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III: 3meMHWMMIH l .333 .3 33,: £533“ H33 4':- wo- ::‘ 4522;: m d-F: 02:: “I.“ 3': “a: “ +fiw -:':‘:.:‘ iém 2W" .H 3%.? .g_ BIOCHEMICAL ANALYSIS OF TRANSFORMATION—SENSITIVE ALTERATIONS IN THE SUBSTRATUM ASSOCIATED MATERIAL OF CHICKEN EMBRYO FIBROBLASTS By John Blenis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1983 ABSTRACT BIOCHEMICAL ANALYSIS OF TRANSFORMATION—SENSITIVE ALTERATIONS IN THE SUBSTRATUM ASSOCIATED MATERIAL OF CHICKEN EMBRYO FIBROBLASTS By John Blenis Removal of chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus (RSV—CEF) from their substrata with the calcium chelator, EGTA, leaves behind the substratum associated material (SAM or extracellular matrix). We have studied the proteins/glycoproteins and the glycosamino- glycan, hyaluronic acid (HA), in SAM during the development of the transformed state and have: (I) detected a novel, low molecular weight protein (MP-221,000) in SAM; (2) biochemically analyzed this 21K protein; (3) characterized transformation- sensitive alterations in HA; and (4) investigated the role of HA in modifying cell-substratum adhesion. Increased synthesis and deposition of the 21K protein into SAM is observed within hours of transfer of RSV-CEF to the permissive (transforming) temperature. Characterization reveals metabolic labeling by amino acids, but not mannose or phosphorous. It is resistant to extraction by EGTA, urea and several detergents except sodium dodecyl sulfate. The 21K protein is sensitive to Pronase and chymotrypsin but insensitive to trypsin or collagenase. It is resistant to removal from the substratum by cell—associated proteolytic or glycolytic enzymes. The 21K protein appears to be shed from the cell surface, possibly as part of a glycoprotein/glycosaminoglycan complex and then becomes strongly associated with the substratum. Elevated amounts of hyaluronic acid are synthesized during the early stages of transformation. We have detected several alterations in both amounts and organization of HA during this process. The greatest alteration is in a high molecular weight species, which is 2—fold higher in SAM and approximately 20-fold higher in a "loosel y-associated" HA-species. Cell surface distribution of HA is also altered upon transformation. In nontransformed RSV—CEF, 75% of the cell surface HA is in SAM. Upon transformation this falls to 62% as a result of increased "loosely—associated" HA. Alterations in cell surface HA upon transformation coincide with the loss of cell-substratum adhesion. Treatment of transforming cells with hyaluronidase (specifically degrades HA) results in stronger cell-substratum adhesion. Exogenously added HA only slightly affects initial attachment of cells to the substratum. HA does not appear to affect cell morphology. Results suggest that HA may be involved in retarding normal cell spreading and promoting the ability of transformed cells to detach. TO MY FATHER This work is also dedicated to my Mother and brother, George, for their unselfish and loving support throughout my education and life, and to my wife, Donna, to whom I dedicate this thesis and the rest of my life. ACKNOWLEDGMENTS To Dr. Susan P. Hawkes, I would like to express my sincere appreciation for her advice and patience during my graduate training. To Dr. John L. Wang, I would like to express my gratitude for his advice and help in many matters. I would like to give special thanks to Dr. Stephen J. Ullrich for a tremendous amount of advice, friendship and patience. I would also like to express my gratitude to all the members of Dr. Hawkes' and Dr. Wang's laboratories for their friendship, cooperation and encouragement. Finally, I would like to thank Dr. JoAnn K. Yamamoto for her friendship and help, and Kathleen E. Studebaker for her friendship and tremendous amount of assistance in the preparation of this dissertation . TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . V LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . V; LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . ix CHAPTER I. INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 ll. TRANSFORMATION- SENSITIVE PROTEIN ASSOCIATED WITH THE CELL— SUBSTRATUM . . . . . . . . . . . 25 Materials and Methods . . . . . . . . . . . . . . . 26 Results . . . . . . . . . . . . . . . . . . . . . . 31 Discussion. . . . . . . . . . . . . . . . . . . . . 50 III. CHARACTERIZATION OF THE TRANSFORMATION-SENSITIVE 21K PROTEIN OF THE CELL-SUBSTRATUM . . . . . . . . 53 Materials and Methods . . . . . . . . . . . . . . . 54 Results . . . . . . . . . . . . . . . . . . . . . . 61 Discussion . . . . . . . . . . . . . . . . . . . . . 110 IV. TRANSFORMATION—SENSITIVE ALTERATIONS IN HYALURONIC ACID 0 O O O O O O I O O O O O O O O O O O O O O 0 121 Materials and Methods . . . . . . . . . . . . . . . 122 Results . . . . . . . . . . . . . . . . . . . . . . 127 Discussion . . . . . . . . . . . . . . . . . . . . . 145 V. ROLE OF HYALURONIC ACID IN CELL-SUBSTRATUM ADHESION 153 Materials and Methods . . . . . . . . . . . . . . . 154 Results . . . . . . . . . . . . . . . . . . . 161 Discussion . . . . . . . . . . . . . . . . . . . . . 180 CONCLUDING STATEMENT . . . . . . . 186 BIBLIOGRAPHY. . . . . . 190 LIST OF TABLES Table Page 1 Summary of extraction properties of the 21K protein......................72 II Ratio of transforming and nontransforming CEF [ H]-—Iabeled HA isolated from a) cellular, cell 5 rface and medium fractions, and b) cell surface [ Hl—labeled HA divided into SAM, Ist EGTA, 2nd EGTA and trypsinate compartments . . . . . . . . 130 III Percent distribution of [3H]-Iabeled HA eluted on Sepharose CL-ZB from cellular, cell surface and medium fractionS. . . . . . . . . . . . . . . . . . 133 IV Comparison of high, intermediate and low molecular weight [ H]-|abeled HA species from cellular, cell surface and medium fractions , , , , , , , , , , 134 V Distribution of [3H]—Iabeled HA from various cell surface compartments . . . . . . . . . . . . . . . . 136 VI Psrcent distribution of fractionated cell surface [H]—|abeled HA on Sepharose CL-ZB . . . . . . . . . 139 VII Comparison of high, intermediate and lo molecular weight species of isolated cell surface [ H]-Iabeled HA 0 O O O I O O O I O O O O O O O O O O O O O O O 14] VIII Effect of hyaluronidase treatment on cell surface hyaluronic acid . . . . . . . . . . . . . . . . . . 164 IX Effect of exogenous hyaluronic acid on growth in agar . 179 LIST OF FIGURES Figure 1 2 10 11 12 13 Ill 15 Schematic diagram of procedure for SAM preparation . Comparison of metabolically labeled substratum associated proteins and EGTA-released cellular proteins 0 O O O O O O O O O O I O O O O O O O C Radioactive profiles of substratum associated proteins. Incorporation of [2—3H]mannose into SAM glycoproteins The effect of cell density on the synthesis of substratum associated proteins. . . . . . . . . . . Comparison of substratum associated proteins from RSV(LA2462)-infected CEF, Prague A wild type RSV- infected CEF and normal, uninfected CEF . . . . . . Turnover of normal cellular proteins during the development of the transformed phenotype . . . . . Analysis of 21K protein synthesis and deposition in SAM as a function of transformation . . . . . . . . Turnover of the 21K protein and total substratum associated proteins at the permissive and nonpermissive temperatures . . . . . . . . . . . Schematic diagram of radioiodination protocol . . . Analysis of substratum associated proteins from NY68— infected chicken embryo fibroblasts . The effect of phorbol myristate acetate on the 21K protein..................... Extraction of substratum associated proteins with SDS. Resistance of substratum associated proteins to extraction with various agents . . . . . . . . . Detection of disulfide bonds in the 21K protein . . . Page 28 33 35 37 40 43 1+6 47 49 60 63 66 69 71 74 Sensitivity of the 21K protein Turnover of substratum associated proteins by cellular Association of the 21K protein with SAM . Binding of conditioned media proteins to various Effect of serum on binding of conditioned media proteins to the substratum Analysis of substratum binding proteins from serum- 0 to SAM-, Effect of colchicine on SAM serum- an d BSA—coa ted to proteases Effect of cAMP on substratum associated proteins . Radioiodination of SAM and cell surface proteins . . . Sepharose CL—ZB elution profiles of [3H]—Iabeled isolated from normal the 21K protein and heat shock proteins and beling of surface—associated hyaluronic acid with [ nglucosamine Sepharose CL-2B chromatography of fractionated cell surface hyal uronic acid Hyaluronidase treatment of EGTA—released [3H]—labeled Schematic diagram of adhesion assay Effect of hyaluronidase treatment of cell monolayers on Effect of transformation on adhesion of cells to Figure 16 17 proteases . . 18 19 substrata . . 20 21 21K protein binding substrata . . 22 free conditioned media 23 214 25 26 Comparison of 27 Long term I 28 hyaluronic acid transforming CEF 29 30 hyaluronic acid 31 32 cell surface GAG 33 substratum . 3h Effect of hyaluronidase on adhesion of LA24G2- infected CEF . Page 78 80 83 88 92 95 98 102 101i 109 117 128 132 I38 143 155 163 166 168 Figure Page 35 Effect of hyaluronidase on attachment . . . . . . . . 171 36 Effect of hyaluronidase on cell spreading . . . . . . . 174 37 Effect of hyaluronidase on cell spreading . . . . . . . I76 viii ACA BSA CEF CM CMISF) CMF-PBS CMF—PBS+ db-cAMP ECM EGTA FN GAG HA HMW I MW LA24G2 LMW LIST OF ABBREVIATIONS e—amino-n-caproic acid bovine serum albumin chicken embryo fibroblasts conditioned medium serum-free CM calcium/magnesium—free phosphate buffered saline CMF-PBS containing 1 mg/ml c—amino—n-caproic acid dibutryl cyclic AMP extracellular matrix ethyleneglycoI—bis(B—amino—ethyl ether)N,N,N',N'—tetra— acetic acid fibronectin glycosaminoglycan hyaluronic acid high molecular weight species of hyaluronic acid intermediate molecular weight species of hyaluronic acid subclone of the LA24 temperature—sensitive mutant of the Prague A strain of Rous sarcoma virus low molecular weight species of hyaluronic acid relative molecular weight normal, uninfected cells NY68 PMA PMSF RSV RSV-CEF SAM SDS-PAGE TLCK TPCK TRU 221+ 35 (x h) temperature—sensitive mutant of the Schmidt-Ruppin A strain of the Rous sarcoma virus phorbol myristate acetate phenyl methyl sulfonyl fluoride Rous sarcoma virus Rous sarcoma virus—infected chicken embryo fibroblasts substratum associated material sodium dodecyl sulfate—polyacrylamide gel electro— phoresis p—tosyl-L—Iysyl chloromethyl ketone L—I—tosy l amide-2—phenylethyl chloromethyl ketone turbidity reducing unit media-I99 containing 2% tryptose phosphate broth, 2% calf serum, 1% chicken serum and 0.1% glucose Refers to the growth of LA24G2—CEF cultures at the permissive temperature (350) for x h INTRODUCTION INTRODUCTION The interaction between a cell and its environment is responsible for many properties of cell growth. In particular, the substratum upon which cells grow and various components of the extracellular matrix appear to play an important role in the behavior of the cell. For purposes of clarity, the term extracellu— lar matrix (ECM) will be used as a general term to define, that material, both endogenous and exogenous, which resides in an organized manner outside the cell but with many intimate associa- tions with the cell membrane and intracellular cytoskeleton in both i_rl vivo and _l__rl vitro systems. In addition, the term substratum associated material (SAM) will be used interchangeably with ECM when discussing _i_rl m cell systems. Other terms used to described the ECM for various cell systems include basement membrane, basal lamina, pericellular matrix and microexudate. It has only recently become clear that the extracellular matrix is more than just an inert structural scaffolding upon which the cells reside. In fact it is now understood that a dynamic interaction occurs between cells and their ECM such that there is not only a structural continuum to the cell interior but a functional one as well. As cells continually synthesize and turnover their own ECM, alterations in components present can result in changes of structural and functional properties. 2 Furthermore, the ECM can be altered by other cells which can then influence the behavior of the original cells. Thus the cell and the ECM appear to communicate and direct the fate of each other as well as other cell types which might contact the original cell's ECM. For example, when cells expressing the transformed phenotype are cultured on ECM from normal cells, the transformed cells develop a normal morphology (1). Furthermore, addition of the ECM glycoprotein, fibronectin, to transformed fibroblasts also results in reversion to normal phenotype (2,3). In one develop- mental system, addition of fibronectin to chondroblasts (epithelioid cells) results in dedifferentiation to a fibroblastic state (4). Thus the ECM can indeed alter cell behavior. Cell proliferation is greatly influenced by the extracellular matrix as is cell-substratum adhesion. As these functions are fund— amental properties of cells, it is apparent that the ECM plays a crucial role in many cellular properties such as cell proliferation and migration during embryogenesis and morphogenesis, providing structural support for cells, tissues and organs, aiding in wound healing, permitting localization and presentation of environmental signals to cells, and in the pathology of disease states such as the invasiveness of metastatic tumors. Excellent reviews covering these structural and functional properties of the ECM have recently been published in a book (5). It is clear that one must understand the role of the ECM in maintaining or directing cell behavior. Biochemical analysis of the ECM has recently provided much information concerning this relationship. The best characterized of these macromolecules include elastin, collagen, fibronectin, Iaminin, chondronectin, and the proteoglycans/glycosaminoglycans. A brief description of these macromolecules and their potential physiological roles will be provided. The glycosaminoglycan, hyaluronic acid will be dealt with in more detail as it is a major part of this thesis. Analysis of the structural ECM protein, elastin (for review see 6), has been hampered by its insolubility and almost infinite molecular weight it obtains as a result of an extensive amount of cross-linking. It is known to be a rubber-like elastomer found largely in the ECM of blood vessels and lung where elasticity is clearly an important structural property. As in collagen, one- third of the amino acids of'elastin are glycine and approximately 11% proline, but in contrast to collagen, there is very little hydroxyproline, no hydroxylysine and large amounts of the nonpolar amino acids alanine, valine, Ieucine and isoleucine. In fact, valine makes up from 10-14% of the elastin polymer. In addition, there is no methionine in elastin. Well over 90% of this protein consists of nonpolar amino acids. Like procollagen, elastin is synthesized as a soluble precursor, tropoelastin. Following its organization or alignment by microfibrils in the ECM, extensive cross—linking occurs resulting in the insoluble elastin. The insoluble elastin is unique in that these cross-links are derived from lysine amino acids which have been derivatized into two unique geometric isomers containing a pyridine nucleus, desrnosine and isodesmosine. These cross—links give the elastin molecules the ability to stretch but not slip by each other. Release of tension allows the individual chains to snap back to their original conformation. The study of elastin and its synthesis has been hampered by the fact that very few culture systems are capable of producing insoluble elastin. The possibility exists that certain cell types in culture produce tropoelastin but because of culture conditions do not form the insoluble elastin (6). Recent develop— ments in the area of tissue culturing of insoluble elastin- producing cells should answer many questions of the role and influence that elastin has in the organization of the ECM (7,8). Collagenous proteins are principal components of most types of extracellular matrices. The biosynthesis, processing, structure and function of collagen has recently received much attention (for reviews see 9-13). Collagen is a rigid, rod-like molecule with a triple—helical region comprising more (than 95% of its length. The triple-helical region consists of three separate chains tightly wrapped around one another in the form of a left-handed helix. This confirmation is stabilized by interchain hydrogen bonds and is only possible due to the fact that every third amino acid residue of the helix is glycine. Thus the chain is composed of a series of triplets with the sequence, GIy—X—Y, where X or Y can be any amino acid. Frequently, X or Y are proline or hydroxypro- line, each of which makes up approximately 10% of the molecule. There are also variable amounts of the unique amino acid, hydroxylysine. Collagen is actually composed of a heterogeneous class of at least five isotypes and different isotypes predominate in matrices of different cell types and at different stages of growth and development. 5 The biosynthesis of a collagen molecule is a very complex process. There are many post—transcriptional and post—transla- tional modifications which occur before the final product is made (10,14). Following transcription, capping and polyadenylation, the pre-mRNA, having 52 exons with introns ranging from 80 to 2,000 base pairs long, must undergo greater than 50 splicing reactions before becoming the translatable, cytoplasmic mRNA (15). The collagen mRNA is then translated on membrane bound polysomes and the translated product immediately hydroxylated at many proline and lysine residues. Carbohydrate sidechains are also added at this time. The hydrophobic signal sequence is rapidly processed in the rough endoplasmic reticulum. Following helix formation the procollagen molecule is secreted. The procollagen molecule, the precursor of collagen, contains amino— and carboxyl-terminal propeptides which are proteolytically removed to give the final triple helical collagen molecule. The amino—terminal propeptide is generally removed upon secretion, the carboxyl- terminal propeptide is removed sometime after secretion allowing the collagen molecules to spontaneously aggregate into fibers. Following deposition into the matrix, the collagen molecules in fibrils are then cross-linked via condensation of aldehydes formed from certain lysyl and hydroxylysyl residues in the molecule. It has generally been assumed that the major function of collagen is a structural one, that is, to provide strength and support to cells, tissues and organs. However, it is now clear that collagen has numerous developmental and physiological functions (for reviews see 11,16). One such function, the ability 6 of collagen to mediate cell-substratum adhesion, is thought to be mediated directly or via collagen—bound glycoproteins such as fibronectin, laminin or chondronectin (13). As this thesis is concerned with the analysis of the cell-substratum during the transformation process, it is significant that the level of procollagen decreases in transformed cells (17-19). This decrease occurs at the level of transcriptional control (20,21). In addition to‘elastin and collagen, three glycoproteins of the extracellular matrix have recently been isolated and function- ally characterized (fibronectin, Iaminin, and chondronectin). These glycoproteins are interesting matrix components in that they have the common property of influencing cell behavior and cell adhesion as well as being able to bind to other matrix components such as collagen. In the past ten years the glycoprotein fibronectin has been extensively characterized (for reviews see 22-25). It is known to exist in two forms, plasma fibronectin and cellular fibronectin, which have very similar molecular properties. Plasma fibronectin exists as a dimer of two subunits with molecular weights of approximately 215,000-220,000 whereas the cellular fibronectin can exist as a dimer with two molecular weight subunits of 220,000- 240,000, as a larger polymer or to a lesser extent, as a monomer. The dimer is cross-linked by disulfide bonds located at the carboxyl end. This unique characteristic probably confers elasticity to the molecule making it a more efficient adhesive and structural component of the ECM. The synthesis of cellular fibronectin _i_n_ vitro has been demonstrated in several cultured 7 cell types including; fibroblasts, myoblasts, undifferentiated chondrocytes, endothelial cells and amniotic epithelial cells. However, production of fibronectin _i_r_1_ .