T c e l l lymphoma LSTRA. C e l l 43: 393-404. Marth, J.D., Cooper, J.A., King, C.S., Ziegler, S.F., Tinker, D.A., Overell, R.W., Krebs, E.G., Perlmutter, R.M. (1988a). Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase ( p p 5 6 l G K ) . Mol. C e l l . B i o l . 8: 540-550. Marth, J.D., Overell, R.W., Meier, K.E., Krebs, E.G., Perlmutter, R.M. (1988b). Translational a c t i v a t i o n of the lck proto-oncogene. Nature 332: 171-173. Martin, G.S. (1986). The erbB gene and the EGF receptor. Cancer Surv. 5: 199-219. Martin, P., Papayannopoulou, T. (1982). HEL c e l l s : a new human erythroleukemia c e l l l i n e with spontaneous and induced globin expression. Science 216: 1233-1235. Martin-Zanca, D., Hughes, S.H., Barbacid, M. (1986). A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature 319: 743-748. Mathey-Prevot, B., Hanafusa, H., Kawai, S. (1982). A c e l l u l a r protein is immunologically crossreactive with and funct i o n a l l y homologous to the Fujinami sarcoma virus 172 transforming protein. C e l l 28: 897-906. Mayes, E.L.V., Waterfield, M.D. (1984). Biosynthesis of the epidermal growth factor receptor in A431 c e l l s . EMBO J. 3: 531-537. McBurney, M.W., Jones-Villeneuve, E.M.v., Edwards, M.K.S., Anderson, P.J. (1982). Control of muscle and neuronal d i f f e r e n t i a t i o n in a cultured embryonal carcinoma c e l l l i n e . Nature 299: 165-167. McCarthy, D.M., San Miguel, J.F., Freake, H.C, Green, P.M., Zola, H., Catovsky, D., Goldman, J.M. (1983). 1,25-dihydroxyvitamin D 3 i n h i b i t s p r o l i f e r a t i o n of human promyelocytic leukaemia(HL-60) c e l l s and induces monocyte-macrophage d i f f e r e n t i a t i o n in HL-60 and normal bone marrow c e l l s . Leuk. Res. 7: 51-55. McCulloch, E.A. (1983). Stem c e l l s in normal and leukemic hemopoiesis. Blood 62: 1-13. McGrath, J.P., Capon, D.J.,Goeddel, D.V., Levinson, A.D. (1984) . Comparitive biochemical properties of normal and activated human ras p21 protein. Nature 310: 644-649. Messner, H.A., Izaguirre, C.A., Jamal, N. (1981). I d e n t i f i c a t i o n of T lymphocytes in human mixed hemopoietic colonies. Blood 58: 402-405. Messner, H.A. (1986). The role of CFU-GEMM in human hemopoiesis. Blut 53: 269-277. Metcalf, D. (1985). The granulocyte-macrophage colony-stimulating factors. Science 229: 16-22. Miles, B.D., Robinson, H.L. (1985). High-frequency transduction of c-erbB in avian leukosis virus-induced erythroblastosis. J. V i r o l . 54: 295-303. Minowada, J., Ohnuma, T., Moore, G.E. (1972). Rosette-forming human lymphoid c e l l l i n e s . I. Establishment and evidence of thymus-derived lymphocytes. J. Natl. Cancer Inst. 49: 891-895. M i t c h e l l , R.L., Zokas, L., Schreiber, R.D., Verma, I.M. (1985) . Rapid induction of the expression of proto-oncogene fos during human monocytic d i f f e r e n t i a t i o n . C e l l 40: 209-217. M i t c h e l l , R.L., Hennlng-Chubb, C , Huberman, E., Verma, I.M. (1986) . c-fos expression is neither s u f f i c i e n t nor obligatory for d i f f e r e n t i a t i o n of monomyelocytes to macrophages. C e l l 45: 497-504. Mougneau, E., Lemieux, L., Rassoulzadegan, M., Cuzin, F. 173 (1984 ). B i o l o g i c a l a c t i v i t i e s o£ v-nay_c_ and rearranged c-myc oncogenes in rat f i b r o b l a s t c e l l s ln culture. Proc. Natl. Acad. USA 81: 5758-5762. Muller, R., Bravo, R., Burckhardt, J., Curran, T. (1984). Induction of c-fos gene and protein by growth factors precedes a c t i v a t i o n of c-myc. Nature 312: 716-720. Neuman-Silberberg, F.S., Schejter, E., Hoffmann, F.M., Shilo, B.