‘LL‘LQ does not always correlate with synthesis _i_n_ vitro. Furthermore, _i_n_ vivo fibronectin is present around many cell types but the source of this fibronectin is not necessarily known. It is now clear that fibronectin contains a series of function— al as well as structural domains. Many of these domains have been identified by the ability of peptides, generated by limited proteolytic digestion, to bind to certain macromolecules. Portions of fibronectin are involved in the binding of fibrinogen/fibrin, collagen/gelatin, heparin/heparan sulfate, transglutaminase substrates, cell surfaces, hyaluronic acid, actin, DNA, and bacteria (S.aureus). Cellular and plasma fibronectin can mediate attachment of cells to collagen and both molecules can bind directly to collagen. The glycosaminoglycan, heparin, binds to fibronectin. The binding of fibronectin to collagen is enhanced by heparin binding (26). Another glycosaminoglycan, hyaluronic acid, also binds to fibronectin (27,28). It has been suggested that heparan sulfate enhances adhesion whereas hyaluronic acid may interfere with this process (29). The existence of separate binding sites for these three extracellular matrix components as well as a binding site for the cell surface suggests that fibronectin may play a critical role in the organization and cellular binding of a variety of extracellular or secreted molecules. 8 Fibronectin is synthesized and processed in the rough endo— plasmic reticulum as are other asparagine-linked glycoproteins. Fibronectin is then thought to be secreted either directly onto the cell surface or into the culture medium. The rate of turnover of cell surface fibronectin is relatively slow (half life of 30-36 hours), however, the half life is shorter in cells treated with tunicamycin (30), a drug which inhibits the addition of aspara— gine-linked oligosaccharides, and on transformed cell surfaces. Interestingly, the lack of asparagine—linked carbohydrates does not interfere with the binding properties described. Many trans- formed cell types have lower amounts of fibronectin. In addition to increased turnover of the cell surface molecule, the amount of mRNA for fibronectin has also been demonstrated to be lower in transformed cells, by use of a cDNA probe (31). Fibronectin has been shown to have several functions i_rl vitro, its functions _i__rl vivo are not as well established. The best established functions of fibronectin are its roles in cell- substratum adhesion, cell spreading and morphology. As mentioned, many transformed cell systems possess lower cell surface fibronectin levels. These cells are less adhesive and possess a rounded or poorly spread morphology. Addition of fibronectin to these culture results in morphological reversion and increased adhesi veness of the transformed cells (2, 3). Interestingly, cellular fibronectin is 50 times more active than plasma fibronectin in causing this reversion. There also appears to be some transmembrane interaction of fibronectin with actin as addition of fibronectin to transformed cells also results in actin reorganization (2,3,32). In addition, culturing of normal cells in the presence of cytochalasin B, a microfilament disrupting agent, results in the rapid release of cell surface fibronectin (33-35). Probably as a result of its involvement in adhesion and cell mor- phology, fibronectin also appears to increase the ability of cells to migrate. The role of the ECM, and therefore potentially fibronectin, in regulating cell growth has recently received much attention. Folkman and Moscona (36) have shown that the degree of cell spreading affects the rate of proliferation of cells as fully spread cells are much more active than those not completely spread. Benecke §_t_ a_l_. (37) have also shown the importance of the interaction of the cell with a substratum on cell proliferation as growth of 3T6 fibroblasts in suspension results is a dramatic de- crease in mRNA levels and protein synthesis. Reattachment of sus— pended cells leads to a rapid recovery of protein synthesis. The importance of the extracellular matrix in promoting cell prolifera- tion has been further demonstrated by Gospodarowics and Lui (38). They have shown that bovine aortic endothelial cells cultured on tissue culture plastic required the presence of fibroblast growth factor for growth. Active proliferation of these cells on corneal endothelial cell ECM occurred in the absence of fibroblast growth factor and regardless of the batch of serum used. The importance of the ECM in cell proliferation was further demonstrated when it was shown that normal rat hepatocytes which do not survive more than a few weeks on tissue culture plastic or on type I collagen gels, could be maintained for more 10 than five months _i_n vitro when cultured on rat liver biomatrix (ECM) which is isolated from rat liver connective tissue fibers (39). Finally, it has been shown that fibronectin, added to serum-free defined culture medium, stimulates proliferation of rat ovarian follicular cells (40) and mouse embryonic carcinoma cells (41), whereas in the absence of fibronectin no growth occurs. Thus the requirement for cell-substratum attachment and spreading and the requirement for certain serum factors or hormones appears to be required for both active cell proliferation and growth. Transformation has in some way freed the transformed cell from these requirements. The phenomena of anchorage-independent growth as a general characteristic of transformed cells was an early observation (42,43) and appears to be a consistently observed parameter of transformation. Fibronectin and the extracellular matrix have also been found to have profound effects on morphogenesis and differentia— tion of cells. For example, rat mammary epithelial cells (44) and chick-embryo sternal chondrocytes (45) retain their differentiated state when cultured on their appropriate ECM. Growth of these cells on tissue culture plastic results in the loss of their differentiated state. The component of the ECM which is at least partially responsible for the maintenance of the differentiated state of the chondrocytes is fibronectin. In this case, it is the absence of fibronectin. It has been shown that as chondrocytes begin to differentiate, fibronectin strands disappear (46). This occurs when chondrocytes in culture begin to accumulate extra- cellular cartilage matrix. Furthermore, addition of fibronectin to 11 chondrocyte cultures induces the loss of the chondroblastic phenotype and dedifferentiation of cells to a fibroblastic state (47,48). Cultured myoblasts exhibit a similar phenotypic change. That is, as myoblasts fuse to form myotubes and differentiation proceeds, ECM fibronectin disappears (49,50). Addition of exogenous fibronectin to rat myoblast cultures inhibits fusion and antibody to fibronectin accelerates this differentiation process I51). Finally, during gastrulation of chicken embryos, the areas of active proliferation and migration overlap with the areas of ECM most intensely stained with fluorescently labeled fibronectin antibodies (52). Thus it is apparent that fibronectin plays an integral part in the structure, organization and functional aspects of cel l—ECM interactions. Recently, two newly described glycoproteins, Iaminin and chondronectin, have been found associated with the cell surface/ECM and like fibronectin are known to possess cell- substratum adhesion properties (53-55). Laminin is a glycoprotein containing 12-15% carbohydrate and is rich in half—cystine residues. It contains many disulfide bonds and exists as a 800,000 dalton macromolecule. After disulfide reduction, two subunits with molecular weights of 220,000 and 440,000 are obtained. This glycoprotein is a constituent of the basal lamina or basement membrane of epithelial cells, endothelial cells and myotubes (56,57). Laminin is not produced by fibroblasts. Due to the large potential for extensive cross-linking by disulfide bonds, it is believed that Iaminin may act as the structural framework within the basement membrane. Laminin has affinity for 12 heparin/heparan sulfate, type IV collagen and cells. Like fibronectin, Iaminin first binds to type IV collagen and then cells can bind to the Iaminin-type IV collagen complex (13,58). Heparan sulfate probably stabilizes this interaction as it does with fibronectin in fibroblasts. Thus Iaminin also acts as an adhesive glycoprotein that can mediate adhesion of certain epithelial cells to basement membrane collagen (59,60). In cells possessing both laminin and fibronectin it has been shown that although they act together in mediating adhesion, they have completely separate cell surface receptors (60,61). Laminin has been described to have possible roles in morphogenesis of embryonic kidneys (62) and in the maintenance of a proper tissue organization during liver regeneration (63). Finally it has been shown that both fibronectin and Iaminin are absent from the surface of transformed rat kidney cells despite the fact that they are both synthesized at rates comparable to normal rat kidney cells (64). Chondronectin is an adhesive glycoprotein found in serum and in cartilage and exists as a 180,000 dalton molecule with subunits of an apparent molecular weight of 80,000 following reduction of its disulfide bonds. This glycoprotein mediates the adhesion of chondrocytes to type I I collagen, which is characteristic of cartilage. Like laminin and fibronectin, chondronectin interacts with heparin. Chondronectin is unique though in that it is specific for chondrocytes and has strong specificity for type II collagen, whereas laminin and fibronectin do not Show such specificity to collagen types (13,53,65). 13 Proteoglycans are a complex set of molecules which are found in almost all mammalian tissues and are especially prominant in connective tissues. The exact molecular structure differs for different tissues and will not be discussed here, other than to state that proteoglycans in general contain a core protein to which is covalently linked at least one glycosaminoglycan (for reviews see 66-70) . The 9 l ycosaminoglycans (GAG) can be chondroitin sulfate and/or keratan sulfate or heparan sulfate. These GAG are covalently attached to a core protein through a glycosidic bond to the hydroxyl group of a serine (chondroitin sulfate and heparan sulfate) or serine/threonine (keratan sulfate) amino acid residue. The core protein ranges in molecular weights from 17,000 to greater than 300,000 and also possesses O- and N—glycosidically linked oligosaccharides. Hyaluronic acid and a hyaluronic acid link protein are also part of some proteoglycan aggregates. Proteoglycans are known to provide structural support for many tissues and appear to be involved in many morphogenetic events. The heparan sulfate proteoglycan is of special interest as it is a major component of epithelial and fibroblast ECM. Cell surface heparan sulfate proteoglycan from rat liver membranes has been isolated and found to have a molecular weight of appro— ximately 75,000. The core protein molecular weights ranged from 17,000 to 40,000 with an average molecular weight of 14,000 for the glycosaminoglycan chains released from the core protein by alkali treatment (71). Endogenous cell surface heparan sulfate proteoglycans are associated with the cell surface of rat 14 hepatocytes by two independent mechanisms. One mechanism involves a noncovalent interaction, as two—thirds of the cell surface heparan sulfate proteoglycans can be displaced from the cell surface by exogenously added heparin. The remaining third is released by mild trypsin treatment (72). The latter population of heparan sulfate proteoglycans can be extracted with detergent and it appears that the core protein of this proteoglycan is embedded in the lipid bilayer of the plasma membrane (73). Heparan sulfate proteoglycans are believed to be partially responsible for the structural integrity of the ECM within which it resides. In addition to its structural properties, heparan sulfate proteoglycan appears to have some functional properties as well. This proteoglycan has only recently been found to be present in and to make up approximately 85% of the glomerular basement membrane proteoglycans and has been proposed to function as a blood plasma filter to anionic or neutral macromolecules larger than 70,000 molecular weight. Hyaluronic acid, although present in small amounts, may also have a role in this process (74—76). It has previously been shown that GAG of mammary epithelial cells cultured on collagenous matrices are not as degraded as GAG of cells cultured on plastic (77). However, transformed mammary epithelial cells do not respond to the collagen substratum and thus, like normal cells cultured on plastic, cannot maintain a basal lamina. The major GAG of this basal lamina is heparan sulfate, which is rapidly degraded by the transformed cells. It has been suggested that the altered ECM allows the neoplastic cells to invade surrounding tissues (78). These results might 15 suggest that heparan sulfate may aid in regulation of cell proliferation and mobility. In addition to epithelial cells, the two forms of heparan sulfate previously described have been found associated with glial cells, endothelial cells and fibroblasts. The heparan sulfate proteoglycan without the hydrophobic core protein was found only in extracts of cells capable of forming a fibrillar ECM but not in extracts of cells devoid of matrix (79). It has been shown that heparan sulfate produced by transformed fibroblasts has a lower sulfate content than that produced by normal fibroblasts (80,81). As previously described, heparan sulfate has been shown to bind to fibronectin. It may well be that lower sulfation of heparan sulfate from transformed fibroblasts results in a weakened interaction with fibronectin. This may result in an unorganized or unstable ECM. The fact that heparin (high sulfate content) displaces heparan sulfate (low sulfate content) gives support to this idea as well as the fact that transformed fibroblasts do not assemble a normal ECM. The molecular alterations in heparan sulfate upon transformation and its potential effect on the normality of the ECM may in part be responsible for the decrease in cell- substratum adhesion that is observed in neoplastic cells. Culp and coworkers (29,82) have analyzed the GAG in substrate— attached material (SAM, the ECM remaining following 21 \_/_i_t_r_(_)_ detachment of cells from their. substratum with the calcium— chelator, ethylene glycol bis (B-aminoethyl ether)-N,N,N',N'- tetraacetic acid) from newly attaching or detaching cells and find new adhesion sites enriched for heparan sulfate whereas mature 16 adhesion sites have increase hyaluronic acid. They have suggested that since new adhesion sites are enriched in heparan sulfate, the proteoglycan may be involved in adhesion of cells. Thus it appears that heparan sulfate, like the other ECM components described thus far, is critical for maintenance and behavior of the cells with which it interacts. As mentioned, the glycosaminoglycan, hyaluronic acid is another major component of most extracellular matrices (for reviews see 67,83). Hyaluronic acid is a linear, nonbranching polymer having a repeating disaccharide unit consisting of D—glucuronic acid linked 8(1-3) to N-acetyI-D—glucosamine which is in turn linked 8(1-4) to D—glucuronic acid. The chemical and physical properties of hyaluronic acid have recently been reviewed (67). This polymer has a reported molecular weight of approximately 106—107. In solution it has the ability to swell to one thousand times its anhydrous volume with an enormous molecular domain of approximately 200 nm in diameter. Despite its relatively simple chemical composition, hyaluronic acid can exist in several conformations including random-coil and helical. In the presence of a calcium counterion, it can exist as a threefold helix. Even in dilute solutions the long, entangled hyaluronic acid chains can form a molecular sieve which behaves as an ion-exchange matrix and appears to be able to enhance the diffusion of certain solutes such as glucose and lysine (84). Thus, even at low concentrations, hyaluronic acid could play a dominant role in the physical, chemical and biological properties of normal cell surfaces. Alterations in concentration and/or 17 organization of hyaluronic acid during the transformation process may therefore significantly influence cell behavior. Hyaluronic acid is found at its highest concentrations in embryonic tissues and it is well established that this polymer plays an important role in morphogenesis and differentiation. For example, hyaluronic acid is known to be involved in the migration and proliferation of embryonic chick corneal mesenchymal cells and their subsequent differentiation to corneal keratocytes. Levels of hyaluronic acid are highest during migration and proliferation and then it is removed just prior to differentiation (85). Hyaluronic acid has also been found to be involved in the migration of sclerotomal cells to the developing chicken embryo notochord during chondrogenesis. When these cells reach the notochord, HA is then removed. This is followed by synthesis of a cartilage matrix by these cells (86—88). There are many more examples where a correlation has been made between the presence of hyaluronic acid and morphogenetic movement during the development of embryonic tissues. In most these cases, subsequent differentiation has been shown to be associated with decreased amounts of hyaluronic acid. In addition to embryogenesis, similar observations of changes in amounts of hyaluronic acid levels have been observed during salamander limb regeneration, tendon remodeling, bone fracture repair and skin wound healing. The role of hyaluronic acid in morphogenesis has recently been reviewed (83). Alterations in amounts of hyaluronic acid associated with transformed cells have been well documented. Increase hyaluronic 18 acid synthesis and/or amounts in transformed cells has been observed in chicken embryo fibroblasts infected with the wild type strain of Rous sarcoma virus (89) or with its temperature- sensitive mutant, the Bryan strain (90), in Rous sarcoma virus (RSV)-transformed chondrocytes (91) and chondrocytes transformed with the temperature-sensitive mutant, Prague A, LA24 (92), SV40-transformed— hamster embryo fibroblasts (93) and human fibroblasts (94), primary African green monkey kidney cells transformed with herpes simplex type II virus (93), human glioma cells (95), certain carcinomas (96,97,98), Morris hepatomas (99,100), Wilm's tumor (101) and fibrosarcomas (102). Although increased levels of hyaluronic acid upon transformation would seem to be a general rule, exceptions have been found including mouse melanomas (103) and SV40-transformed 3T3 fibroblasts (104), where hyaluronic acid is lower than in their normal counterparts. Thus, there is no absolute consistency even among cells transformed with SV4O virus. However, it is obvious that a great majority of transformed cells have greater amounts of hyaluronic acid than their normal counterparts. Another important factor which should be considered is the cell surface distribution of HA. The distribution could be greatly altered in mouse melanoma and SV40-transformed 3T3 fibroblasts. We have addressed the question of cell surface distribution of HA in Chapter 4. In comparison to the amount of information known about the role of hyaluronic acid in morphogenesis and differentiation, rela- tively little is known concerning its role in transformation. How-— ever, increasing interest in this area has provided information 19 concerning the potential role of hyaluronic acid in influencing cell-cell and cell—substratum interactions. Hyaluronic acid has the potential of interfering with cell—cell interactions as it has been shown to form a "barrier" on some transformed cell surfaces. Previous work by Hawkes and coworkers (105,106) has demonstrated that the nonfluorescent reagent fluorescamine, can react approximately 3—fold more with available primary amines (to give a fluOrescent adduct) in normal cells than in transformed cells. It was proposed that a "barrier" might exist which prevented this nonphysiological reagent from reaching the cell surface. Subsequently, we found that pretreat— ment of cells with the hyaluronic acid—specific endoglycosidase from Streptomyces hyalurolyticus, resulted in equivalent fluorescamine labeling of normal and transformed cells at a level slightly higher than found with untreated normal cells (107). These results supported our "barrier" hypothesis. Further evidence to support this idea has come from the work of Burger and Martin (108) who have observed that RSV-transformed chicken embryo fibroblasts required much less wheat germ agglutinin or concanavalin A for maximal agglutination if they were first treated with Streptomyces hyaluronidase, presumably because hyaluronic acid had been covering up the agglutinin binding sites. Furthermore, lymphocyte-mediated cytolysis of adherent fibrosarcoma cells is enhanced following removal of a hyaluronidase—sensitive "halo" around the cells (109). Cell—substratum interactions, in particular adhesion, also appear to be influenced by changes in amount of hyaluronic acid 20 present. Kraemer and coworkers (110,111) have shown that loosely attaching and tightly attaching adhesion variants contain more and less cell surface hyaluronic acid, respectively, than their parental CHO cell line. However, they found that these variants did not differ in their ability to attach to the substratum. Thus hyaluronic acid does not appear to be required for cell attachment but was at least partially responsible for the observed alterations in adhesion (detachment). Biochemical analysis of cellular footpads remaining associated with the cell substratum following EGTA—mediated cell detachment has also provided information concerning the role of hyaluronic acid in adhesion. Culp and coworkers (29) have found that footpads or adhesion sites from mature, migrating cells contain much more hyaluronic acid than that found in new adhesion sites of attaching cells. They have suggested that as hyaluronic acid accumulates in the mature adhesion sites, it destabilizes the footpad allowing it to detach from the cell as it migrates. Schubert and LaCorbiere (112,113) have isolated an adhesion mutant from the anchorage-dependent L6 skeletal muscle myoblast cell line which they designate, M3A. They have isolated shed glycoprotein/glycosaminoglycan complexes containing several proteins including fibronectin and collagen and the glycosamino- glycans, hyaluronic acid, heparan sulfate and chondroitin sulfate. Addition of complexes from L6 cultures to M3A cells leads to increased adhesion, whereas M3A complexes do not possess this activity. In addition, the M3A complexes have been found to 21 contain elevated amounts of hyaluronic acid in comparison to amounts of hyaluronic acid from L6 complexes. Finally, it has been demonstrated that SV-3T3 possess high affinity binding sites for hyaluronic acid (114). The ability of hyaluronic acid to bind to cell surfaces may account for the recent observation that addition of hyaluronic acid to cultured 3T3 cells facilitates their detachment from the substratum (115). As described, hyaluronic acid is involved in migration and proliferation of cells during morphogenesis. Recently, Toole _e_t_ _a_l_. (97) has provided evidence that hyaluronic acid may be involved in migration and proliferation of tumor cells during the process of metastasis. They have analyzed hyaluronic acid content in the V2 carcinoma grown in rabbits, where it is invasive, to that found in the V2 carcinoma grown in the nude mouse, where it is noninvasive and have found 3-4 times more hyaluronic acid in the rabbit tumor. In the process of morphogenesis, it has been proposed that hyaluronic acid exerts a swelling pressure which can give rise to separation of cell and collagen layers, thus providing avenues for cell migration (83). Finally, analysis of several tumors isolated from mammalian sources has revealed several variations of intercellular glycosaminoglycans when compared to the corresponding normal tissue. These differences were small for benign and noninvasive tumors while invasive tumor glycosaminoglycans were significantly different from glycosaminoglycans isolated from normal tissues. The most notable alteration described was the increased amount of hyaluronic acid found in many tumors (96). 22 The components described above are the major and most completely characterized components of extracellular matrices in general. The ECM is made up of many more proteins, glyco— proteins, mucins, glycosaminoglycans, proteoglycans and lipids which have yet to be characterized. Characterization of these components will shed light upon their potential structural and/or functional roles and provide additional information about the role of the ECM in modulating cell behavior. Furthermore, although changes in the ECM have been monitored during morphogenesis and differentiation, such an analysis of the ECM during the development of the transformed state has to our knowledge not been examined. The system we have chosen for our studies makes use of chicken embryo fibroblasts infected with the temperature—sensitive mutant of the Prague A strain of Rous sarcoma virus, LA24 subclone G2. The virus has been cloned from virus given to us by Dr. Steven Martin, U.C. Berkeley. Isolation of the temperature- sensitive mutants of RSV and their use in investigating cell transformation has been reviewed (116). In our system we use freshly derived embryonic cells, instead of established lines of normal and transformed cells, which eliminates variations which arise during the passage of cell lines. Furthermore, we do not encounter differences due to viral infection as both nontransformed and transformed cells are infected and release virus. This system gives us the advantage of being able to "turn-on" and/or "turn—off" transformation and thus by altering the growth temperature, we can monitor the process of transformation as a 23 function of time after temperature shift. Growth of cells infected with the temperature-sensitive mutant of Rous sarcoma virus at the permissive temperature (350) allows for the expression of the oncogene product, pp6OSFC. This protein has been found to be a tyrosine protein kinase (for review see 117). This activity is expressed at normal levels in infected cells cultured at the nonpermissive temperature (410). The ability to monitor changes which occur during the process of oncogenesis not only allows us to measure early changes which result during the development of the transformed phenotype but also allows us to compare temporally, changes we observed, to other already characterized altered parameters of transformation. For example, several differ- ences between virally-transformed and nontransformed cells at the level of the cell surface have been described. Some of the changes include: (a) loss or reduction of fibronectin (118,119), (b) increase in the rate of hexose transport (120), (c) changes in membrane glycolipids (121), (d) alterations of cytoskeletal microfilaments (122), (e) increased agglutinability by Iectins (123), (f) decreased synthesis of collagen (17,18,19), and the changes in cell surface GAG described above. Using this system our goal has been to analyze changes in the substratum associated material (SAM) which occur during the early stages of the transformation process. We have pursued this goal by analyzing changes in substratum associated proteins/glyco- proteins and in the glycosaminoglycan, hyaluronic acid, and have examined its potential importance in cell-substratum adhesion. SAM from 3T3 and SV3T3 murine fibroblasts have been extensively 24 characterized by Culp and his coworkers. They have examined the proteins (124), glycoproteins (1,125), phospholipids (126), proteoglycans (127), and glycosaminoglycans (128) in SAM. Furthermore, biochemical characterization of these macromolecules in SAM from old or mature adhesion sites and new adhesion sites has made it possible to confirm predictions of interactions of these components with one another and their potential role in cell-substratum adhesion (28,29,129). They have proposed that heparan sulfate and fibronectin function as adhesive molecules as they are particularly enriched in new adhesion sites. Old adhesion sites (detaching) have greater amounts of hyaluronic acid and they have proposed that this somehow destabilizes the heparan sulfate/fibronectin interaction with the cell substratum (29). However, their work has concentrated on old and new adhesion sites and not on differences between normal and fully transformed cells. Furthermore, we have concentrated on changes occurring during the development of the transformed state. The work contained in this thesis has thus been divided into four parts: (a) the initial characterization of substratum associated proteins and identification of a transitory, low molecular protein which is increased in SAM during the early stages of transforma— tion; (b) characterizatio‘n of this low molecular weight protein; (c) analysis of the cell surface distribution of hyaluronic acid during the development of the transformed state; and (d) the potential role of hyaluronic acid in modulating cell-substratum adhesion. TRANSFORMAT ION-SENSI T IVE PROTEIN ASSOCIATED W I TH THE CELL—SUBSTRATUM Use of a cell culture system which allows us to "turn—on" transformation has made it possible to study alterations of normal cell properties during the development of the transformed state. Using such a system, i.e., chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus, we have analyzed changes in substratum associated proteins following transfer of infected cells to the permissive (transforming) temperature. This analysis has resulted in the detection of a low molecular weight substratum associated protein. In this chapter I have characterized some of the transformation properties of this protein. 25 MATERIALS AND METHODS Cell Culture Chicken embryo fibroblasts (CEF) were prepared as previously described (130), using specific pathogen—free eggs from SPAFAS, Norwich, CT. In general, primary cultures (390) were seeded at 1x106 cells/100 mm plastic tissue culture dish (Falcon, Oxnard, CA) and infected with approximately 2.5 x104 focus forming units per dish of wild-type or a temperature- sensitive mutant of the Prague A strain of Rous sarcoma virus, LA24, clone G2 (LA24 was a gift from Dr. Steve Martin, U.C. Berkeley). All experiments were completed with secondary or tertiary cultures which were generally seeded at 1-1.5 x 106 cells per 100 mm dish in 10 ml medium 199 (Gibco, Grand Island, NY) supplemented with 2% tryptose phosphate broth (Difco, Detroit, MI), 2% calf serum (Flow Laboratories, McLean, Virginia), 1% chicken serum (Flow Laboratories, McLean, Virginia) and 0.1% glucose (medium 221+). The cells were used within 48h of seeding. Chicken embryo fibroblasts infected with the temperature sensitive mutant, LA24, clone GZ (LA24G2—infected CEF), were grown in a humid atmosphere of 5% CO and 95% balanced air at 2 o . . . . . 41 (nonpermlsSlve temperature) untll temperature shift, at which time cells to be transformed were placed in a 350 incubator 26 27 (permissive temperature). SAM from LA24G2—infected CEF was prepared at various times after temperature shift. Preparation of SAM The experimental procedure for preparation of SAM is an adaptation of the method described by Culp (125) and is schematically shown in Figure 1. Basically, radioactively labeled, subconfluent monolayers of cells, cultured as described, were washed 2—3 times with 5—10 ml of Ca2+, MgZ+ free—phosphate buffered saline (CMF-PBS, Gibco, Grand Island, NY) containing 1 mg/ml e-amino-n-caproic acid and 1mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4 (CMF—PBS+). The cell monolayers were then incubated with 5 ml of 5 mM EGTA (Sigma, St. Louis, MO) in CMF—PBS+ per 100 mm tissue culture dish at 390 for 15 min. Cells were gently removed by pipetting and collected by centrifugation (HN—Sll, Damon/IEC LTK, Bedfordshire, England) at 400 x g for 5 min. The material remaining attached to the culture dishes was then washed by pipetting 2-3 times with CMF—PBS+, at first gently and then vigorously, and finally washed once with ice cold, distilled H20. Material remaining attached to the culture dish at this point is defined as SAM. SAM and cells were solubilized in an electrophoresis sample buffer containing 80 mM Tris-Cl, pH 6.8, 10% glycerol, 10% 2—mercaptoethanol and 2% sodium dodecyl sulfate (SOS-PAGE sample buffer). SAM was removed from four tissue culture dishes (100 mm) in a total volume of 100-200 111 by scraping the dishes with a 1 1/4 inch wide rubber policeman. Samples were then boiled for 10 min before electrophoresis. This 28 EXPERIMENTAL PROCEDURE Secondary or tertiary chicken embryo fibroblasts intected with Rous sarcoma virus (LA 24) 12-20 hours temperature shitt / \ 41° 35' , radioactive precursors ~ .\ 4-12 hours V radialsotope removed — 5 mM EGTA. ACA in CMF-PBS added -— incubated 15 minutes extenswe washing released substrate attached material released cells (SAM) cells solublllzation. SOS-PAGE and tluorography Figure 1. Schematic diagram of SAM preparation procedure. 29 procedure solubilized approximately 85% of the total radiolabeled material in the culture dishes. Solubilized SAM and cell proteins were analyzed by gel electrophoresis. Prior to solubilizing SAM in SOS-PAGE sample buffer, drained dishes were stored at 40. Storage for long periods of time does not appear to affect the 21K protein whereas higher molecular weight proteins were not as stable. Metabolic Labeling LA24G2-infected CEF were incubated for various lengths of time (4—12 h) with several radioactive precursors. These included H-amino acids, [2-3H]mannose (15 pCi/mmol), 14C-amino acids, [355]methionine (936 uCi/mmol), H332POA (carrier free) and [y—32P]ATP (3100 uCi/mmol). All radioactive products were purchased from New England Nuclear, Boston, MA. Phosphorus—32 experiments were carried out in 221+ or medium with reduced phosphate. Other details of the labeling procedure are described further in the text or figure legends. Gel Electrophoresis Gradient polyacrylamide gel electrophoresis -was performed using the buffer system of Laemmli (131). Slab gel apparati, model no. SE 520 or 500 (Hoeffer Scientific, San Francisco, CA), were used in all experiments. The dimensions of the gels were 28 x 14 x 0.15 cm (model 520) or 11 x 14 x 0.15 cm (model 500). Stacking gels were 1 cm in length. Acrylamide gradients (5-16% or 7—17%) were poured using a Hoeffer gradient maker (model no. XPO 77). Unless specified, the amounts of radioactivity loaded per 30 lane were in the range of 20,000—50,000 cpm for 3H—Iabeled proteins and 7,000-15,000 cpm for 35S—labeled proteins. Proteins used as molecular weight standards were cytochrome c (12.3K), B—lactoglobulin (18.4K), d-chymotrypsinogen (25.7K), ovalbumin (43K), bovine serum albumin (68K), phosphorylase B (92.5K) and myosin H chain (200K) from Bethesda Research Laboratories, Gaithersburg, MD. Immediately after electrophoresis, gels were fixed in 10% trichloroacetic acid or methanol:water:acetic acid (5:5:1, v/v/v). Gels were then stained in 0.125% Coomassie blue in 25% isopropanol/10% acetic acid overnight followed by destaining in 40% methanol/10% acetic acid. Prior to drying, gels containing 3H—, 1AC- or 35S-labeled proteins were impregnated with 2,5—diphenyloxazole (PPO) as described by Bonner and Laskey (132). Dried gels were then exposed to Kodak XR-Omat film at -750. Immediately after destaining and drying, gels containing 32P—labeled proteins were exposed to film at room temperature using DuPont lightning plus intensifying screens. Incorporation of radioactivity into proteins was determined by cutting out the Coomassie blue stained bands from the dried gel, dissolving the cross—linked acrylamide in 1 ml of 30% H202 at 550 for 6 h and measuring radioactivity in a liquid scintillation counter (LS9000, Beckman, Irvine, CA). RESULTS Comparison of Substratum Associated Proteins Chicken embryo fibroblasts infected with the temperature- sensitive mutant of Rous sarcoma virus, LA24G2, were incubated with 3H-labeled amino acids for 12h beginning 4 h after temp— erature shift. The cells were then detached from the culture dishes with EGTA as described in Figure I and both cellular proteins and those remaining with the substratum associated material (SAM) analyzed by gradient polyacrylamide gel electrophoresis and fluorography. A comparison of 3l-I-labeled proteins from normal (410) and transforming (350) cultures indicate many apparent differences in both cellular and SAM fractions (Figure 2). Of these differences, which represent both increases and decreases in the levels of incorporation of tritium into specific polypeptides, the most significant and consistent is an elevation in the incorporation of tritium into a small protein present in the SAM of transforming cells. This protein, which is designated the 21K protein, migrates with an apparent molecular weight of 20,750 as determined by comparison with protein stan- dards (10 determinations). This protein comigrates on SDS- polyacrylamide gels with a similar protein in SAM from normal, uninfected chicken embryo fibroblasts as well as LA24G2—infected CEF cultured at the nonpermissive temperature (410). It is 31 32 Figure 2. Comparison of metabolically labeled substratum associated proteins and EGTA-released cel ular proteins. LA24GZ-infected CEF were labeled with H—amino acids (25 uCi/dish) for 12 h beginning 4 h after temperature shift from 410 and 350. Samples containing equivalent amounts of radioactivity were analyzed by gradient polyacrylamide gel electrophoresis, with separating and stacking gels comprising 6-15% and 3% acrylamide, respectively, and fluorograph . Lanes A and B, EGTA- generated SAM proteins at 41 and 350, respectively; C and D, the corresponding released cellular proteins. FN, fibronectin. ' 33 molecular weight standards 200K _92.5K ——68K ——25.7K —18.4K 34 therefore apparently not a protein induced by viral infection. The 21K protein is clearly enriched in the SAM fraction of transforming cells as evidenced by both increased tritium incorporation and also by Coomassie Blue staining of the polypeptides (data not shown). Quantitative information concerning the alteration in amounts of 21K protein was obtained by measuring the incorporation of radioactivity labeled amino acids into the polypeptides of SAM. To do this, LA24GZ-infected CEF were labeled with 3H-amino acids for 12 h beginning 3 h after temperature shift. Substratum associated proteins from 410 and 350 (15h) cultures were then separated by polyacrylamide gradient gel electrophoresis. Lanes containing Coomassie blue stained polypeptides were then cut out of the slab gel and sliced into 2 mm pieces. These were then solubilized and associated radioactivity determined. As illustrated in Figure 3, a 0 (350-SAM), with a protein in SAM from cells grown at 35 molecular weight of approximately 21,000 has accumulated much more radioactivity than the corresponding protein from 410-SAM. This alteration represents a 4.4-fold increase in incorporated radioactivity. Characterization of the 21K Protein We have Shown that the 21K protein can be metabolically _ . 3 14 . . 35 . . labeled With H- and C-amino aCIdS and [ S]methionine. To further characterize the 21K protein, cells were incubated with 3 [2- H]mannose and SAM proteins analyzed by gradient polyacrylamide gel electrOphoresis as described. Figure 4 35 V V V V U 21K , 20' 4f l 2 I 9 .51 60K ‘JK 2 7K 10‘“ l" l l l l l « Tar at «>7 s . O 3 3 l '7'. °l - 20» . 3 35 1 \ E Q U isi » , 10 . 5i . 25 so 75 ion .25 gel slice number Figure 3. Radioactive profiles of substratum associated proteins6 Secondary LA24GZ-infected CEF seeded at 1 x 10 cells/100 mm dish were labeled 3 h after temperature shift with 25 uCi/culture dish H-amino acids and substratum associated proteins prepared 15h after temperature shift. Proteins were then separated by electrophoresis on 5—16% gradient polyacrylamide gels in thg presence of SDS. Coorgassie blue stained lanes for 41 (top profile) and 35 (bottom profile) substratum associated proteins were cut out of the slab gel and sliced into 2 mm pieces. These were then solubilized in 30% H202 and associated radioactivity determined. Figure 4. 36 Incorporation of [2-3H]mannose into SAM glycoprot ins. Secondary LA24GZ-infected cCEF, seeded at 1x10/100 mm dish, were grown at 41 for 2c; h and then half the dishes were transferred to 35 . Two hours after temperature shift the medium (221+) was replaced 3with 5ml of fresh medium containing 15 tiCi of [ S]- methionine (control dishes) or 850 uCi of [2- H]num1nose. SAM was prepared by EGTA treatment 12h after temperature shift and the protein samples, containing equivalent eunounts cfi radioacthnty, analyzed by $05 gradient polyacrylamide (7-17%) gel electrophoresis and fluorography. Lane 1, molecular weight standards; lanes 2 and 3, [2- H]mannose-—labeled SAM proteins at 41 and 35 , respectively (5 day exposure of autoradio- gram); lanes 4 and 5, same as lanes 2 and 33) (12 day exposure of autoradiogram); lanes 6 and 7, [ S]methio- nine labeled SAM proteins at 350 (12 h) and 410, respectively. IFN, fibronectin. 37 i-FN I 21': 43K ‘wl “ 25.7K- 21K— "-— —21K 18.4K - 12.3K - 38 indicates that the 21K protein in SAM, detectable by incorporation [35 of S]methionine (lanes 6 and 7), does not incorporate mannose (lanes 2-5) and is therefore not a mannose-containing glyco— protein. Similar experiments designed to determine whether the . . 