-Z. (1984). The Drosophila ras oncogenes: structure and nucleotide sequence. C e l l 37: 1027-1033. Nicola, N.A., Peterson, L. (1986). I d e n t i f i c a t i o n of d i s t i n c t receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical c r o s s - l i n k i n g . J. B i o l . Chem. 261: 12384-12389. Nilsen, T.W., Maroney, P.A., Goodwin, R.G., Rottman, F.M., Crittenden, L.B., Raines, M.A., Kung, H.-J. (1985). c-erbB a c t i v a t i o n in ALV-induced erythroblastosis: novel RNA processing and promoter insertion r e s u l t in expression of an amino-truncated EGF receptor. C e l l 41: 719-726. Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and i t s implications for c e l l u l a r regulation. Nature 334: 661-665. Nusse, R. (1986). The ac t i v a t i o n of c e l l u l a r oncogenes by r e t r o v i r a l i n s e r t i o n . Trends Genet. 2: 244-247. Owen, A.J., Pantazis, P., Antoniades, H.N. (1984). Simian sarcoma virus-transformed c e l l s secrete a mitogen i d e n t i c a l to platelet-derived growth factor. Science 225: 54-56. Palaszynski, E.W., Ihle, J.N. (1984). Evidence for s p e c i f i c receptors for interleukin 3 on lymphokine-dependent c e l l lines established from long-term bone marrow cultures. J. Immunol. 132: 1872-1878. Papkoff, J., Nigg, E.A., Hunter, T. (1983). The transforming protein of Moloney murine sarcoma virus is a soluble cytoplasmic protein. C e l l 33: 161-172. Patschinsky, T., Hunter, T., Esch, F.S., Cooper, J.A., Sefton, B.M. (1982). Analysis of the sequence of amino acids surrounding s i t e s of tyrosine phosphorylation. Proc. Natl. Acad. S c i . USA 79: 973-977. Pawson, T., Guyden, J . , Kung, T-H., Radke, K., Gilmore, T., Martin, G.S. (1980). A s t r a i n of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and c e l l u l a r transformation. C e l l 22: 767-775. Payne, G.S., Bishop, J.M., Varmus, H.E. (1982). Multiple 174 arrangements of v i r a l DNA and an a c t i v a t e d h o s t oncogene i n b u r s a l lymphomas. Nature 295: 209-214. P e g o r a r o , L., Abrahm, J . , Cooper, R.A., L e v i s , A., Lange B., Meo, P., Rovera, G. (1980). D i f f e r e n t i a t i o n of human leukem i a s i n response t o 1 2 - 0 - t e t r a d e c a n o y l p h o r b o l - 1 3 -a c e t a t e i n v i t r o . B l o o d 55: 859-862. P e l i c c i , P.