32 protein is phosphorylated are also negative. Incorporation of P into the 21K protein from transforming cells, incubated with either [y—32P]ATP or H3 32PO“, could not be detected (data not shown). The Effect of Cell Density on 21K Protein Synthesis and Deposition in SAM Cell density and rate of growth can substantially influence the synthesis of ECM macromolecules (133,134). It was therefore important to determine whether the increase in the 21K protein is related to a transformation—specific alteration or results from differences in growth conditions of the cultures at the two temperatures. A comparison of cells growing at a range of densities (sparse to confluent) at both 410 and 350 demonstrates that the incorporation of tritium into the 21K protein is enhanced in the SAM of transforming cultures at all densities examined (Figure 5) and is particularly apparent at subconfluent and confluent densities. Smaller differences at low density may be due to much slower growth rate of cells cultured at this density (data not shown). This is likely due to an underdeveloped ECM which has been shown to be required for normal cell proliferation (38). All subsequent experiments reported in this paper were therefore performed with subconfluent cultures under conditions where the Figure 5. 39 The effect of cell density on the synthesis of substratum associated proteins. Secondary LAZgGZ-infected CEF were seeded in 100 mm dishes at 1 x 10 cells/disg (sparse), 1 x 10 cells/dish (subconfluent) and 4 x 10 cells/dish (confluent). After 22 h at 410, half the cultures were transferred to t permissive temperature and 3 h later incubated with H-amino acids (25 uCi/dish) for 8 h. Samples containing equivalent amounts of radioactivity for each density (sparse, 7,100 cpm; subconfluent and confluent, 15,700 cpm) were analyzed by 505 polyacryla- mide gel (7—17%) electrophoresis and fluorography. SAM proteins from cultures which had been maintained at 41 and 350 respectively for 11 h are represented by lanes A and B (sparse), C and D (subconfluent) and E and F (confluent). The autoradiogram of lanes A and B was exposed for a longer period of time than C-F to compensate for the lower tritium content in the samples. FN, fibronectin. 40 41 O and 410, as cells were growing at equivalent rates at 35 previously determined (106). The synthesis and deposition of the 21K protein in the SAM of LA24GZ—infected cells therefore appear to be stimulated by the process of transformation and not by variations in the growth conditions of actively growing cells. This is in sharp contrast to other components of SAM such as fibronectin which is clearly influenced by cell density. At confluent densities in both 410 and 350 cultures (Figure 2, lanes E and F), fibronectin does not migrate at its usual position in the polyacrylamide gel (compare with lanes A—D). It remains to be determined whether this is because fibronectin is no longer being synthesized and deposited in SAM or because fibronectin is cross linked, either to itself or other SAM components. As Coomassie blue staining also indicates that fibronectin (Mr =220,000) is decreased in SAM at high cell densities and a significant amount of radioactive material is present at the top of the separating gel, the latter explanation is likely. Is 21K Protein a Turnover Product of a Normal Cellular Protein Induced by 'Transformation? The 21K protein is not enriched in the SAM of normal, uninfected chicken embryo fibroblasts grown at 410 or 350 (data not shown). Interestingly, the 21K protein is also not elevated in the SAM of cells infected with the wild type Rous sarcoma virus (Prague A strain) and which are therefore fully transformed (Figure 6). To examine the p0ssibility that this protein is Figure 6. 42 Comparison of substratum associated proteins from RSV(LA24G2)-infected CEF, Prague A wild type RSV— infected CEF and normal, uninfected CEF. Lanes A and B are substratum associated proteins fom secondary LA24G2-infected CEF seeded at 1.5 x 10 cells/100 mm culture dish, and cultured at 410 and 350 (15 h), respectively. Secondary Prague A wild type RSV—infecteg CEF and normal, un' fected CEF were seeded at 2 x 10 cells and 1.5 x 10 cells/100 mm dish, resp ctively, and 22 h later labeled for 15 h with 50 iiCi H—amino acids/dign. Lanes C and D are the EGTA-generated substratum associated proteins of wild type RSV-infected CEF and normal, uninfected CEF, respectively. 43 w .‘H. . U.” V I 1.0. O . .. ~ ‘ ,1... . “‘i' p a.» «r- "‘"" -— 21 K — ”a ""4“ r . J. 44 derived from a higher molecular weight precursor of normal cells, which is processed or degraded upon transformation, normal cell proteins were labeled with [35$]methionine and the cells trans— ferred to 350 in the absence of radioisotope. The levels of incorporation of 355 into the 21K protein after a 2 or 4 h period at 350 in the absence of label (Figure 7, lanes A and B) are significantly lower than the control which results from a 4 h pulse of [35$]methionine at 350 (lane C). The 21K protein is therefore not produced by turnover of a normal cellular protein upon transformation . The Kinetics of 21K Protein Synthesis and Deposition into SAM Following Temperature Shift The synthesis and deposition of the 21K protein in SAM was investigated as a function of time after shift to the permissive [35$]methio— temperature (350) by measuring the incorporation of nine into the protein during incubation periods of 4 h prior to preparation of SAM (Figure 8). The data are expressed as the ratio of radioactivity in the 21K protein from SAM of 350 cells to that in the 21K protein from 410 cells. The synthesis and deposition of 21K protein in SAM are stimulated significantly during the early stages of transformation, peaks around 8 h after temperature shift and by 20 h declines to levels 1.3-fold that of 21K protein found in the SAM of control cells at 410. Again, this is in distinct contrast to the ratio of amounts of incorporation of [35$]methionine into fibronectin in SAM at 350 and 410, which do not alter appreciably during the 36 h of the experiment. 45 Figure 7. Turnover of normal cellular proteins during the development of the transformed phenotyrge. Secondary LA24GZ-infected CEF were seeded at 1 x 10 cells/100 mm dggh and maintained at 410. Cells were incubated with [ S]methionine (15 uCi/dish) for 12 h (lane A) and 10 h (lane B) before tempeg‘gture shift. At temperature shift medium containing [ S]methionine was replaced with mediu lacking radioactively labeled methionine and the S label was chased at the permissive temperature for 2 h (lane A) and 4 h (lane B). L C represents SAM proteins from cells incubated with [ S]- methiocnine (30 pCi/dish) for 4 h after temperature shift to 35 . The data represent an analysis of samples, containing equivalent amounts of radioactivity, by gradient polyacrylamide gel electrophoresis (7-17%) and fluorography. —200K ’ ' —92.5 K —25.7 K 47 e. -l V e‘ in 8 . E Q 0 law 4 0,, ——o q a 4 8 l2 16 20 24 28 32 36 Time alter temperature sniit (hours) Figure 8. Analysis of 21K protein synthesis and deposition in SAM as a function of transformation . Seocondaryo LA24GZ- infected CEF were transferred from 41 to,5 35 at zero time. Cells were labeled with [ S]methionine (15 “Ci/100 mm) dish for 4 h periods prior to prepara— tion of SAM. For example, data for the zero time point was derived using cells which were incubated with radioisotope for the 4 h before temperature shift. Cultures were seeded so that cell densities were approxi— mately equal at the time of SAM preparation. Equal amounts of radioactivity were applied to each lane of SDS gradient polyacrylamide gels (separating and stacking gels comprising 7-17% and 4% acrylamide, respectively). The 21K protein and fibronectin bands were cut out and processed for radioactivity determina- tions as described. Data for the 21K protein (O-——-—-O) represents the average of two experiments and for fibronectin (l—-—-l) a single experiment. 48 Turnover of the 21K Protein The rates of turnover of the 21K protein at 410 and 350 were investigated by labeling cultures for 6 h with [355]methio— nine at 350 to incorporate isotope into the 21K protein, followed by removal of labeling medium and a chase period at 410 and 350 for 4, 8 and 12 h. SAM was prepared as described starting at 0 h and polypeptides separated by electrophoresis. After drying the stained gels, the 21K protein was cut out, solubilized and radioactivity determined. Total radioactivity associated with SAM was also determined. It is apparent from Figure 9 that the 21K protein represents approximately 10% of the total [35$]methionine incorporated into SAM proteins. Note the 10—fold difference in radioactivity scales. It is evident that turnover of the 21K protein is much slower at the transforming temperature (350, right panel) than at 410 (left panel). However, its rate of turnover at either temperature is comparable to the rate of turnover of total SAM proteins. Thus, even though synthesis and deposition of the 21K protein decline at later stages in the transformation process, the slower rate of turnover must contribute to its net accumu- lation in the SAM of transforming cells. Indeed, long term labeling indicates that by 15 h after temperature shift, the level of incorporation of label is 4.4-fold higher in the 21K protein of 350 cells than of 410 cells (Figure 3). It should be noted though that in fully transformed cells (Prague A-infected CEF), turnover of SAM is apparently more rapid (Chapter 3). 49 20 n ‘2‘ 2 .5." 5 u I ‘ H 9 E I a “ o S .s' u 3 ' o 3 to a m x r—a g" «'3 —d ('0'? in J." 5 )- ul [- ‘l 5 A 1‘ L L L L 4 8 12 4 8 12 Chase time (hours) Figure 9. Turnover of the 21K protein and total substratum associated proteins at the permissive and nonpermissive temperaturgs. Secondary LA24GZ—infected CEF were sgeded at 1 x10 cells/100 mm dish and cultured at 4135for 20 h. They were then incubated for 6 h with [ S]— methionine (15 uCi/dish) at 350. The medium containing radioisotope was then removed and replaced with fresh medium. Half the cultures were transferred to 410 and SAM proteins were prepared from both the 350 and 410 cultures immediately and at 4, 8, and 12 hours after Hag beginning of the chase period. Incorporation of [ S]methionine into total SAM proteins was determined by liquid scintillation counting of samples in electro— phoresis buffer. SAM proteins (total protein from 4 x 100 mm culture dishes) were then analyzed by $05 gradient polyacrylamide (7-17%) gel electrophoresis and stained with Coomassie blue. The 21K protein band was removed from the dried gels and its associated radio- activity determined as dggcribed in Materials and Methods. Incorporation of [ S]methionine into total SAM proteins (l—-—-—I) and the 21K protein (D——--—D) is plotted as a function of time after chase at the nonpermissive and permissive temperatures. Right panel-—turnover at 350; left paneI--turnover at 41 . DISCUSSION In this chapter we report the analysis of substratum associated proteins during the development of the transformed state (as opposed to the fully transformed state). We have analyzed the d_e_ 22312 synthesis of a small protein which is deposited in the SAM of cultured chicken embryo fibroblasts during the early stages of oncogenic transformation. To our knowledge this protein, which has a nominal molecular weight of 21,000, has not been described previously. The 21K protein is not a mannose—containing glycoprotein and under our experimental conditions does not appear to be phosphorylated. The observed alteration of the 21K protein in SAM is not the result of differences in growth rate at 350 and 410 as LA24G2—infected CEF cultured at the permissive (350) or nonpermissive (410) temperature have approximately equivalent doubling times (data not shown). This protein is present in SAM of normal, uninfected CEF at levels equivalent to that found in SAM of CEF infected with the temperature—sensitive mutant of Rous sarcoma virus, LA24G2 and cultured at the nonpermissive (nontransforming) temperature (41°). Initiation of transformation, by transfer of LA24GZ-infected CEF to the permissive temperature (350) leads to a rapid and dramatic increase of this protein as observed by both Coomassie blue 50 51 staining and radioisotope incorporation data. Measurement of total protein in SAM from four 100 mm tissue culture dishes containing a total of 12 million cells demonstrated the presence of approximately 70-100 119 protein. We have shown that the 21K protein is readily visible with Coomassie blue staining and that it makes up approximately 10% of the total radioactively labeled substratum associated proteins. It is therefore likely that the 21K protein may be present in quantities of around 10 119 per 12 miHion ceHs. Increased synthesis of the 21K protein can be induced by culturing LA24GZ-infected CEF at 350 for only 1h (data not shown). The increase in 21K synthesis and deposition into SAM appears to peak by 8 h following temperature shift to 350. This is followed by an eventual decline to a level which is 1.3-fold higher in transforming cells, 20 to 36 h after temperature shift (Figure 8). The transitory nature of the 21K protein in SAM is not the result of turning on the degradation of larger, normal cell proteins following temperature shift as shown in Figure 7. We have measured a 4.4-fold increased accumulation of the 21K protein by 15 h following temperature shift. In light‘of the 4 h synthesis data presented in Figure 8, this would suggest that the 21K protein is turned over more slowly at the permissive temperature during the early stages of transformation. Indeed, this is what we find. The apparent transitory nature of 21K protein synthesis and deposition into SAM are particularly interesting in light of the fact that the SAM of fully transformed CEF infected with the 52 related wild-type virus does not contain increased amounts of the 21K protein. This stresses the potential importance of the 21K protein in the development of the transformed state. Although many extracellular components have been shown to be transiently involved in morphogenesis and differentiation (83), no such components have been demonstrated to be involved in the development of the transformed phenotype. Thus understanding the function of this protein may provide additional information about the transformation process, per se. CHARACTERIZATION OF THE TRANSFORMATION-SENSITIVE 21K PROTEIN OF THE CELL-SUBSTRATUM In the previous chapter, the detection and initial characterization of the transformation properties of the 21K protein were described. In this chapter I have further characterized the 21K protein for: (1) its transformation—sensitive properties; (2) its association with the cell—substratum; and (3) the manner in which it is deposited into the substratum. 53 MATERIALS AND METHODS Cell Culture Chicken embryo fibroblasts were cultured as described earlier except that we have also analyzed CEF infected with the temperature—sensitive mutant of Rous sarcoma virus, NY68, the A strain of Schmidt-Ruppin. In certain experiments, uninfected and LA24GZ—infected CEF were incubated in media containing 100 ng/ml phorbol myristate acetate (PMA, PL Biochemicals, Milwaukee, WI) in dimethyl sulfoxide (DMSO, final concentration in medium, 0.1%). Previous work (135,136) has demonstrated that the concentration of PMA used is sufficient for the development of the transformed phenotype. PMA was added at zero hour (time of temperature shift) followed by addition of [355]methionine (140 mCi/mmol, Amersham, Arlington Heights, IL) to normal CEF and LA24G2— infected CEF cultured at the permissive (350) and nonpermissive (410) temperatures. Analysis of Substratum Associated Material by Alternative Extraction Procedures As described in Chapter 2, radioactively labeled cells are removed from their culture substratum with 5mM EGTA in CMF-PBS+ followed by solubilization of SAM in SDS-PAGE sample 54 55 buffer. In several experiments, SAM was also solubilized in 0.1%-0.2% $05 as described by Culp (1). Various methods have been utilized to prepare and analyze the extracellular matrix . We were therefore interested in analyzing and comparing proteins from SAM prepared by EGTA treatment, as described, to substratum associated proteins prepared with several alternative extraction agents. It was anticipated that the data obtained from this analysis would also provide additional information concerning the nature of the association of the 21K protein with the substratum. The resistance of the 21K protein to extraction from SAM was determined by treating cell monolayers or SAM with the following agents: octylglucoside (0.5% (w/v) in PBS, pH 7.4); Triton X-100 (0.5% (v/v) in PBS, pH 7.4); Nonidet P40 (NP40) (0.5% (v/v) in PBS, pH 7.4); deoxycholate (0.5% (w/v) in distilled and deionized H20); 0.1 N NaOH; 0.1 N NHAOH; 1.0 N NHAOH; 0.5% NP40 plus 0.5 M KCI; 0.5% NP40 plus 1.0% 2-mercaptoethanol; 4 M urea in PBS, pH 7.4; 6 M urea; 6 M urea plus 10% 2—mercaptoethanol; and 6 M guanidine HCI. The reagents were obtained from the following sources: Triton X-100 and 2-mercaptoethanol (Bio-Rad Lab., Richmond, CA), NP40 (Particle Data Lab., LTD., Elmhurst, IL), deoxycholate and octylglucoside (Sigma, St. Louis, MO), urea and guanidine HCI (Bethesda Research Lab., Inc., Rockville, MD) and all others were reagent grade (Mallinckrodt, Paris, KY). The same protocol for SAM preparation (Chapter 2 and Figure 1) was used in all extractions with the exception of replacing 5 mM EGTA in CMF-PBS+ with the appropriate extracting 56 agent. Unless otherwise noted, the remaining substratum associated proteins were solubilized with SDS-PAGE sample buffer and analyzed by polyacrylamide gradient gel electrophoresis and fluorography . Sensitivity of the 21K Protein to Proteolysis Sensitivity of the 21K protein to both exogenous and endogenous proteases was determined. Sensitivity to exogenous proteases was determined by incubating EGTA-SAM (this abbrevia- tion will be used to describe SAM prepared by extraction of cells with 5 mM EGTA in CMF-PBS+, pH 7.4) in 3 ml PBS/100 mm tissue culture dish containing: tosylamide—phenylethyl chloromethyl ketone (TPCK)-trypsin (2 units/ml, Worthington, Freehold, NJ); tosyl—lysine chloromethyl ketone (TLCK)—chymotrypsin (2 units/ml, Worthington, Freehold, NJ); Protease XIV (Pronase, 0.5 mg/ml, Sigma, St. Louis, MO) and TPCK, TLCK—treated collagenase (clostridiopeptidase A, 0.83 units/ml, Worthington, Freehold, NJ). TPCK and TLCK (Sigma, St. Louis, M0) were added from a stock solution of 5 mg/ml ethanol to a final concentration of 50 ug/ml. All incubations were for 15 min at 390. This was followed by extensive washing of plates with ice cold distilled and deionized H20. The remaining proteins were analyzed by electrophoresis as described. TPCK and TLCK were used to inhibit potentially contaminating enzymes. Earlier we had found that contaminating enzymes in Difco trypsin (Difco, Detroit, MI) gave conflicting results. 57 Sensitivity of the 21K protein to endogenous cellular proteases was determined by seeding EGTA-released normal and Prague A—transformed CEF onto [35$]methionine—labeled SAM from normal cells. The unlabeled cells were then cultured on the 3(SS-labeled SAM at 390 for 24 h. The remaining radioactively labeled substratum associated proteins. were analyzed by polyacrylamide gel electrophoresis after removal of cells with EGTA as described . BindinLof conditioned media components to SAM To assess the ability of conditioned medium (CM) components to bind to SAM, medium from LA24GZ-infected CEF was conditioned in the presence of [35S]methionine at 410 and 350. The CM was then removed, centrifuged at 900 x g for 10 min, supernatant fractions pooled (4lo—CM and 350-CM were kept separate) and equal aliquots incubated for 4 h at 390 and/or 40 as follows: (1) in empty plastic tissue culture dishes, (2) in dishes containing 410- or 350(12h)-SAM (prepared as described in Chapter 2), (3) with monolayers of LA24G2—infected CEF, (4) in serum-coated, plastic tissue culture plates or (5) in BSA-coated, plastic tissue culture plates. Conditioned medium could be stored at —800 to 4 o with no apparent effect on 21K protein binding to the substratum. o . However, storage at 4 does lead to some breakdown of higher molecular weight components as determined by electrophoretic analysis. After the incubation period, the medium was removed, the dishes washed extensively as in the preparation of SAM (Figure 1) and the proteins solubilized in SOS—PAGE sample buffer 58 and analyzed by SDS—PAGE as described. Details for individual experiments are described in the figure legends. Radioiodination of cultures and SAM lodination was performed basically as described previously (118). Briefly, SAM (prepared as previously described) or cell monolayers were washed 3 times with cold CMF—PBS, pH 7.4. This was followed by the addition of 4ml cold glucose solution (9 mg/ml in CMF-PBS) per 100 mm tissue culture dish, to which 125 1 mCi of Na I (13—17 mCi/pg, Amersham, Arlington Heights, IL) was added. Enzyme cocktail (200 pl) containing glucose oxidase (0.1 unit/ml, Sigma, St. Louis, MO) and lactoperoxidase (0.1 mg/ml, Sigma, St. Louis, MO) was .then added and culture dishes incubated for 10 min on ice with swirling. Tissue culture dishes were then washed twice with cold CMF-PBS. 350(13 h)-SAM was iodinated directly and analyzed by electrophoresis. lodinated monolayers were processed in two ways, (1) monolayers cultured at 410 and 350 for 12 h and labeled as described, were washed 2 times with warm CMF-PBS+ followed by removal of cells from the substratum with EGTA as described previously. lodinated cellular and SAM proteins were then analyzed separately by electrophore- sis. (2) Alternatively, following iodination, fresh medium 221+ (5 ml) was added to labeled monolayers which had been previously cultured at 350 for 6 h, and further incubated at 350 for 7h. 125l-labeled SAM and cellular proteins were then prepared and analyzed as described above (data not shown). In addition, medium conditioned by iodinated monolayers was 59 analyzed for ”SI—labeled components capable of binding to unlabeled SAM as described. A schematic diagram of the lodination protocol is illustrated in Figure 10. 6O RADIOIODINATION PROTOCOL 125' 123‘ '2‘I (__S,AM___I lmonolayerl monola er I 1 can L2:_;;asufl . ‘2‘ - ‘2N'-cefls ' l Iresh medium see - no: I“ 803 - no: sea - no: monola er EGTA ‘" - '“l - cells SDS- PAGE 505 'PAOE conditioned medium + SAM 0h -CM EGTA Emacuzsuu SOS -PAOE Figure 10. Schematic diagram of radioiodination protocol. SAM was prepared as described in Chapter 2. Analysis substratum binding proteins from conditioned medium described in Materials and Methods. Radioiodination was performed as described by Hynes (118). RESULTS Association of the 21K Protein with the Cell Substratum in Other Transformation Systems In the previous chapter we reported the identification of a transformation-sensitive protein in CEF infected with the temperature-sensitive mutant of the Prague A strain of Rous sarcoma virus, LA24G2. To determine if this phenomenon was unique to our particular virus clone, we have analyzed SAM isolated from chicken embryo fibroblasts during the development of the transformed state initiated by other viruses or by a tumor promoter. We have identified this increase in 21K protein levels in the SAM of CEF infected with another clone of LA24, G4. In addition, as shown in Figure 11, the 21K protein is increased in SAM of CEF infected with the temperature-sensitive mutant of the Schmidt-Ruppin A strain of Rous sarcoma virus, NY68. Thus, the 21K proteinlis not a transformation-sensitive protein of a single clone or even a single strain of Rous sarcoma _virus, but its increased synthesis and association with SAM appear to be general properties of CEF infected with temperature-sensitive mutants of avian sarcoma viruses during the early stages of transformation. Tumor promoters such as phorbol myristate acetate (PMA) have been shown to cause the reversible oncogenic transformation of treated cells (135,136). This thus provided us with another 61 Figure 11. 62 Analysis of substratum associated proteins from NY68- infected chicken embryo fibroblasts. Tertiary LA24G2- infecte CEF and NY68—infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish and cul red at 410 for 21 h. At temperature shift 15 uCi [ S]- methionine was added to all cultures and they were further incubated at 410 or 350 for 12 h. SAM from NY68-infected CEF was prepared as described in Chapter 2. Radiolabeled conditioned media from LA24GZ— infected CEF cultured at 350 (12 h) and NY68-infected CEF cultured at 410 and 350 (12 h) were collected and radiolabeled SAM-binding proteins prepared and analyzed as described in Materials and Methods. Unlabeled SAM used for binding of radiolabeled conditioned media components was obtained from LA24GZ—infected cells cultured at 350 for 12 h. Lane 1, 35° (12 h) NY68-SAM. Lane 2, 41° NY68-SAM. Lane 3, SAM binding proteins from 350-CM of LA24G2-infected CEF. Lane 4, SAM binding proteins from 410-CM of NYO68-infected CEF. Lane 5, SAM binding proteins from 35 -CM of NY68-infected CEF. Total radioactivity labeled proteins were analyzed per sample by gel electrophoresis. 63 3 4 5 10'3 (it Q ' I » 92.5 i. 4 68 ...... . n“ .. inn-au- ‘ ‘43 .............. " i 25.7 I 18.4 64 agent with which to analyze the development of the transformed state. Initiation of transformation by PMA at 100 ng/ml leads to increased amounts of 21K protein in SAM following PMA-treatment of both normal CEF and LA24GZ-infected CEF cultured at 410 in comparison to untreated controls (Figure 12). Controls treated with 100 ng/ml of the PMA analog, phorbol, which is not a tumor promoter, also did not result in increased levels of the 21K protein in SAM (data not shown). Visual analysis of autoradio- grams from electrophoretic profiles of LA24G2-infected CEF cultured at 350 and incubated with 100 ng/ml PMA does not indicate an additive or synergistic effect on the amount of 21K protein synthesized, as has been described for certain transformation- sensitive parameters (137,138). Thus it appears that transforma- tion induced by expression of the avian sarcoma virus oncogene or induced by the action of the tumor promoter PMA, both lead to increased 21K protein in SAM of CEF. Comparison of Substratum Associated Proteins Prepared by Various Extraction Protocols Substratum associated proteins have been routinely solubilized by scraping the tissue culture plates in a minimal volume (100—200 pl) of SOS-PAGE sample buffer and analyzed directly by electrophoresis. Components which comprise SAM in murine cells have been extensively analyzed by Culp and coworkers (29). They have analyzed SAM by a slightly different protocol. Components are removed by extraction for 30 min in 0.2% SDS at 390 followed by lyophilization and electrophoresis (125). Figure 12. 65 The effect of phorbol myristate acetate on the 21K protein. Secondary LA24GZ-infected CEF and normal, uninfected CEF were seeded at 1x10 ocells/100 mm tissue culture dish and grown at 41 and 39, respectively, for 22 h. At zero hour, 8 LA24GZ-infected CEF cultures were shifted to 350. At the same time, 10 I stock phorbol myristate acetate (PMA, 100 ug/ml DMSO) was added to 4 LA24GZ-infected CEF cultures at 410 and 350, and to 4 normal, uninfected CEF at 390. Final concentration PMA was 100 ng/ml. One half hour later, 15 thi [ S]methionine was added to each culture dish. SAM from LA24G2—infected cultures incubated with or without PMA was prepared 6.5 h after temperature shift. SAM from normal, uninfected cultures incubated with or without PMA was prepared 7.25 h after PMA addition. Total substratum associated proteins from 4 culture dishes/sample were analyzed by SOS-gradient polyacrylamide (7-17%) gel electrophoresis and fluorography. Lanes 1 and 2 represent SAM proteins from 410 cells (29,700 cpm) and 350 cells (27,900 cpm), respectively. Lanes 3 and 4 are SAM proteins from PMA-treated 410 cells (30,100 cpm) and 350 cells (24,900 cpm), respectively. Lanes 5 and 6 are SAM proteins from normal, uninfected CEF (26,500 cpm) and identical cultures incubated at 390 with PMA (25,700 cpm), respectively. FN, fibronectin. 66 '{ 5 I \ \ D ‘f s i a ‘i L,‘ \ I ' I ~15. ,Q ,3. 4'7 ‘1!— y 4»:- ‘t't- u "'7”? ‘t 'N e d i. Wt 4““ n all." - .q - r 3 ‘ ..--21 K Md?) _M, ‘ ' «t ‘ \ ' II I I I ‘ ' 67 Comparison of substratum associated proteins analyzed by both methods is shown in Figure 13. The protein patterns are essentially identical with the 21K protein significantly elevated in 350-SAM prepared by both extraction procedures. Several other reagents have also been monitored for their ability to solubilize or extract the 21K protein from the substratum. As described, 5 mM EGTA does not solubilize the 21K protein. We have found that 0.1—O.2% SDS will solubilize it. Several other detergents have also been analyzed; 0.5% octylgluco— side, 0.5% Triton X-100, 0.5% NP40 and 0.5% deoxycholate, and have all been found to be ineffective in solubilizing this protein (Figure 14, lanes 4-7). Thus the association of the 21K protein with the substratum is not mediated solely by lipid. However, when cells are extracted with 0.5% NP40 containing 1.0% 2—mercaptoethanol or 0.5M KCI, the 21K protein is removed, although 1.0% 2-mercaptoethanol or 0.5 M KC1 alone are ineffective (Table I). An electrophoretic comparison of substratum associated proteins solubilized in sodium dodecyl sulfate-polyacrylamide gel sample buffer, with or without 10% 2-mercaptoethanol, revealed that in the absence of the reducing agent the 21K protein migrates with a faster mobility, indicating the presence of one or more internal disulfide bonds (Figure 15). Apparently the secondary structure of the 21K protein, which can be altered by 2-mercaptoethanol or high salt, is important to its removal by detergents alone. Figure 13. 68 Extraction of substratum associated proteins with 5056 Secondary LA24GZ—infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish. After 20 h at 41°, 10 culture dishes were shifted to 350 and 10 left at 410. Three hours later, all cultures were labeled with 50 tiCi/IOO mm dish H-amino acids for 12 h. EGTA—SAM was prepared as described in Chapter 2 from 5 dishes per sample. All samples were analyzed by SOS-gradient polyacrylamide (5-16%) gel electrophoresis and fluorography. Lanes 1 and 2 represent 410—SAM and 350-SAM, respectively, extracted with 0.2% (w/v) SDS as described by Culp and coworkers (29). Lanes 3 and 4 represent 410-SAM and 350-SAM, respectively, prepared as demufibed in Chapter 2. 4. I- . .63; .2: 3' I a 91’s. .2: 2.» Ill . 1'. Figure 14. 70 Resistance of substratum associated proteins to extrac- tion with various agents. Secondary RSV (LA2462)- infected CEF were seeded at l x 10 cells/100 mm culture dish and cultured at the nonpermissive temperature (410) for 23 h. At temperature shift all but four plates were switched to the permissive tgigperature (350) . Cultures were labeled with [ S]methioninJe (15 uCi/culture dish) and 12 h after shift to 35, substratum associated proteins were isolated by extraction of cells with the following agents and solubilization of the remaining substratum associated proteins in SOS-PAGE sample buffer: 1, 410 — 5 mM EGTA; 2, 35° - 5 mM EGTA; 3, 35° - 0.05% Difco trypsin; 4, 350 - 0.5% octylglucoside; 5, 35° — 0.5% Triton X—100; 6, 35° — 0.5%, NP40; 7, 35° — 0.5% deoxycholate; 8, 35° - 1 M NHAOH and 9, 35° — 4 M urea. All extractions were carried out as described for SAM preparation with 5mM EGTA in Figure l. TABLE 72 Summary of Extraction Properties of the 21K Protein. T rea tmen t of SAM or cells with: 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) It) )2) 13) 14) SDS-PAGE sample buffer 0.) — 0.2% SDS 0. .5% .5% .5% .5% .5% .0 — 10% B-mercaptoethanol .5M 4M 6M 6M GM 00000 5% NP40 Triton X-100 Octylglucoside Deoxycholate NP40 + 1.0% B-mercaptoethanol NP40 + 0.5M KCI KCI Urea Urea Urea + 10% B—mercaptoethanol Guanidine HCl 15) 1.0 N NHAOH )6) 0.1 N NHAOH l7) 0.] N NaOH Removal of the 21K Protein from the Substratum: Figure 15. 73 Detection of disulfide bonds in the 21K protein5 Secondary LAZAGZ-infected CEF were seeded at S x 10 cells/100 mm tissue culture dish and cultured at 41° for 38 h. At temperature shift all cultures were sgg'tched at 35° (8 plates). After 8.5 h, 15 tic: [ S]methionine was added per 100 mm culture dish for 5 additional hours. EGTA-SAM was prepared as descrHoed. Four SAM—coated dishes were scraped in SOS—PAGE sample buffer as described in Chapter 2 (lane 1). The remaining 4 dishes were extracted with SDS-PAGE sample buffiy‘ nflnus 10% 2—mercapuxflhanol (lane 2). Proteins were analyzed by SOS-gradient (546%) gel electrophoresis and fluorography. Equivalent amounts of radioactivity were loaded on each sample lane. The arrow indicates the location of the 21K protein. 74 1 2 tfi‘ ' 75 In addition to detergent extraction, we have found that extraction with 1. M urea is ineffective (Figure 14, lane 8), 6 M urea partially effective, and 6 M urea containing 10% 2—mercaptoethanol completely effective in destabilizing the interaction of the 21K protein with the substratum. In addition, 6 M guanidine HCl completely solubilizes the 21K protein (Table I). Finally, 1M NH OH removes the 21K protein from the 4 substratum (Figure Ho, lane 7). When radiolabeled SAM was extracted with l M NHAOH, followed by neutralization with acetic acid, lyophilization and polyacrylamide gel electrophoresis, the 2lK protein was not recovered (data not shown). Apparently, the 21K protein is not stable or recoverable under these conditions. Extraction with O.I N NHAOH or O.) N NaOH was unsuccessful (Table I). Sensitivity of the 21K Protein to Proteases The sensitivity of the 21K protein to several proteases was examined. We had originally found that the 21K protein is selectively resistant to digestion and removal from the substratum when LAZAGZ-infected CEF cultured at the permissive temperature were removed with 0.05% Difco trypsin (Figure IA, lane 3). However, we later found this not to be true when using a separate lot of Difco trypsin (Figure 16, lane 2). Apparently, the second batch had a higher concentration of contaminating proteases capable of digesting the 21K protein. As a result, we subsequently used purified enzymes and added inhibitors to any potential contaminating proteases. When EGTA-SAM is incubated with 2 U/mI (3 ml) TPCK—trypsin, the 21K protein is found to be 76 resistant. (Figure I6, lane 3). However, the 21K protein is sensitive to 2 U/ml (3 ml) TLCK—chymotrypsin (Figure 16, lane 4) and the proteases present in Pronase (data not shown). Furthermore, although the 21K protein appears to incorporate a significant amount of [3H]proline (data not shown), it is insensitive to collagenase (2.5 units/100 mm plate, Figure 16, lane 5) in both the presence or absence of TPCK (50 ug/ml) and TLCK (50 ug/ml). Therefore it does not appear to be collagen— Iike. The 21K protein is also not released from the substratum by Streptomyces hyaluronidase (5 TRU/ml, 3 ml), a hyaluronic acid specific endoglycosidase (data not shown). This result suggests that the 21K protein is not linked to the substratum via hyaluronic acid, which is also elevated in transforming SAM (following chapter). An increase in cell-associated and secreted proteases in transformed cells has been described (I39). Therefore, the sensitivity of the 21K protein to proteases or glycosidases associated with normal and Prague A—transformed CEF was analyzed. Normal CEF were radioactively labeled with [35$]meth ion i ne for )6 h . SAM was then prepared as described. Unlabeled normal and Prague A—infected CEF were removed from their substrata with 5 mM EGTA, centrifuged and seeded in 5 ml medium 221+ upon [35$]methionine-labeled SAM from normal cells. Cells were cultured for 24 h at 390 and then removed with EGTA to prepare SAM as described in Chapter 2. The remaining 355— labeled substratum associated proteins were analyzed by electrophoresis (Figure )7). During the 24 h incubation period, Figure 16. 77 Sensitivity of the 21K protein to proteases. Secondarg LA2462—infecuuj (CEF were seeded at I x 10 cells/100 nun tissue culture dish ancl cultured at 410 for 21 h. All culture dishes were shifted to 3503§nd 2 h later labeled with 15 uCi/culture dish of [ S]- methionine for an additional 11 h. Radiolabeled SAM was then prepared as described in Chapter 2. Four plates each were incubated for 15 min at 390 with 3 U” of the folHMNing: lane I, PBS, prI 7.4 (34,500 cpm); lane 2, 0.05% (w/v) Difco trypsin (12,800 cpm); lane 3, 2 U/ml TPCKrtrypshw (18,400 (xnn); lane h, 2 U/ml TLCK—chymotrypsin (16,100 cpm); lane 5, 0.83 units/ml TPCK, TLCK—clostridiopeptidase A (collagenase) (39,300 cpm). All enzymes were in PBS, pH 7.4. Figure 17. 79 Turnover of substratum associated proteins by cellular proteases. Tertiary normal CEF and Prague A-infected CEF were seeded at 5 x 10 cells/100 mm tissue culture dish and cultured at 390. After 34 h, normal CEF cultures were radiolabeled with 15 uCi [ S]methionine per 100 mm dish for 16 h. Radiolabeled SAM was prepared as described in Chapter 2. EGTA—released norrrtgl CEF and Prague A-infected CEF was then seeded on S—Iabeled N—SAM (2 dishes each) in medium 221+ and cultured at 390 for 24 h. The remaining 2 radio— labeled SAM-coated dishes were incubated with medium 221+ alone. After 24 h SAM was again prepared and remaining substratum bound proteins analyzed by gradient (7-17%) gel electrophoresis as described in Chapter 2. Lane 1, Prague A—infected CEF on 355- labeled armal CEF—SAM (3300 cpm). Lane 2, normal CEF on —labeled normal CEF-SAM (6000 cpm). Lane 3, control S-Iabeled normal CEF—SAM (7000 cpm). 80 van-.4 9 w-rn I 200— 92.5- 68— 43- 25.7— 18.4“ 12.3— ,,,. 81 Prague A—infected CEF—associated proteases remove most substratum associated proteins (lane 1) whereas in the presence of normal CEF, alterations of the 35S-—labe|ed polypeptides in SAM (lane 2) were minor compared to the untreated, normal SAM (lane 3). The most significant observation is the relative resistance of the 21K protein to digestion by the Prague A-infected CEF-associated proteases. Secretion of the 21K Protein into the Growth Medium To investigate the manner in which the 21K protein is deposited in SAM, radioactively labeled proteins in conditioned medium (CM) from LA2462-infected CEF were tested for their ability to bind to various surfaces as described in the legend to Figure 18. Medium was conditioned in the presence of [3SS]methio- nine for 7 h at 410 and 350 following temperature shift. The 4IO—CM and 350-CM were removed, centrifuged to remove any cells in suspension, and the supernatant fractions incubated with the following: (1) unlabeled 41°-5AM (EGTA-SAM from LA2462-infected CEF cultured at 41°); (2) unlabeled 35°(13n)—5AM (EGTA-SAM from LA24C52-infected CEF cultured at 350 for the specified time); and (3) empty plastic tissue culture 'dishes. The results of this experiment, shown in Figure 18, provide the following information: (i) the 21K protein is secreted into the medium of both normal and transforming cultures; (ii) more 21K protein associates with all substrata incubated with 35°-CM than 410-CM suggesting that there is more 21K protein in 350—CM (compare lanes 1 and 2, 3 and 4, 5 and 6); (iii) more 21K protein from both 350- and 41o-CM associates with SAM than with plastic when analyzing Figure 18. 82 Association of the 21K protein with SAM. LAglsGZ- infected CEF were incubated with 1.5 uCi/ml [ S]- methionine at 41 and 35 for 7 h following temperature shift (10 mI/culture dish). The) conditioned media (CM) were removed, centrifuged at 900 x g for 10 min and 10 ml aliquots added to empty plastic culture dishes and dishes containin SAM prepared from cultures grown at 41 and 35 for 12 h, as described in Figure 1. After a n at 35° (35°-CM) or 410 (4lo-CM), the media were removed, the dishes washed extensively and the proteins solubilized in SDS—PAGE sample buffer and analyzed by SOS-PAGE, as described in Figur 1, and fluorography. The auto- radiogram shows [ S]methionine labeled proteins from CM which remained associated with the various surfaces. Lanes 1 and 2, 410-CM and 35°-CM, respec- tively, incubated in empty plastic culture dishes; lanes 3 and 4, 410-CM and 350-CM, resgectively, incubated with BSD—SAM; lanes 5 and 6, 41 -CM and 350—CM, respectively, incubated with lilo-SAM. 200K— 92.5 K— 68 K— 43K— 25.7K— 18.4K— _~,-, 12.3K— 84 equal amounts of radioactively labeled proteins by gel electrophoresis, suggesting that there may be specificity of 21K protein interaction with components in SAM (for example, for 35°-CM, compare the 21K protein in lanes 4 and 6 to lane 2); and (iv) SAM from either normal or transforming cells appears to be equally capable of binding the 21K protein. Furthermore, the 21K protein from conditioned medium of NY68-infected CEF also binds to SAM in the same manner as described for LA24GZ-infected CEF (Figure 11). It thus appears that at least one mechanism by which the 21K protein reaches the substratum is through secretion or shedding followed by adsorption or binding to SAM. The 21K protein could reach the substratum by an alternative pathway, through adhesion sites left behind when cells are removed with EGTA. In this case, the 21K protein would be concentrated in the adhesion sites of transforming cells. However, it does not appear that the 21K protein is in new adhesion sites. Prelabeled CEF, cultured at 410 and 350 for 7 h following temperature shift, were removed with EGTA, collected by centrifugation and seeded on unlabeled 410-SAM, 350 (12 h)-SAM and empty plastic tissue culture dishes for 2 h (at their appropriate temperature). This procedure allowed for full spreading of cells and the formation of new radioactively labeled adhesion sites. The cells were removed with EGTA and labeled SAM analyzed as described. No alteration in the incorporation of [3SSlmethionine into the 21K protein was observed. In fact, very little 35S-labeled 21K protein was found associated with SAM (data not shown). There is also the possibility that the 21K protein is 85 directed only to old or mature adhesion sites. We have not investigated this possibility . Analysis of the Binding of the 21K Protein to SAM We have shown that the 21K protein is not increased in SAM of fully transformed, Prague A-infected CEF. We have also shown that the 21K protein synthesis and deposition in SAM in the later stages of transformation are decreased (Figure 8). To account for these observations we have investigated the possibility that the protein is synthesized by Prague A cells but the receptors necessary for 21K protein binding to Prague A-SAM or cells are fewer in number or less active. Therefore, medium from Prague A-infected CEF was conditioned in the presence of [35$]methionine for 8 h. The CM was prepared as described and incubated with unlabeled Prague A-SAM and SAM from normal, uninfected CEF for 4 h at 390. 