-G., L a n f r a n c o n e , L., B r a t h w a i t e , M.D., Wolman, S.R., D a l l a - F a v e r a , R. (1984). A m p l i f i c a t i o n of the c-myb oncogene i n a case of human a c u t e myelogenous l e u k e m i a . S c i e n c e 224: 1117-1121. P e p i n s k y , R.B., S i n c l a i r , L.K. (1986). E p i d e r m a l growth f a c t o r - d e p e n d e n t p h o s p h o r y l a t i o n of l i p o c o r t i n . Nature 321: 81-84. P e t e r s , G., Lee, A.E., D i c k s o n , C. (1984). A c t i v a t i o n of c e l l u l a r gene by mouse mammary tumour v i r u s may occur e a r l y i n mammary tumour development. Nature 309: 273-275. P i e r c e , J.H., R u g g i e r o , M., F l e m i n g , T.P., Di F i o r e , P.P., Gre e n b e r g e r , J.S., V a r t i c o v s k i , L., S c h l e s s i n g e r , J . , Rovera, G., Aaronson, S.A. (1988). S i g n a l t r a n s d u c t i o n t h r o u g h the EGF r e c e p t o r t r a n s f e c t e d i n IL-3-dependent h e m a t o p o i e t i c c e l l s . S c i e n c e 239: 628-631. P i k e , L . J . , K u e n z e l , E.A., C a s n e l l i e , J.E., K r e b s , E.G. (1984). A comparison of the i n s u l i n - and e p i d e r m a l growth f a c t o r s t i m u l a t e d p r o t e i n k i n a s e s from human p l a c e n t a . J . B i o l . Chem. 259: 9913-9921. Piwnica-Worms, H., Saunders, K.B., R o b e r t s , T.M., Smith, A.E.,, Cheng, S.H. (1987). T y r o s i n e p h o s p h o r y l a t i o n r e g u l a t e s t h e b i o c h e m i c a l and b i o l o g i c a l p r o p e r t i e s of pp60° — C e l l 49: 75-82. P o l l a r d , J.W., B a r t o c c i , A., A r c e c i , R., O r l o f s k y , A., Ladner, M.B., S t a n l e y , E.R. (1987). Apparent r o l e of the macrophage growth f a c t o r , CSF-1, i n p l a c e n t a l development. Nature 330: 484-486. P u r c h i o , A.F. (1982). E v i d e n c e t h a t pp60mx:°, the p r o d u c t of the Rous sarcoma v i r u s s r c gene, undergoes a u t o p h o s p h o r y l a t i o n . J . V i r o l . 41: 1-7. Q u i n t r e l l , N., Lebo, R., Varmus, H., B i s h o p , J.M., P e t t e n a t i , M.J., Le Beau, M.M., D i a z , M.O., Rowley, J.D. (1987). I d e n t i f i c a t i o n of a human gene(HCK) t h a t encodes a p r o t e i n - t y r o s i n e k i n a s e and i s e x p r e s s e d i n h e m o p o i e t i c c e l l s . M o l . C e l l . B i o l . 7: 2267-2275. Rauscher, F . J . , Cohen, D.R., C u r r a n , T., Bos, T.J., Vogt, P.K., Bohmann, D., T j i a n , R., F r a n z a , B.R. (1988). Fos-a s s o c i a t e d p r o t e i n p39 i s the pr o d u c t of the j u n p r o t o -175 o n c o g e n e . S c i e n c e 240: 1010-1016. R e t t e n m i e r , C.W., S a c c a , R., Furman, W.L., R o u s s e l , M.F., H o l t , J . T . , N i e n h u i s , A.W., S t a n l e y , E.R., S h e r r , C . J . ( 1 9 8 6 ) . E x p r e s s i o n o f t h e human c-fms p r o t o - o n c o g e n e p r o d u c t ( C o l o n y - s t i m u l a t i n g f a c t o r - 1 r e c e p t o r ) on p e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s and c h o r i o c a r c i n o w a c e l l l i n e s . J . C l i n . I n v e s t . 77: 1740-1746. Reymond, C D . , Gomer, R.H., Mehdy, M . C , F i r t e l , R.A. ( 1 9 8 4 ) . D e v e l o p m e n t a l r e g u l a t i o n o f a D i c t y o s t e l i u m gene e n c o d i n g a p r o t e i n homologous t o mammalian r a s p r o t e i n . C e l l 39: 141-148. R i c h e r t , N.D., D a v i e s , P.J.A., J a y , G., P a s t a n , I.H. ( 1 9 7 9 ) . C h a r a c t e r i z a t i o n o f an immune complex k i n a s e i n i m m u n o p r e c i p i t a t e s of a v i a n sarcoma v i r u s - t r a n s f o r m e d f i b r o b l a s t s . J . V i r o l . 