35S—labeled proteins, bound to SAM, were analyzed by electrophoresis and fluorography (Figure 19, lanes 1—3). In this experiment, t_<_)_t_a__l. radioactively-labeled components which have bound to the substratum were loaded onto the gel. No difference is observed in the amount of radioactivity associated with the 21K protein from Prague A—CM associated with empty, plastic tissue culture dishes (lane 1), Prague A—SAM (lane 2) or normal-SAM (lane 3). In contrast, large amounts of radioactively labeled 21K protein from medium conditioned at 350 for 8 h in LA24G2-infected CEF can bind to plastic, tissue culture dishes (lane 4), Prague A-SAM (lane 5) and N-SAM (lane 6). Thus there are no differences in the amount of Prague A— and normal-SAM binding sites for the 86 21K protein as 21K protein from CM of LA2462-infected CEF binds equally well to all substrata tested. The presence of unlabeled Prague A—transformed cells or normal, uninfected cells showed no difference in the binding at 40 of the 21K protein from Prague A—CM as shown in lanes 7 and 8, respectively. Binding of the 21K protein to cell monolayers was performed at 40 to prevent metabolic incorporation of radioactive isotope from the CM. In addition, the presence of Prague A infected—CEF do not affect binding of 21K protein from CM from LA2462—infected CEF at 4° (lane 9). Thus there is no difference in SAM or cell-associated binding sites for the 21K protein in Prague A-infected or normal, uninfected CEF. The low level of the 21K protein in SAM of Prague A-infected cells compared to cells infected with the temperature- sensitive mutant and cultured at 350 during the early stages of transformation is therefore probably the consequence of changes in the synthesis and/or secretion of the 21K protein. Additional information concerning the specificity of binding of the 21K protein to SAM was obtained from results shown in Figure 19 where fetal radioactively labeled proteins which bind to the substratum were analyzed. This is in contrast to the analysis described in Figure 18 where equal amounts of radioactivity were analyzed by electrophoresis. Earlier (Figure 18), the analysis of equivalent radioactivity/gel lane revealed that much more 21K protein associated with SAM than with the empty plastic tissue culture dishes. In a subsequent experiment the total radioactively labeled proteins in 35°18 h)-—CM of LA2462-infected CEF which had bound to empty plastic tissue culture dishes (lane 4), Prague Figure 19. 87 Binding of conditioned media proteins to various substrata. Secondary LA24G2—infected CEF and PraguS A-infected CEF were seeded at 3 x10 cells/150 cm flasks and .cultured at 410 and 390 for 25.5 h, respectively. Fresh medium 221+ (10 ml) was then added to all flasks and LA2462-' fected CEF transferred to 350. 2 h later 40pCi [ S]methionine was added per flask and cells were further cultured for 7 h. CM from Prague A-infected CEF and LA24GZ- infected CEF was collected, centrifuged at 900 x g for 10 min and supernatant fractions pooled. CM (5 ml) was added to various substrata (4 plates each) as described below. Secondary normaé and Prague A—infected CEF were seeded at 1 x 10 cells/100 mm culture dish and grown at 390 for 32 h. Normal—SAM and Prague A—SAM were prepared as described in Chapter 2. Radiolabeled CM from Prague A-infected CEF was incubated for 4 h at 390 with: empty plastic tissue culture dishes (lane 1); Prague A-SAM (lane 2); N—SAM (lane 3); and for 4 h at 4 with monolayers of Prague A—infected CEF (lane 7); and normal CEF (lane 8). Radiolabeled CM from LA24C32—infected CEF was incubated for 4 h at 390 with: empty plastic tissue culture dishes (lane 4); Prague A-SAM (lane 5); N-SAM (lane 6); and for 4 h at 4 with monolayers of normal CEF (lane 9). Following the incubation period, radiolabeled CM was removed and all dishes prepared and analyzed as described in Figure 1. Total substratum bound radio- actively labeled proteins in each sample were analyzed by SOS-polyacrylamide gradient (7-17%) gel electrophoresis. 88 123456789 r x 10'3 — 200 --92.5 I-—43 —-25.7 -——21 -—18.4 —-12.3 89 A-SAM (lane 5) and N-SAM (lane 6) were analyzed by electrophoresis. Greater than 4—fold more radioactively labeled proteins adsorbed to the empty plastic culture dishes than to the Prague A— or N-SAM. In spite of the greater amount of protein adsorbing to plastic, the amount of 21K protein binding to SAM was identical. This is true even though 4—fold less radioactively- labeled proteins have associated with SAM. From these results it is apparent that there are several possible ways in which the 21K protein can interact with the substratum. These include: (1) specific binding to an endogenous SAM component from CM, (2) specific binding to a serum component in SAM, (3) a requirement for binding to serum and/or other conditioned medium components for subsequent binding to SAM or (4) the possibility that its interaction with the tissue culture substratum is so strong that it is selectively removed from the conditioned medium. To differen- tiate between these possibilities several experiments were performed. In an attempt to prevent nonspecific adsorption to plastic tissue culture dishes, they were pretreated with 221+ (serum containing medium). This procedure (see legend to Figure 20) results in the coating of the dish with serum proteins (140,141). 0 Dishes coated with SAM (35, 12 h) were also incubated with medium 221+ in order to coat any exposed areas of the culture [35 dishes. S]methionine—labeled conditioned medium was then incubated with the serum-treated plates for 4 h at 390. Radioactively bound components were analyzed by SDS-PAGE and fluorography. As shown in Figure 20 (lanes 2 and 3), several 90 proteins from 410 and 350 conditioned medium still bind to plastic coated with serum proteins. However, the protein profile now has distinct bands (compare to Figure 19, lanes 1 and 4) and therefore indicates that there is reduction of nonspecific binding. The binding of the 21K protein to the substratum is not affected by serum coating the plastic or SAM (compare lanes 3 and 5). Note that incubating SAM with serum also reduces the number of proteins bound (compare to Figures 18 and 19), yet as mentioned, does not affect the binding of the 21K protein. These results suggest that the 21K protein, either as a complex with other conditioned medium components or by itself, has an affinity for a specific adsorbed serum protein. However, these results do not rule out the possibility that the 21K protein may simply have a high affinity for the plastic of the tissue culture dish to which it could bind by displacing serum proteins or other adsorbed components. To test the possibility that serum proteins might be involved in the binding of the 21K protein to the substratum, we analyzed the ability of the 21K protein from conditioned medium to bind to tissue culture dishes coated with bovine serum albumin and the ability of the 21K protein from serum—free conditioned medium to bind to tissue culture dishes and SAM. In an attempt to prevent the deposition of the 21K protein onto the substratum, tissue culture dishes were preincubated with 5 ml’ bovine serum albumin (BSA, 10 mg/ml) to precoat the tissue culture plastic. [35Slmethionine—labeled conditioned medium from 350 cultures was then incubated for 4 h at 390 (Figure 21) with Figure 20. 91 Effect of serum on binding of conditioned media proteins to the substratum. Secon ary LA24C32—infected CEF which were seeded at 1 x 10 cells and cultured a3541o for 22 h. 1.5 h after temperature shift, 15 uCi [ S]methionine was added per 100 mm dish and cells at 410 and 350 further cultured for 10.5 h. Conditioned medium (CM) was collected and processed as described in Materials and Methods. 10 ml 350—CM and 410-CM was added to the substrata described below and incubated for 3 h at 390. Serum-coated SAM and plastic were prepared as follows‘ Secondary LA24GZ-infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish and cultured at 410 for 32 h. SAM was prepared as described in Chapter 2. SAM and empty plastic tissue culture dishes were incubated with 2 changes of medium 221+ (10 mI/dish) for 1 h and 4 h to serum—coat all substrata. Dishes were then rinsed twice with CMF—PBS+. 350-CM or 4IO-CM were incubated with the substrata as described above and processed as described in Materials and Methods. Lane 1, molecular weight standards (Chapter 2). Radiolabeled proteins from 41 -CM which are associated with serum-coated plastic (lane 2, 5,800 cpm) and serum—coated SAM (lane 4, 2,800 cpm). Radiolabeled proteins from 35°-CM which are associated with serum-coated plastic (lane 3, 5,500 cpm) and serum—coated SAM (lane 5, 3,500 cpm). 92 93 SAM-coated dishes (lane 1), serum-coated dishes (lane 2) and BSA-coated dishes (lane 3). The details of preparation of the various substrata are described in the legend to Figure 21. Preparation of radioactively bound components and analysis by gel electrophoresis and fluorography has been described. As shown in Figure 21, although the electrophoretic profile of bound components differs for each substratum analyzed, equivalent amounts of the 21K protein have bound to the various substrata. In this analysis, we are assuming that the 21K protein does not bind to BSA and that precoating with BSA should prevent nonspecific adsorption. Therefore, it appears that the interaction of the 21K protein with the substratum does not require a substratum—associated component with which to interact, in fact, its association with the substratum is not prevented by precoating the tissue culture dish with BSA, suggesting that its interaction with the substratum is much stronger than that of BSA, binds to sites not occupied by BSA or that it does in fact bind to BSA. To determine if serum in the conditioned medium was necessary for binding, we investigated the interaction of the 21K protein from serum-free conditioned.medium (CM(SF)) with the substratum. Medium 221+ from LA24GZ-infected CEF cultured at 410 was removed and replaced with serum—free medium containing [35$]methionine. All cultures were then transferred to 350 for 12 h. The CM(SF) was collected and centrifuged as described for CM and equal aliquots added to empty tissue culture dishes and dishes containing SAM. In addition, calf serum and chicken serum was added to CM(SF) to a final concentration of 2% and 1%, Figure 21. 94 21K Protein binding to SAM—, Serum- and BSA—coated substrata. Secondary LA24GZ-infected CEF were seeded at 1 x 10 cells/100 mm culture dish and cultured at 41 for 22 h. All cultures were trartgferred to 350 for 13 h in the presence of 15 uCi [ S]methionine per culture dish (10 ml medium). Radiolabeled 350-CM was collected and incubated with the various substrata described below for 4h at 390 and analyzed as described in Materials and Methods. SAM-coated substrata were prepared from LA2462- infected CEF as described in Chapter 2. Serum-coated substrata were prepared by 2, two hour incubations with 5 ml of a solution containing 50% calf serum/50% chicken serum. BSA—coated substrata were prepared by 2, two hour incubations with 5 ml of BSA in PBS, pH 7.4 (10 mg/ml). Following the incubation periods, all substrata were washed twice with CMF-PBS+, pH 7.4. Total radiolabeled substratum binding proteins were analyzed as described in Materials and Methods. Lane 1, 350-CM proteins binding to SAM-coated dishes. Lane 2, 35O-CM proteins binding to serum-coated dishes. Lane 3, 350-CM proteins binding to BSA—coated dishes. 96 respectively, to determine if the presence of serum in medium had an effect on 21K protein binding to empty tissue culture dishes. Analysis of radiolabeled SAM from cultures grown in serum— free medium (Figure 22, lane 1) revealed an abundance of the 21K protein. However, very little radiolabeled 21K protein bound to plastic tissue culture dishes from CM(SF) (lane 2) or CM(SF) plus 2% calf serum and 1% chicken serum (lane 3). More 21K protein from CM(SF) bound to SAM than to plastic although the amounts were small in comparison to previous experiments designed to analyze the binding of the 21K protein from CM to SAM or plastic (compare Figure 22, lane 4 to Figures 18 and 19). Thus it appears that the presence of serum components may be involved in deposition of the 21K protein into ‘SAM from conditioned medium. However, subsequent addition of serum to CM(SF) does not restore the ability of the 21K protein to bind to the substratum. One explanation for this could be a requirement for the presence of serum during the initial shedding process. To summarize, the amounts of the 21K protein in SAM of cells cultured in medium with or without serum are similar, whereas the amounts of the protein, in media collected from these cultures, which are capable .of binding to various surfaces are different (decreased in CM(SF)). Therefore, the following possibilities for the deposition of the 21K protein in SAM are: (1) the 21K protein can be deposited directly in SAM by the cells cultured in the absence of serum (for example, via adhesion sites); (2) the secretion/binding process is very rapid in the absence of serum; and (3) the binding capacity of the 21K protein is retained for longer periods Figure 22. 97 Analysis of substratum binding proteins from serum- free conditioned mediur . Secondary LA2462—infected CEF were seeded at 1 x 10 cells/100 mm culture dish and cultured in medium 221+ for 21 h. At temperature shift all cultures were washed one time with serum—free medium followed b addition of serum-free medium containing 20 pCi [ Slwmthionine (5 nH/100 nun cmflture dish). Cells were cultured at 350 for 12 h. Serum-free conditioned medium (CM(SF)) was collected and prepared as described in Materials and Methods. SAM front 4 dishes of 350 cells cultured iri serunhfree medium (lane 1, 62,700 cpm) was prepared as described in Figure 1. Radiolabeled CM(SF) was incubated for 4 h at 390 with tissue culture plastic (lane 2, 24,800 cpm) and SAM-coated dishes (lane 4, (“800 cpm). Calf smnun and (fiflcken semmn were added to CM(SF) to a final concentration of 2% and 1%, respectively, prior to incubation with tissue culture plastic (lane 3 13,300 cpm). Substratum binding proteins from 35 —CM(SF) were prepared and analyzed as described in Materials and Methods. 99 of time in the presence of serum. Furthermore, as more 21K protein from CM(SF) binds to the SAM-coated dishes than to plastic, it is still possible that some component of SAM is involved in the binding of the 21K protein to the substratum. The role of serum and endogenous conditioned medium components in the shedding and deposition of the 21K protein is currently under further investigation . Analysis of the Mode of the 21K Protein Release into the Medium The possibility that the 21K protein is shed from the cell surface in the form of membrane vesicles or as glycoprotein/glycos— aminoglycan complexes (112) was investigated. Medium was [-3551methionine by 1) LA2462- conditioned in the presence of infected CEF cultured at 410 for 6.5 h, 2) LA24GZ—infected CEF cultured at 350 for 6.5 h, 3) normal, uninfected cells for 7.25 h and 4) cultures identical to 3) but containing 100 ng/ml PMA. All conditioned media were then centrifuged to pellet large debris and cells at 900 x g for 15 min followed by centrifugation of the supernatant fractions at 20,000 x g for 15 min. The 20,000 x g supernatant fractions were then centrifuged for 2 h at 100,000 x g to pellet membrane vesicles (142) or glycoprotein/GAG complexes (112,113). Pelleted material from the 20,000 x g and 100,000 x g centrifugations was solubilized in SDS-PAGE sample buffer and analyzed by electrophoresis. The 21K protein was not present in the electrophoretic profiles of radioactively labeled proteins obtained from the 20,000 x g pellets, however, a protein migrating with a mobility equal to that of the 21K protein in SAM 100 was identified in the 100,000 x g pellets (data not shown). In addition, there appears to be a slight increase in the amount of this protein from 350 CM and CM from cells cultured in the presence of PMA. The 21K protein in the 100,000 x g pellet is not related to the viral structural proteins since it does not migrate with molecular weights described for these proteins and the 100,000 x 9 21K protein is present in CM from uninfected CEF. Thus it appears that the 21K protein is shed into the medium as a glycoprotein/GAG complex or in the form of a membrane vesicle. To determine if the 21K protein was shed in the form of a membrane vesicle, two approaches were tried. First, uninfected CEF and LA24C32-infected CEF were cultured in the presence of 1 uM colchicine, a microtubule disrupting agent which has been demonstrated to lead to increased shedding of membrane vesicles (143). Analysis of radiolabeled SAM from colchicine treated cells did not reveal an increase in the level of the 21K protein (Figure 23) compared to the corresponding controls. Second, cyclic nucleotides have been shown to stabilize cytoskeletal components and as a result lead to decreased shedding of membrane vesicles (144). When LA24G2-infected CEF were cultured at 350 in the presence of dibutyryl cyclic AMP (db—CAMP), no effect was observed in the 21K protein levels in SAM. This is demonstrated in Figure 24, where 1.2 mM db-cAMP plus 1.0 mM theophilline, an inhibitor of CAMP phosphodiesterase, was added to 350 cultures and 410 cultures and SAM prepared 14.5 h later, lanes 2 and 4, respectively. The 21K protein is not . o . . affected, most notably in 35 cultures, whereas other proteins in Figure 23. 101 Effect of Colchicine on SAM. Secondary normal ang LA24G2-infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish and cultured at 390 and 410, respectively, for 22 h. At temperature shiftb 8 LA2462-infected CEF cultures were switched to 35 and 10 ul freshly prepared colchicine (1 mM in medium) added to 4 dishes (final concentration was 1 pM). Col icine was also added to 4 normal CEF cultures. [ S]methionine (15 “Ci/100 mm culture dish) was added to all cultures (8 LA2462-infected CEF at 350 and 8 normal CEF at 390). After 7.5 h, SAM was prepared as described in Chapter 2. Lane 1, normal— SAM (26,500 cpm). Lane 2, 02(350)-SAM (27,900 cpm). Lane 3, SAM from colchicine treated normal cells (36,900 cpm). Lane 4, SAM from colchicine treated LA2462-infected CEF culture at 35° for 7.5 n (36,100 cpm). FN, fibronectin. 102 1234 Figure 24. 103 Effect of cAMP on substratum associated proteins5 Tertiary LA24G2—infected CEF were seeded at 7 x 10 cells/60 mm tissue culture plates and cultured at 410 for 12 h. At temperature shift 10 culture dishes were switched to 35°. Theophylline and dibutyr I cyclic AMP were added to 5 dishes at 35° and Al to a final concentration of 1.2 mM and 1.0 mM, resaectively (145). All cultures were labeled with 10 uCi H-amino acids and further cultured for 15 h. SAM was prepared as described in Chapter 2 with the exception that one half the volume was used for 60 mm culture dishes. Lane 1, 35° (15 h)-SAM. Lane 2, 35° (15 h)-SAM from CAMP-treated cells. Lane 3, 410—SAM. Lane 4, 41o-SAM from CAMP—treated cells. 1000 cpm loaded per sample and exposure to x—ray film was 2 months. 104 4 _ 0 2 1 FN-* II-f‘. M Pm" . ti ...fl.x.,.~. 