31: 695-706. R o e b r o e k , A.J.M., S c h a l k e n J.A., V e r b e e k , J . S . , Van den Ouweland, A.M.W., On n e k i n k , C , B l o e m e r s , H.P.J., Van de Ven, W.J.M. ( 1 9 8 5 ) . The s t r u c t u r e o f t h e human c - f e s / f p s p r o t o - o n c o g e n e . EMBO J . 4: 2897-2903. R o e b r o e k , A.J.M., S c h a l k e n , J.A., L e u n i s s e n , J.A.M., O n n e k i n k , C , B l o e m e r s , H.P.J., Van de Ven, W.J.M. ( 1 9 8 6 a ) . E v o l u t i o n a r y c o n s e r v e d c l o s e l i n k a g e of t h e c - f e s / f p s p r o t o -oncogene and g e n e t i c s e q u e n c e s e n c o d i n g a r e c e p t o r - l i k e p r o t e i n . EMBO J . 5: 2197-2202. R o e b r o e k , A.J.M., S c h a l k e n , J.A., Bu s s e m a k e r s , M.J.G., van Heer i k h u i z e n , H., On n e k i n k , C , Debruyne, F.M.J., B l o e m e r s , H.P.J., Van de Ven, W.J.M. ( 1 9 8 6 b ) . C h a r a c t e r i z a t i o n o f human c - f e s / f p s r e v e a l s a new t r a n s c r i p t i o n u n i t ( f u r ) i n t h e i m m e d i a t e l y u p s t r e a m r e g i o n o f t h e p r o t o - o n c o g e n e . M o l e c . B i o l . Rep. 11: 117-125. R o h r s c h n e i d e r , L ., Rosok, M.J. ( 1 9 8 3 ) . T r a n s f o r m a t i o n p a r a m e t e r s and pp6Qax° l o c a l i z a t i o n i n c e l l s i n f e c t e d w i t h p a r t i a l t r a n s f o r m a t i o n m utants o f Rous sarcoma v i r u s . M o l . C e l l . B i o l . 3: 731-746. R o t h b e r g , P.G., E r i s m a n , M.D., D i e h l , R.E., R o v i g a t t i , U.G., A s t r i n , S.M. ( 1 9 8 4 ) . S t r u c t u r e and e x p r e s s i o n o f t h e oncogene c-myc i n f r e s h tumor m a t e r i a l f r o m p a t i e n t s w i t h h e m a t o p o i e t i c m a l i g n a n c i e s . M o l . C e l l . B i o l . 4: 1096-1103. R o u s s e l , M.F., D u l l , T . J . , R e t t e n m i e r , C.W., R a l p h , P., U l l r i c h , A., S h e r r , C . J . ( 1 9 8 7 ) . T r a n s f o r m i n g p o t e n t i a l of t h e c-fms p r o t o - o n c o g e n e ( C S F - 1 r e c e p t o r ) . N a t u r e 325: 549-552 . R o v e r a , G., O ' B r i e n , T.G., Diamond, L. ( 1 9 7 9 a ) . I n d u c t i o n of d i f f e r e n t i a t i o n i n human p r o m y e l o c y t i c l e u k e m i a c e l l s by tumor p r o m o t e r s . S c i e n c e 204: 868-870. 176 Rovera, G., Santoli, D., Damsky, C. (1979b). Human promyelocyte leukemia c e l l s in culture d i f f e r e n t i a t e into macrophage-1ike c e l l s when treated with a phorbol diester. Proc. Natl. Acad. S c i . USA 2779-2783. Rudd, C.E., T r e v i l l y a n , J.M., Dasgupta, J.D., Wong, L.L., Schlossman, S.F. (1988). The CD4 receptor i s complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human lymphocytes. Proc. Natl. Acad. S c i . USA 85: 5190-5194 Ruley, E. (1983). Adenovirus early region IA enables v i r a l and c e l l u l a r transforming genes to transform primary c e l l s in culture. Nature 304: 602-606. Sachs, L. (1987) The molecular control of blood c e l l development. Science 238: 1374-1379. Sadowski, I., Stone, J.C., Pawson, T. (1986). A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming a c t i v i t y of Fujinami sarcoma virus P130 < 3 a < 3 - C E" B. Mol. C e l l . B i o l . 6: 4396-4408. Samarut, J., Mathey-Prevot, B., Hanafusa, H. (1985). P r e f e r e n t i a l expression of the c-fps protein in chicken macrophages and granulocytic c e l l s . Mol. C e l l . B i o l . 5: 1067-1072. Sariban, E., M i t c h e l l , T., Kufe, D. (1985). Expression of the c-fms proto-oncogene during human monocytic d i f f e r e n t i a t i o n . Nature 316: 64-66. Schalken, J.A., Roebroek, A.J.M., Oomen, P.P.C.A., Wagenaar, S.S., Debruyne, F.M.J., Bloemers, H.P.J., Van de Ven, W.J.M. (1987). fur gene expression as a discriminating marker for small c e l l and nonsmall c e l l lung carcinomas. J. C l i n . Invest. 80: 1545-1549. Scolnick, E.M., Papageorge, A.G., Shih, T.Y. (1979). Guanine nucleotide-binding a c t i v i t y as an assay for src protein of rat-derived murine sarcoma viruses. Proc. Natl. Acad. S c i . USA 76: 5355-5359. Sedmak, J.J., Grossberg, S.E. (1977). A rapid, s e n s i t i v e , and v e r s a t i l e assay for protein using Coomassie b r i l l i a n t blue G250. Anal. Biochem. 79: 544-552. Sherr, C.J., Rettenmier, C.W., Sacca, R., Roussel, M.F., Look, A.T., Stanley, E.R. (1985). The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. C e l l 41: 665-676. Shibuya, M. Hanafusa, H., Balduzzi, P.C. (1982). C e l l u l a r sequences related to three new one genes of avian sarcoma virus(fps f yes, and ros) and their expression in normal and 177 transformed c e l l s . J. V i r o l . 4 2 : 1 4 3 - 1 5 2 . Shtlvelman, E., L i f s h l t z , B., Gale, R.P., Canaani, E. (1985). Fused tra n s c r i p t of abl and bcr genes in chronic myelogenous leukaemia. Nature 315: 550-554. Slamon, D.J., Cline, M.J. (1984a). Expression of c e l l u l a r oncogenes during embryonic and f e t a l development of the mouse. Proc. Natl. Acad. USA 81: 7141-7145. Slamon, D.J., deKernion, J.B., Verma, I.M., Cline M.J. (1984b). Expression of c e l l u l a r oncogenes in human malignancies. Science 224: 256 -262. Smith, L.J., Cu r t i s , J.E., Messner, H.A., Senn, J.S., Furthmayr, H., McCulloch, E.A. (1983). Lineage i n f i d e l i t y in acute leukemia. Blood 61: 1138-1145. Smithgall, T.E., Yu, G., Glazer, R.I. (1988). I d e n t i f i c a t i o n of the di f f e r e n t i a t i o n - a s s o c i a t e d p93 tyrosine protein kinase of HL-60 leukemia c e l l s as the product of the human c-fes locus and i t s expression in myelomonocytic c e l l s . J. B i o l . Chem. 263: 15050-15055. Snyder, M.A., Bishop, J.M., McGrath, J.P., Levinson, A.D. (1985). A mutation at the ATP-binding s i t e of pp60 v— B X ; C 5 abolishes kinase a c t i v i t y , transformation, and tumorigenicity. Mol. C e l l . B i o l . 5: 1772-1779. Soderquist, A.M., Carpenter, G. (1984). Glycosylation of the epidermal growth factor receptor in A-431 c e l l s . J. B i o l . Chem. 259: 12586-12594. Solanki, V., Slaga, T.J., Callaham, M., Huberman, E. (1981). Down regulation of s p e c i f i c binding of (20- 3H)phorbol 12,13-dibutyrate and phorbol ester-induced d i f f e r e n t i a t i o n of human promyelocytic leukemia c e l l s . Proc. Natl. Acad. S c i . USA 78: 1722-1725. Southern, E.M. (1975). Detection of s p e c i f i c sequences among DNA fragments separated by gel electrophoresis. J. Mol. B i o l . 