105 35° SAM are altered. Furthermore, as db-cAMP has been shown to increase adhesion of transformed cells (145), it therefore appears that the- 21K protein is not involved in CAMP-dependent alterations which influence cell-substratum adhesion upon transformation. These results suggest that the 21K protein in the 100,000 x g pellet is probably shed in the form of a large complex of the type described by LaCorbiere and Schubert (112). We have observed the presence of polypeptides migrating with molecular weights equal to fibronectin and similar to certain procollagens in the 100,000 x g pellet. These proteins have been described to be part of the glycoprotein/glycosaminoglycan complex (112). Analysis of Cell Surface and Substratum Associated Proteins by lodination. SAM and cell monolayers were labeled with iodine-125 (1251) and labeled proteins analyzed as described in Materials and Methods and as illustrated in Figure 10. Figure 25 demonstrates that iodination of 35°-SAM results in intense labeling of the 21K protein (lane 1). Thus the 21K protein in SAM is readily accessible to lactoperoxidase—catalyzed iodination. There were no Coomassie blue stained proteins migrating with a molecular weight equivalent to fibronectin in the SAM-coated dishes used. This was possibly the result of long term storage of the unlabeled SAM at 40. When SAM was prepared from 125l—labeled cultures grown at 410 (lane 2) and 350 for 12 h (lane 3), the 21K protein was also strongly labeled. Moreover, there was a greater degree of 106 iodination of the 21K protein from 350-SAM. Thus the presence of cells at the time of labeling does not totally interfere with the iodination of the 21K protein. As the extent of iodination is less than 125l-labeled 21K protein from labeled SAM, this suggests that some of the 21K protein may indeed be covered by the cells. To determine if the 21K protein was a cell surface molecule, 410 and 350 iodinated—cells released from .the substratum in preparation of SAM were analyzed by electrophoresis. Results show that there are two lines of evidence suggesting that it is a cell 0 and 350 cells) surface molecule. First, lanes 4 and 5 (41 indicate that iodination of a protein with an approximate molecular weight of 21,000 appears to comigrate with the 21K protein in SAM. This iodinated protein was only observed after long exposure (note overexposure of higher molecular weight proteins), indicating a low level of iodination. This may be due to (1) low levels of cell surface 21K protein, (2) to the possibility that the 21K protein is cryptic on the cell surface, or (3) to the possibility that it is a different protein. The second line of evidence supporting its surface location comes from the results shown in lane 6. Cell monolayers cultured at 350 for 6 h were iodinated and following several washes in CMF-PBS and addition of 5 ml 221+ per 100 mm tissue culture dish, placed back in culture for 6 h at 350. Conditioned medium was removed, centrifuged at 400 x g for 10 min and the supernatant fraction incubated with unlabeled 35°(lz h)—SAM for 4 h. Conditioned media components which had bound to SAM were then prepared and analyzed as described in Materials and Methods. As shown in‘ 107 lane 6, iodinated 21K protein has bound to the substratum. Therefore, it appears to have been shed by the 125l-labeled cells or released from the 125l—labeled SAM during the conditioning process. The latter possibility is unlikely, however, in light of the very tight association of this protein with the substratum (see extraction data, Figure 14 and Table 1). Furthermore, as shown in Figure 17, the 21K protein is selectively resistant to release by cells. Thus, the 125l—labeled 21K protein appears to be shed from the cell surface. Figure 25. 108 Radioiodination of SAM and cell surface proteins6 Secondary LA24C52-infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish and grown at 41 for 22 h. The radioiodination procedure is described in the Materials and Methods and illustrated in Figure 10. Each sample is from 2 iodinated culture dishes. Briefly, lane 1 epresents 350 (13 h)-SAM iodinated directly (Pzg x 10 cpm,, 1.5 h exposure); lanes 2 and 3, I-labeled SAM prepared from iodinated monolayers of 410 cells and 350 2 h) cells, respectively (2.3 x10 cpm a9? 2.9 x10 cpm, 5.5 h exposure); lanes 4 and 5, l-Iabeled EGTA-released cells from lanes 2 and 3, cu tured at 410 and 35; (12 h), respectively (1.7 x 10 cpm and 2.0 x 10 cggw, 40% of total sample, 5 day exposure); lane 6, l-labeled componentslfiinding to urglabeled SAM frorg medium conditioned by l—labeled 35 cells (1.4 x 10 cpm, 14 day exposure with DuPont Lightning Plus intensifying screen). DISCUSSION This chapter extends the characterization of the 21K protein, a novel transformation—sensitive protein which may play an integral role in the development of the transformed state in chicken embryo fibroblasts infected with the LA24GZ temperature- sensitive mutant of Rous sarcoma virus. Furthermore, culture of chicken embryo fibroblasts infected with a temperature-sensitive mutant (NY68) from a different strain of Rous sarcoma virus, Schmidt—Ruppin A strain, at the permissive temperature also leads to increased amounts of the 21K protein in SAM and in conditioned medium having the ability to bind to SAM. In addition, we have demonstrated that the 21K protein is increased in SAM of chicken embryo fibroblasts incubated in the presence of the tumor promoter, phorbol myristate acetate. Once again, changes in SAM were monitored at the early stages of the transformation process which is induced in this case by PMA. Thus, the increase in substratum associated 21K protein during the development of the transformed state is not due solely to transformation by the LA2462 or NY68 temperature—sensitive mutants of Rous sarcoma virus but is related to some common transformation—sensitive pathway also induced by the tumor promoter, phorbol myristate acetate. 110 111 The association of the 21K protein with the substratum has been extensively analyzed in CEF infected with LA2462. This pro- tein is resistant to extraction from the substratum by several detergents with the exception of SDS. However, 0.5% NP40 containing 1.0% 2-mercaptoethanol or 0.5M KCI will remove the 21K protein from its substratum whereas 2-mercaptoethanol or KCI alone will not. The 21K protein appears to have internal disulfide bonds, as demonstrated in Figure 15. Thus secondary structure apparently is crucial to the resistance of the 21K protein to extraction by detergents alone. This is also illustrated by the fact that 6 M urea only partially solubilizes the 21K protein whereas 6 M urea plus 2—mercaptoethanol completely solubilizes it. In addition, 6M guanidine HCl» completely extracts the 21K protein. Recently, certain growth factors have been described whose extraction properties are remarkably similar to those described here for the 21K protein. The growth factors also associate tightly with the substratum under which conditions they are still active (146). The sensitivity of the 21K protein in SAM to several proteases has also been examined. The 21K protein is sensitive to the proteases present in Pronase and the mixture of enzymes found in Difco trypsin. In addition, it is sensitive to TLCK- treated chymotrypsin. However, this protein is resistant to TPCK- trypsin. Originally, we found the 21K protein to be insensitive to Difco trypsin, however, use of a different lot resulted in its sensitivity to this mixture of proteases. Apparently, the first batch was relatively pure trypsin. Since the 21K protein is 112 accessible to enzymes, it must be lacking arginine and lysine amino acids, substrates for trypsin activity, or these amino acids must be sequestered to the center of the protein and thus protected. This might suggest that the 21K protein has a hydrophobic exterior. In addition, although the 21K protein incorporates a significant level of radioactively labeled proline, it is insensitive to collagenase and is therefore not collagen—like. Finally, a key feature of the 21K protein is that it appears to be shed in increased levels into the medium during the early stages of transformation and subsequently binds to the substratum. At this time we have not proved that the 21K protein in SAM and CM are identical. We are presently analyzing SAM and CM components which bind to the'substratum by two—dimensional gel electrophoresis. The binding of the 21K protein appears to be specific in the sense that it has a greater affinity for the substratum than other components of conditioned medium which bind to the substratum. For example, when equivalent counts of radioactively labeled proteins are tested for their ability to bind to various substrata, much more 21K protein binds to SAM-coated tissue culture dishes than to empty tissue culture dishes. However, when t_o_t_a_l_ radio- actively labeled substratum bound proteins are analyzed, four times more radioactivity binds to the empty tissue culture dishes than to SAM whereas the amounts of radioactively labeled 21K protein which bind are equivalent. The interaction of the 21K protein from conditioned medium is not altered by precoating tissue culture dishes with serum or BSA, yet serum—free 113 conditioned medium does not appear to have much soluble 21K protein which is capable of binding to the substratum, even after addition of serum. As 21K protein is present in SAM of cells grown in serum-free medium, the possibility exists that the soluble form of the 21K protein is stabilized by the presence of serum proteins such that upon collection of conditioned medium there is a larger amount of soluble form which slowly binds to the substratum than in serum—free medium. The possibility also exists that under conditions of serum deprivation, the 21K protein reaches the substratum by alternative routes. We are currently investigating these possibilities. We find that the 21K protein is shed in the form of a complex which is precipitable by centrifugation at 100,000 x g for 2 h. Thus, this is much like the 16S particle described by Schubert and LaCorbiere (112). Two lines of evidence demonstrate that the 21K protein is not shed in the form of a vesicle. First, treatment of infected- or uninfected-CEF with colchicine, a microtobule disrupting agent which leads to increased shedding of vesicles, does not lead to increased levels of the 21K protein in SAM. Second, treatment of LA24GZ-infected CEF at the permissive or nonpermissive temperature with dibutyryl cyclic AMP, which stabilizes m icrotobu 1 es and therefore decreases shedding of vesicles, does not lead to decreased 21K protein levels in SAM of LA24G2-infected CEF cultured at the permissive temperature. Extraction data and its resistence to TPCK—trypsin suggest that the 21K protein may be hydrophobic in nature. In such a case, this protein would have to be surrounded by or complexed with 114 hydrophilic proteins (for example, FN and the procollagens which are also pelleted by centrifugation at 100,000 x g for 2 h) in order to leave the cell membrane and exist in conditioned medium. In the preceding chapter we demonstrated that SAM from fully transformed Prague A-infected CEF did not contain increased amounts of 21K protein compared to SAM from normal, uninfected CEF. We have demonstrated that this is not the result of an inability to bind to an altered substratum as the binding of 21K protein from Prague A-conditioned medium to N-SAM and Prague A-SAM is not appreciably different. It remains to be determined if 21K protein synthesis is increased in Prague A cells but that the protein has lost the ability to bind to the substratum. The use of specific antibodies should answer this question. We have provided evidence that the 21K protein is present on the cell surface as it is 125l—labeled by glucose oxidase/lecto— proxidase-catalyzed iodination. The level of iodination though is low, which might suggest that it is at a low concentration on the cell surface, but turned over rapidly in transforming cells or that most of the 21K protein is cryptic. However, the 21K protein is intensely labeled when SAM alone is iodinated or when cells in culture are iodinated followed by removal of cells with EGTA and analysis of SAM by electrophoresis. Thus, the 21K protein in SAM is readily accessible to iodination by lactoperoxidase. Furthermore, the incubation of conditioned medium from iodinated cells with unlabeled SAM results in the binding of 125I--labeled 21K protein. It has therefore been shed from the iodinated cell surface or released from the iodinated substratum of the cultured 115 cells into the medium. As the 21K protein appears to be tightly associated with the substratum, the former is more likely. This provides at least one mechanism by which this protein can be released into the medium. Finally, it must be noted that there are significant differences in labeling of SAM and EGTA-released 125I. Radioiodination of cell monolayers, cellular proteins by followed by solubilization of total proteins and electrophoresis, apparently not only detects cell surface proteins but also these proteins associated with the substratum. These may include, (1) serum proteins, (2) extra- and intracellular footpad material (adhesion sites described by Culp SEQ" (29)), (3) material shed from the cell surface and/or (4) material secreted by cells. We have considered several proteins as possible candidates for the identity of the 21K protein. We were particularly interested in the possibility that the 21K protein was related to the p22 heat shock protein described by Kelley and Schlesinger (147), particularly because transformation in our cell—virus system is turned on by a change in temperature, albeit by a decrease in temperature. However, synthesis of the 21K protein is not induced by heat shock treatment of normal or virus—infected chicken embryo fibroblasts (Figure 26). Furthermore the 21K and p22 proteins can be distinguished by differences in electrophoretic mobility (Figure 26) and unlike the p22 protein the 21K protein does not incorporate radiolabeled mannose. Erikson gt _aI_. (148) have described an avian sarcoma virus polypeptide p19, which exists in two forms, phosphorylated and nonphosphorylated. The phosphorylated form has a slightly lower Figure 26. 116 Comparison of the 21K protein and heat shock proteins. Secondary germal and LA24GZ-infected CEF were seeded at 1 x 10 cells/100 mm tissue culture dish and cultured at 410 for 27 h. All cultures were labeled with 10 uCi [ S]methionine per culture dish for a total period of 6 h and cells or SAM obtained from cultures grown under the followéng conditions: lane 1, EGTA-released normal CEF at 45 -3 h (heat shock) and then 41 -3 h; lane 2, EGTA—released normal CEF cultured at 410-6 h; lane 3, EGTA—released LA2462- infected CEF at 450-3 h (heat shock) and then 410-3 h; lane 4 EGTA-released LA2462-infected CEF at 350-3 h and 41 —3 h; lane 5, SAM from normal CEF at 450—3 h (heat shock) and 410-3 h; lane 6, SAM from normal CEF at 410—6 h; lane 7, SAM from LA2462-infected CEF at 450—3 h (heat shock) and 410—3 h; lane 8, SAM from LA2462-infected CEF at 35°-3 h and 410-3 h; and lane 9, SAM from LA2462—infected CEF at 41°45 h. Lanes 1-4, 15,000 cpm loaded/lane. Lanes 5-9, 10,250 cpm loaded/lane. 117 84.5K— —- M. .— 74 K— 68K—l —4 47K—--—-—-—-— " *5 -O' "a 24 K—' ~ 118 mobility on one dimensional gels and there is twice as much of the nonphosphorylated form. Therefore the nonphosphorylated form is a good candidate for the 21K protein. However, the 21K protein is also present in SAM of normal, uninfected CEF and is increased in SAM from CEF transformed by PMA. Thus it does not appear to be related. Recently , Bloom and Lockwood (149) described the dephosphorylation of a protein of molecular weight 20,000 upon morphological reversion of transformed Chinese hamster ovary cells with dibutyryl cAMP. This protein, pp20 was found to comigrate with the 20,000 molecular weight myosin light chain. However, the phosphorylated form was extractable with 0.5% Triton X-100 while the unphosphorylated (morphological revertant) form was not, suggesting that the phosphorylated (transformed) form may not be associated with SAM. This is in direct contrast to the 21K protein which is not only increased by transformation but also cannot be extracted with 0.5% Triton X-100. Thus to our knowledge, the 21K protein has not been described previously. Other examples of components found in conditioned medium which bind to the substratum have been described. A substratum— conditioning factor from medium conditioned by growth of chicken embryonic heart cells has been found to induce rapid neurite outgrowth from isolated neurons in culture. Only substratum-bound factor possesses activity and it is proposed that this factor possesses sites to which the nerve cell surface components adhere (150,151). As mentioned, Schubert and LaCorbiere have isolated and characterized a 165 glycoprotein complexe which they have 119 termed "adherons". This binds to the substratum and promotes adhesion of cells. Adherons isolated from adhesion deficient mutants have a much reduced ability to promote adhesion (112). The 21K protein has many properties in common with growth factors: (1) its molecular weight is in the range described for many growth factors (152), (2) it is released by cells into the medium, and (3) its extraction properties are similar to those described for substratum associated platelet-derived growth factor and pituitary—derived fibroblast growth factor (146). In addition, many transformed cell systems have been shown to secrete transforming growth factors (152). It has been suggested that the role of the extracellular matrix i_n_ m is to sequester growth factors and provide localized and persistant growth stimulation (146). Since one of the characteristic properties of oncogenically transformed cells is loss of growth control, we are actively pursuing the possibility that the 21K protein may have mitogenic activity. In addition, we are intrigued by the fact that loss of substratum adhesion, mediated by hyaluronic acid (Chapter five), correlates temporally with the deposition of the 21K protein in SAM during the early stages of the transformation process. As SAM is a specialized compartment of the cell surface which is enriched for components most probably involved in cell-substratum adhesion, we are also investigating the potential role of the 21K protein in loss of cell—substratum adhesion. Finally, we are continuing attempts to raise antibodies to the 21K protein. Earlier attempts to raise antibodies to 120 polyacrylamide gel purified protein have been unsuccessful when injecting the 21K protein subcutaneously or surgically into the popliteal lymph nodes of rabbits. Purification of the protein by alternative methods as well as modification may be required to induce an immune response to this protein. The production of antibodies will greatly facilitate the characterization of this small protein and the determination of its relevance to the development of the transformed state. TRANSFORMAT lON—SENSI T IVE ALTERATIONS IN HYALURON 1C ACID In the previous chapters 1 have described alterations in the amount of 21K protein during the development of the transformed state. In this chapter I have analyzed changes in the organiza- tion and amounts of hyaluronic acid following initiation of transformation with particular interest in the cell surface and substratum—associated glycosaminoglycan . 121 MAT ER IALS AND METHODS Cell Culture and Metabolic Labeling LA2462-infected chicken embryo fibroblasts (CEF) were seeded at 1x106 cells/100 mm tissue culture dish and cultured as described. Hyaluronic acid (HA) from secondary cultures was radioactively labeled with [3H]acetate for 4 h periods. Medium was changed at the time of temperature shift (0 h) to fresh medium 221+ (5 ml). At various times after temperature shift ImCi [3H]acetate (2 Ci/mmol, New, England Nuclear, Boston, MA) was added to each 100 mm tissue culture dish. The time of addition of label after temperature shift varied for different experiments. To monitor the incorporation of glucosamine into surface associated hyaluronic acid during the development of the transformed state, tertiary LA2462-infected CEF were seeded at 5 x105 cells/60 mm tissue culture dish. After 36 h in culture 50 uCi D-(6-3H)glucosamine hydrochloride (22.6 Ci/mmol, Amersham, Arlington Heights, IL) was added to all cultures in medium 221+ (5 ml). After 6 h at 410, half the cultures were switched to 350. Duplicate cultures at 410 and 350 were utilized for HA isolation at 0, 4, 8, 12 and 16 h following temperature shift. 