98: 503-517. Struhl, K. (1987). The DNA-binding domains of the 1un oncoprotein and the yeast GCN4 t r a n s c r i p t i o n a l activator protein are f u n c t i o n a l l y homologous. C e l l 50: 841-846. Sugimoto, Y., Whitman, M., Cantley, L.C., Erikson, R.L. (1984). Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and d i a c y l g l y c e r o l . Proc. Natl. Acad. S c i . USA 81: 2117-2121. Sunami, S., Fuse, A., Simizu, B., Eguchi, M., Hayashi, Y., Sugita, K., Nakazawa, S., Okimoto, Y., Sato, T., Nakajima, H. (1987). The c - s i s gene expression in c e l l s from a 178 patient with acute megakaryoblastic leukemia and Down's syndrome. Blood 70: 368-371. Sundstrom, C , Nilsson, K. (1976). Establishment and characterization o£ a human h i s t i o c y t i c lymphoma c e l l l i n e (U937). Int. J. Cancer 17: 565-577. Swartwout, S.G., P r e i s l e r , H., Guan, W., Kinniburgh, A.J. (1987). Re l a t i v e l y stable population of c-myc RNA that lacks long poly(A). Mol. C e l l . B i o l . 7: 2052-2058. Sweet, R.W., Yokoyama, S., Kamata, T., Feramisco, J.R., Rosenberg, M., Gross, M. (1984). The product of ras is a GTPase and the T24 oncogenic mutant i s d e f i c i e n t in this a c t i v i t y . Nature 311: 273-275. Tachibana, N., Raimondi, S.C., Lauer, S.J., Sartain, P., Dow, L.W. (1987). Evidence for a multipotential stem c e l l disease in some childhood Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 70: 1458-1461. Takeya, T., Hanafusa, H. (1983). Structure and sequence of the c e l l u l a r gene homologous to the RSV src gene and the mechanism for generating the transforming v i r u s . C e l l 32: 881-890. Tamanoi, F., Walsh, M., Kataoka, T., Wigler, M. (1984). A product of yeast RAS2 gene i s a guanine nucleotide binding protein. Proc. Natl. Acad. S c i . USA 81: 6924-6928. Taparowsky, E., Shimizu, K., Goldfarb, M., Wigler, M. (1983). Structure and a c t i v a t i o n of the human N-ras gene. C e l l 34: 581-586. Taub, R., Moulding, C , Battey, J., Murphy, W., Vaslcek, T., Lenoir, G.M., Leder, P. (1984). Activation and somatic mutation of the translocated c-myc gene in Burkitt lymphoma c e l l s . C e l l 36: 339-348. T e r r i t o , M.C, Cline, M.J. (1977). Monocyte function in man. J. Immunol. 118: 187-192. Thomas, P.S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to n i t r o c e l l u l o s e . Proc. Natl. Acad. S c i . USA 77: 5201-5205. T i l l , J.E., Mcculloch, E.A. (1961). A d i r e c t measurement of the radiat i o n s e n s i t i v i t y of normal mouse bone marrow c e l l s . Radiat. Res. 14: 213-222. Tushinski, R.J., Oliver, I.T., Guilbert, L.J., Tynan, P.W., Warner, J.R., Stanley, E.R. (1982). Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the d i f f e r e n t i a t e d c e l l s s e l e c t i v e l y destroy. C e l l 28: 71-81. 179 Twardzik, D.R., Todaro, G.J., Marquardt, H., Reynolds, F.H.,Jr., Stephenson, J.R. (1982). Transformation Induced by Abelson murine leukemia virus involves production of a polypeptide growth factor. Science 216: 894-897. U l l r i c h , A., Coussens, L., Hayflick, J.S., D u l l , T.J., Gray, A., Tarn, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger, J., Downward, J., Mayes, E.L.V., Whittle, N., Waterfield, M.D., Seeburg, P.H. (1984). Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma c e l l s . Nature 309: 418-425. U l l r i c h , A., B e l l , J.R., Chen, E.Y., Herrera, R., P e t r u z z e l l i , L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C, Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C , Rosen, O.M., Ramachandran, J. (1985). Human i n s u l i n receptor and i t s re l a t i o n s h i p to the tyrosine kinase family of oncogenes. Nature 313: 756-761. U l l r i c h , A., Gray, A., Tarn, A.W., Yang-Feng, T., Tsubokawa, M., C o l l i n s C , Henzel, W., Le Bon, T., Kathuria, S., Chen, E., Jacobs, S., Francke, U., Ramachandran, J., F u j i t a -Yamaguchi, Y. (1986). I n s u l i n - l i k e growth factor I receptor primary structure: comparison with i n s u l i n receptor suggests s t r u c t u r a l determinants that define functional s p e c i f i c i t y . EMBO J. 5: 2503-2512. Van de V i j v e r , M., van de Bersselaar, R., Devilee, P., Cornelisse, C , Peterse, J., Nusse, R. (1987). Amplification of the neu (c-erbB-2) oncogene in human mammary tumors is r e l a t i v e l y frequent and i s often accompanied by amplification of the linked c-erbA oncogene. Mol. C e l l . B i o l . 7: 2019-2023. Varmus, H.E. (1984). The molecular genetics of c e l l u l a r oncogenes. Ann. Rev. Genet. 18: 553-612. Varmus, H.E. (1987). Oncogenes and t r a n s c r i p t i o n a l control. Science 238: 1337-1339. V e i l l e t t e , A., Bookman, M.A., Horak, E.M., Bolen, J.B., Rosen, N. (1988). The CD4 and CD8 T c e l l surface antigens are associated with the internal membrane tyrosine-protein kinase P561-3*. C e l l 55: 301-308. Verma, I.M., Sassone-Corsi, P. (1987). Proto-oncogene fos: complex but v e r s a t i l e regulation. C e l l 51: 513-514. Vogt, P.K., Bos, T.J., D o o l i t t l e , R.F. (1987). Homology between the DNA-binding domain of the GCN4 regulatory protein of yeast and the carboxyl-terminal region of a protein coded for by the oncogene iun. Proc. Natl. Acad. S c i . USA 84: 3316-3319. Wakelam, M.J.O., Davies, S.A., Houslay, M.D., McKay, I., 180 Marshall, C.J., H a l l , A. (1986). Normal p21 N- , r- B , couples bombesin and other growth factor receptors to i n o s i t o l phosphate production. Nature 323: 173-176. Warner, N.L., Moore, M.A.S., Metcalf, D. (1969). A transplantable myelomonocytic leukemia in BALB/c mice: cytology, karyotype and muramidase content. J. Natl. Cancer Inst. 43: 963-982. Waterfield, M.D., Scrace, G.T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J.S., Deuel, T.F. (1983). Platelet-derived growth factor is s t r u c t u r a l l y related to the putative transforming protein p28" 1 - of simian sarcoma vi r u s . Nature 304: 35-39. Weinberg, R.A. (1985). The action of oncogenes in the cytoplasm and nucleus. Science 230: 770-776. Weinmaster, G., Hinze, E., Pawson, T. (1983). Mapping of multiple phosphorylation s i t e s within the s t r u c t u r a l and c a t a l y t i c domains of the Fujinami avian sarcoma virus transforming protein. J. V i r o l . 46: 29-41. Weinmaster, G., Z o l l e r , M.J., Smith, M., Hinze, E., Pawson, T. (1984). Mutagenesis of Fujinami sarcoma vi r u s : evidence that tyrosine phosphorylation of p i 3 o « A , 3 - I : P ' ! S modulates i t s b i o l o g i c a l a c t i v i t y . C e l l 37: 559-568. Westin, E.H., Wong-Staal, F., Gelmann, E.P., Dalla Favera, R., Papas, T.S., Lautenberger, J.A., Eva, A., Reddy, E.P., Tronick, S.R., Aaronson, S.A., Gallo, R.C. (1982). Expression of c e l l u l a r homologues of r e t r o v i r a l one genes in human hematopoietic c e l l s . Proc. Natl. Acad. S c i . USA 79: 2490-2494. Williams, L.T. (1986) Surveys 5: 233-241. The s i s gene and PDGF. Cancer Wilimzig, M. (1985). L i C l - b o i l i n g method for plasmid mini-preps. Trends Genet. 1: 158. Willingham, M.C., Jay, G., Pastan, I. (1979). Localization of the ASV sre gene product to the plasma membrane of transformed c e l l s by electron microscopic immunocytochemistry. C e l l 18: 125-134. Wiseman, R.W., Stowers, S.J., M i l l e r , E.C, Anderson, M.W., M i l l e r , J.A. (1986). Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F i mouse. Proc. Natl. Acad. S c i . USA 83: 5825-5829. Wolf, N.S., Trentin, J.J. (1968). Hemopoietic colony studies. V. Ef f e c t of hemopoietic organ stroma on d i f f e r e n t i a t i o n of pluripotent stem c e l l s . J. Exp. Med. 127: 205-214. 181 Wong, A.J., Bigner, S.H., Bigner, D.D., Klnzler, L.W., Hamilton, S.R., Vogelstein, B, (1987). Increased expression of the epidermal growth factor receptor gene in malignant gliomas i s invariably associated with gene amplification. Proc. Natl. Acad.Sci. USA 84: 6899-6903. Woolford, J., Rothwell, V., Rohrschneider, L. (1985). Characterization of the human c-fms gene product and i t s expression in c e l l s of the monocyte-macrophage lineage. Mol. C e l l . B i o l . 5: 3458-3466. Yam, L.T., L i , C.Y., Crosby, W.H. (1970). Cytochemical i d e n t i f i c a t i o n of monocytes and granulocytes. Am. J. C l i n . Path. 55: 283-290. Yasamura, Y., Buonassisi, V., Sato, G. (1966). Clonal analysis of d i f f e r e n t i a t e d function in animal c e l l cultures. I. Possible correlated maintenance of d i f f e r e n t i a t e d function and the d i p l o i d karyotype. Cancer Res. 26: 529-535. Young, J . C , Martin, G.S. (1984). C e l l u l a r l o c a l i z a t i o n of c-fps gene product NCP98. J. V i r o l . 52: 913-918. Yukota, J., Tsunetsugu-Yukota, Y., B a t t i f o r a , H., Le Fevre, C , Cline, M.J. (1986). Alterations of myc, myb, and ras"" proto-oncogenes in cancers are frequent and show c l i n i c a l c o r r e l a t i o n . Science 231: 261-264. Yunis, J.J. (1983). The chromosomal basis of human neoplasia. Science 221: 227-236. Ziegler, S.F., Marth, J.D., Lewis, D.B., Perlmutter, R.M. (1987). Novel protein-tyrosine kinase gene(hck) p r e f e r e n t i a l l y expressed in c e l l s of hematopoietic o r i g i n . Mol. C e l l . B i o l . 7: 2276-2285. 

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