122 123 Isolation of Hyaluronic Acid Hyaluronic acid was isolated from cellular, surface and media fractions. The medium fraction represents the labeled HA shed and/or secreted into the culture medium. The surface fraction is defined as radioactively labeled HA released from viable cultures incubated with 0.05% (w/v) Difco trypsin for 15 min at 390 following one wash with CMF-PBS+, pH 7.4. Under these conditions all cells are released from the substratum upon gentle washing. The trypsinized cells and one wash of the tissue culture dishes are then centrifuged at 400 x g for 5 min. The superna- tant. and pellet fractions are the source of surface and cellular HA, respectively. Radioactively labeled hyaluronic acid was then isolated as described by Underhill and Toole with the omission of the DEAE cellulose chromatography step (114). All results reported in this chapter are obtained from radioisotope incorporation data. Measurement of mass, especially of subfractionated cell surface HA, will not be possible until much greater quantities are isolated or a much more sensitive assay is devised. Cell surface HA was further fractionated into SAM—HA, lst EGTA-HA, 2nd EGTA-HA and Trypsinate-HA fractions as follows. After removal of the media and one wash of cell monolayers with CMF-PBS+, LA2462-infected CEF at the permissive and nonpermis- sive temperatures were incubated for 15 min at 390 in 5 ml of 5 mM EGTA in CMF—PBS+ per 100 mm tissue culture dish. Hyaluronic acid released from the cell monolayers under these conditions is defined as the 1st EGTA—HA. Following removal of this fraction, an additional 5 ml of 5 mM EGTA in CMF—PBS+ was 124 added to each tissue culture dish and the cells detached from the tissue culture substratum by gentle pipetting. The 2nd EGTA wash was then removed and combined with one wash of the tissue culture dishes with CMF—PBS+. The cells were then pelleted by centrifugation at 400 x g for 5 min. The supernatant fraction is defined as the hyaluronic acid released by the 2nd EGTA incubar tion, i.e., 2nd EGTA-HA. The remaining cell pellet was resuspended in 0.05% Difco trypsin and incubated for 5 min at 390. Hyaluronic acid still remaining on the cell surface but released by trypsin treatment is defined as trypsinate-HA. Hyaluronic acid remaining associated with the cell substratum following both EGTA incubations is defined as SAM-HA. The SAM-HA was removed directly from the substratum by incubation with Pronase, type XIV (Sigma, St. Louis, MO). Other fractions were treated with Pronase and HA isolated as described by Underhill and Toole (114). In one experiment, HA was labeled with [3H]glucosamine as described and cell surface HA isolated at each time point by wash- ing the cell monolayers one time with 25 mM Tris-Cl buffer, pH 7.4 containing 5mM NaZHPO4’ 5 mM KCI and 137 mM NaCI (5 ml), followed by removal of cell surface GAG by treatment with 0.1 mg/ml 2X crystallized trypsin (Gibco, Grand Island, NY). Cells were pelleted by centrifugation at 400 x g for 5 min and the GAG in the supernatant fraction was isolated by the method of Cohn 351' (153). 125 Size Analysis of Isolated [3H]—labeled Hyaluronic Acid The size distribution of the isolated [3H]-labeled HA was analyzed by Sepharose CL-ZB' column chromatography. Hyaluronic acid, isolated from the various fractions described, was applied to a 0.9 x 55 cm Sepharose CL—ZB column and eluted with 0.05 M Tris-Cl buffer, pH 8.0 containing 0.15 M NaCl and 0.02% sodium azide. One ml aliquots were collected and counted in 10 ml of AquasoI-2 (New England Nuclear, Boston, MA) in a Beckman LS9000 scintillation counter. Sepharose CL—ZB has been reported to have an exclusion limit of 2 x 107 for polysaccharides. Elution profiles represent total radioactivity associated with each isolated fraction and are divided into three size distributions, high molecular weight (HMW, fractions 12—17), intermediate molecular weight (IMW, fractions 18-27) and low molecular weight (LMW, fractions 28—37) species. Hyaluronidase Digestion of Hyaluronic Acid To confirm that the elution profiles obtained represented 3 . . [H]-labeled hyaluronlc aCld, selected fractions were incubated with protease-free hyaluronidase from Streptomyces hyalurolyticus (Miles, Elkhart, IN), an endoglycosidase which is specific for hyaluronic acid (154), and the resulting product analyzed by Sepharose CL-ZB column chromatography as described above. Digestion was performed in a total volume of 0.5 ml with 25 turbidity reducing units (TRU) of hyaluronidase in 0.05 M sodium acetate, pH 5.5 containing 0.15 M NaCl for 3 hours at 390. The 126 reaction was stopped by boiling for 2 min. Under these conditions hyaluronic acid is completely digested. Cellulose Acetate Electrophoresis of Hyaluronic Acid Cellulose acetate electrophoresis was performed on 94 x 76 mm Zip Zone cellulose acetate plates (Helena Laboratories, Beaumont, TX) wetted. with 0.2M calcium acetate buffer, pH 2.0 with excess buffer blotted from the plate. Samples (20 ul) from the [3H]glucosamine labeling experiment were spotted and electrophoresed for 1 h at a constant voltage of 300 V, at 40. Under these conditions hyaluronic acid is well separated from the sulfated glycosaminoglycans. Plates were stained sequentially with 0.1% Alcian blue (5 min) and 0.1%, Toluidine blue (5 min) in 10% acetic acid followed by destaining with 10% acetic acid. The areas comigrating with standard hyaluronic acid and staining sky blue, which is characteristic for HA under these staining conditions (155), was cut out, placed in a 15 ml scintillation vial with 10 ml Aquasol—2 and radioactivity determined. RESULTS Changes in [3H]Glucosamine—Iabeled Cell Surface Hyaluronic Acid During the Development of the Transformed State As described, many transformed cell types exhibit elevated levels of hyaluronic acid in comparison to their normal counter- parts. We were interested in analyzing this alteration during the development of the transformed state. To do this, we isolated hyal- uronic acid from the cell surface of [3H]glucosamine-labeled cul- tures grown at the nonpermissive and permissive temperatures for various times as described in the legend to Figure 27. In this experiment, glycosaminoglycans were isolated as described by Cohn et al. (153). Isolated glycosaminoglycans were then analyzed by cellulose acetate electrophoresis. Hyaluronic acid was identi- fied by staining and co-migration with standard HA, cut out and radioactivity determined by liquid scintillation counting as described in Materials and Methods. As demonstrated in Figure 27, 4 h after initiating the transformation process by switching cultures to 350, a greater than 2—fold increase in cell surface [3H]glucosamine-labeled HA was measured. By 8h, there is greater than 6—fold more surface [3H]g|ucosamine—labeled HA from cells cultured at the permissive temperature compared to those remaining at the nonpermissive temperature. This is followed by a decline to a level which is 2.3-fold higher in transforming cells, 127 128 l I l I 20 l- "1 MA '2 16 ~ '1 ; 3521 :3 12 " k . ‘l a a. e 3 " d \. 0 lll'-lll s , __ a... 1 r _- k a—I I L 4 1 A ll 8 12 16 TIME AFTER TEMPERATURE SHIFT (HOURS) Figure 27. Long term Ijabeling of surface-associated hyaluronic acid with [ H]g| cosamine. LA2462-infected CEF were seeded at 5 x 10 / 0 mm culture dish and maintained at 410 for 36 h. [H]glucosamine (50 uCi/culture dish) was added and after 6h half the cultures were transfered to 350. At various times after temperature shift the cells were washed and the trypsin releasable surface fraction processed for glycosaminoglycan isola— tion according to the method of Cohn (153). Conditions of trypsinization were 0.1 mg/ml trypsin (2 x crystal— lized) for 15 min at 390. Glycosaminoglycans were separated by cellulose acetate electrophoresis (155) and hyaluronic acid identified by staining with Alcian blue and Toluidine blue and co-migsation with an HA standard. The incorporation of H into surface- associated HA was Odetermined by liquid scintillaction counting (O), 35 -T, transforming, (I), 41 —N, nontransformed. 129 16 h after temperature shift. This decline may be the result of depletion of [3H]glucosamine by the "more rapidly HA synthesizing transforming CEF and greater turnover of cell surface HA as greater differences have been observed in several experiments, including a 8-fold increase in the mass of cell surface hyaluronic acid 16 h after temperature shift (data not shown) as determined by the uronic acid assay of Bitter and Muir (156). Analysis of Cellular, Cell Surface and Media Hyaluronic Acid in Normal and Transforming CEF Having demonstrated an early change in cell surface [3H]glucosamine—labeled hyaluronic acid, we were interested in characterizing molecular differences which might occur as a result of transformation. To do this, LA24G2-infected CEF cultured at the nonpermissive and permissive temperatures were labeled for 4 h, 8 h after temperature shift, with 1 mCi [3H]acetate/100 mm culture dish in 5 ml of medium 221+. Tritium labeled hyaluronic acid was then isolated as described in Materials and Methods from cellular, cell surface and media fractions. Total cell surface HA is repre- sented here as the composite of several cell surface subfractions. Alterations in radioactively labeled HA isolated from transforming (350) and nontransforming (410) cultures in these three fractions was measured (Table Ila). During this 4 h period of radioactive labeling, cultures grown at the permissive temperature (8-12 h after temperature shift) incorporated 2.3—, 2.0- and 3.8-fold more radioactivity into cellular, cell surface and medium fractions of . 3 . . . isolated [H]—labeled hyaluronic aCId, respectively, compared to 130 TABLE II. Ratio of transforming and nontransforming CEF [3H]- labeled HA isolated from a) cellular, cell surface and medium fractions, and b) cell surface [3H]—labeled HA divided into SAM, Ist EGTA, 2nd EGTA and trypsinate 1 compartments. Cell a. Cellular Surface Medium 0 o2 35 /41 2.3 2.0 3.8 b. SAM lst EGTA 2nd EGTA Trypsinate o o2 35 /41 1.7 9.9 4.8 0.77 1. Hyaluronic acid was isolated as described in Materials and Methods. 2. Represents the ratio of [3H]—labeled HA isolated from 35° 0 cultures and 41 cultures per 106 cells. 131 cultures grown at the nonpermissive temperature (410). Thus total [3H]-labeled HA is elevated in all fractions in the transforming‘ cells. Molecular weight distribution of these various fractions was then analyzed by Sepharose CL—ZB chromatography (Figure 28). Analysis of cellular and surface fractions at both temperatures reveals the presence of 2 distinct molecular weight species. In addition, comparison of 410 and 350 elution profiles demonstrated differences in [3H]-labeled HA isolated from cellular, surface and medium fractions. Compare panels a, c, and e from 410 cells to panels b, d, and f from 350 cells. These differences in the distribution of [3H]-labeled HA have been quantified in Table III. In this table fractions 12—17 represent the high molecular weight (HMW)-species, 18-27 the intermediate molecular weight (lMW)-species and 28—37 the low molecular weight (LMW)-species. In all fractions, the molecular weight distribution of [3H]-labeled HA for 350 and 410 cultures is altered. The major alterations include the larger percentage of high molecular weight species in 350 cultures and a reduction in the percentage of the low molecular weight species. In addition to monitoring changes in the molecular weight distribution of hyaluronic acid, we compared the amount of incorporation of radioactivity into HA isolated from 350 and 410 cultures, for the various fractions and for the different molecular weight species (Table IV). As shown, there is a 3.7-, 2.9- and 6.2-fold increase of HA from 350 cultures compared to 410 cultures, from the cellular, cell surface and medium fractions, 3H. dpm X 10" Figure 28. 132 Cellular - HA Surface - HA Medium - HA a C e ‘0, 5) 40' °“’ " :lot 0.6f :W 20’ 0.4) 2' 02' l) "1 A A A A A A 4b .90.---‘.. 10 Te 22 2‘8 34 to 16 22 213 a} lo 16 22 {a 34 :0 F ractlon (ml) Sepharose CL-ZB elution profiles of [3H]—labeled hyaluronic acid isolated from normal and transforming CEF . econdary LA24GZ—i nfected CEF were seeded at 1 x 10 cells/tissue culture dish and grown at 410 for 21 h. 480 h after temperature shift, 5 dishes at 350 and 5 at were beled for 4 h with 1 mCi [ H]acetate/- culture dish. [H]—labeled HA was isolated from media, surface and cells as described in Materials and Methods. Panels a and b, cellular-HA from 410 cells and 35 cells, respectively. Panels c and d, surface-HA from 410 cells and 350 cells, respectively. Panels e and f, medium-HA from 41 cells and 350 cells, respectively. ( ----- ) represents equivalent samples of medium-HA incubated with 25 TRU/ml hyaluronidase as described in Materials and Methods. Surface-HA profiles represent the sum of the cell 5 rface fractions isolated and analyzed in Figure 29. [ H]—labeled HA was run on a 0.9 x 55 cm Sepharose CL-ZB column and eluted in 0.05 M Tris-Cl buffer, pH 8.0, 0.15N NaCl and 0.02% sodium azide at room temperature. 133 TABLE III. Percent distribution of [3H]—labeled HA eluted on Sepharose CL-ZB from cellular, cell surface and medium fractions. ’ Fraction: 12—17 18—27 28-37 Cellular HA 0 35 48.7 22.7 28.6 41° 30.3 16.3 53.4 Cell Surface HA 35° 61.6 20.6 17.8 41° 43.0 18.9 38.1 Medium HA 35° 67.1 24.1 41° 41.3 41.4 17.3 1. Hyaluronic acid was isolated as described in Materials and Methods. 2. Percent distribution is: total dpm associated with selected fractions of column total dpm analyzed by Sepharose CL—ZB chromatography x 100' 134 TABLE IV. Comparison of high, intermediate and low molecular weight [3H]—labeled HA species from cellular, cell sur— face and medium fractions. 2 Cell 35°/41° Cellular Surface Medium HMW 3.7 2.9 6.2 le 3.2 2.2 2.2 LMW 1.2 0.9 2.0 Hyaluronic acid was isolated as described in Materials and Methods. Represents ratio of [3H]—isolated HA dpm/106 cells at 350 to 410 for each molecular weight species. This was determined by multiplying total isolated [3H]-labeled HA dpm by percent distri- bution determined by Sepharose CL—ZB chromatography for each species and taking ratio (350/410). 135 respectively. There is also 2 to 3-fold increase in [3H]—labeled HA of intermediate size from transforming cells in all fractions. Changes in the LMW-species of [3H]-labeled HA are not as pronounced. Thus it appears that upon transformation greater amounts of the high and intermediate molecular weight species of HA are synthesized. This in turn alters the distribution of hyaluronic acid in the various fractions. Fractionation and Analysis of Cell Surface Hyaluronic Acid Cell surface [3H]—labeled HA was divided into several fractions designated SAM—HA, lst EGTA-HA, 2nd EGTA-HA, and trypsinate-HA. The method of isolation and definition of these subfractions (or compartments) of the cell surface fraction has been described in the Materials and Methods. The distribution of [3H1-labeled HA among these various compartments for transforming and nontransforming cultures is shown in Table V. As shown, a large percentage of cell surface hyaluronic acid is located in SAM, 74.7% in 410 cultures and 61.5% in 350 cultures. Because of the method used to separate these subfractions, the lst and 2nd EGTA—released fractions are considered "loosely—associated" HA. Furthermore, the association of this hyaluronic acid with the substratum or cell membrane is cation—dependent as it is released in the presence of the calcium-chelator, EGTA. SAM—HA and trypsinate-HA (HA remaining associated with suspended cells following their removal from the cell-substratum by EGTA and released by trypsinization of suspended cells) are considered "tightly—associated" hyaluronic acid. Table 5 demonstrates that a 136 TABLE V. Distribution of [3H]—labeled HA from various Cell Surface 1,2 compartments. SAM Ist EGTA 2nd EGTA Trypsinate 35° 61.5% 12.8% 20.7% 5.3% 41° 74.7% 2.6% 8.7% 13.9% 1. Hyaluronic acid was isolated as described in Materials and Methods. 2. Percent distribution is represented as: dpm associated with a particular compartment total dpm associated with all cell surface compartments 100. 137 large percentage of hyaluronic acid from 410 cultures is of the "tightly associated" type, with 88.6% of the cell surface HA in SAM and trypsinate subfractions. In transforming cultures (35°), 66.8% of the cell surface HA is in these subfractions. Thus a much larger percentage of the cell surface HA in 350 cells is of the ”loosely—associated" type. It is therefore apparent that the distribution of cell surface hyaluronic acid on nontransforming (410) and transforming (350) cultures is altered. Analysis of the relative levels of total [3H]-Iabeled HA isolated from these subfractions between 350 and 410 cells is shown in Table Ilb. Transforming cells under the conditions of this experiment have 1.7—fold more [3H]-labeled HA in SAM, 9.9—fold more [3H]—labeled HA in the lst EGTA incubation and 4.8—fold more [3H]—labeled HA in the 2nd EGTA wash. In the trypsinate fraction, there is 1.3-fold more [3H]-labeled HA in the 410 cultures. Thus this type of analysis indicates that major alterations in cell surface HA also occur in the "loosely—associa- ted" material. Figure 29 shows the Sepharose CL—ZB elution profiles of [3H]labeled HA isolated from these various fractions from 410 cultures (panels a, c, e, g) and 350 cultures (panels b, d, f, h). Obvious differences are observed in the elution profiles of the two EGTA treatments between 410 and 350 cultures. The distribu- tion of [3H]-labeled HA in these elution profiles in quantified and summarized in Table VI. The distribution of HA in SAM, lst EGTA and 2nd EGTA fractions of 350 cultures has shifted to the high molecular weight species, especially in the EGTA released 138 SAM - HA 1$l EGTA - HA 2nd EGTA ° HA Tfypsinate - HA gal a 0 C 5' OOr 3 9' 3‘!) 2 3’ N am 21 9 lo ‘ ‘ X E .1 8: 101“ b to d 20» I 0 eat 11 W 00) ll 10) .q' ‘[ "\ 2W .' \f bl to to 2273734410 it. 22 {a a} A to 1‘6 2‘2 23 3‘4 10 10 2A2 20 :14 £0 Flacllon (ml) Figure 29. Sepharose CL-ZB chromatography of fractionated cell surface hyaluronic acid. A24GZ-infected CEF were cultured and labeled with [ H]acetate as described in Figure 28. Cell surface-HA was fractionated into SAM—HA, lst EGTA-HA, 2nd EGTA—HA and Trypsinate—HA as described in Materials and Methods. Panels a and b, SAM—HA from 410 cells and 350 cells, respectively. Panels c and d, lst EGTA-HA from 410 cells and 35 cells, respectivel . Panels e and f, 2nd EGTA—HA from 41 cells and 35 cells, respectively. Panels 9 and h, trypsiglate—HA from 410 cells and 350 cells, respective— ly. [ HJ—labeled HA was analyzed by Sepharose CL-ZB column chromatography (0.9 x 55 cm) in 0.05 M Tris-Cl pH 8.0, 0.15 N NaCl and 0.02% sodium azide at room temperature. 1 ml fractions were collected. 139 TABLE VI. Percent distribution of fractionated cell surface [3H]- labeled HA on Sepharose CL—ZB.1 Fracthw: 12—17 18-27 28—37 SAM HA 35° 84. 11.6 3.6 41° 63. 19.8 16.7 lst EGTA HA 35° 34. 43.0 22.5 41° 19. 20.7 60.1 2nd EGTA HA 35° 50.4 28.0 21.6 41° 9. 12.0 79.0 Trypshune HA 35° 3. 18.7 78.3 41° 3. 19.5 77.3 1. Hyaluronic acid was isolated as described in Materials and Methods. 2. Percent distribution is: total dpm in selected fractions x 100. total dpm analyzed the Sepharose CL-ZB column 140 fractions. No significant difference in the distribution of the trypsinate fraction between 410 and 350 cultures is observed. Indeed, it appears that most of this fraction is of the low molecular weight species. Moreover, approximately 85% of 350 SAM-HA is of the HMW species whereas less than 4% is of the LMW species. In 410 cultures the distribution of high and low molecular weight species is approximately 64% and 17%, respectively. Thus it appears that a much higher percentage of the HMW-HA species in transforming cells is found at the site where the cell interacts with its substratum. This difference could possibly lead to many abnormal cell—substratum interactions obser— ved in transformed cells. It also appears that normal cells organize cell surface HA such that most of the HMW-HA is segre- gated to the SAM fraction with very little in the remaining frac— tions. This is not true for transforming cultures which also have HMW-HA in the EGTA fractions (see Figure 29). Finally, we have also made a comparison of the ratio of [3H]labeled HA from 350 and 410 cultures for each cell surface subfraction. These results are shown in Table VII. In the SAM fraction, 2.2-fold more HMW—species is found in transforming CEF, essentially no change is observed in the IMW—species whereas there is 2.8-fold more LMW—HA in 41° than 35° cultures. In Table llb, the ratio (35°/41°) of total SAM—HA is 1.7, although this value demonstrates an increase in total HA, molecular weight analysis indicates that a more complex change occurs in SAM-HA, that is, an increase in HMW—HA and a decrease in LMW—HA in SAM o . . of 35 cultures compared to 410 cultures. Such lnformatlon may be 141 TABLE Vll. Comparison of high, intermediate and low molecular 1 weight species of isolated cell surface [3H]—Iabeled HA. 2 35°/41° SAM 1st EGTA 2nd EGTA Trypsinate HMW 2.2 17.8 26.9 0.71 IMW 0.97 20.6 11.2 0.74 LMW 0.36 3.7 1.3 0.78 Hyaluronic acid was isolated as described in Materials and Methods . Represents ratio of counts at 350/410 determined by multiplying total counts from each fraction 106 cells by percent distribution as determined for high, intermediate and low molecular weight species. 142 important in defining different possible functions of the high and low molecular weight species of hyaluronic acid. The lst and 2nd EGTA—released HA fractions or "loosely-associated" HA, show dramatic alterations of HMW-HA, 17.8— and 26.9-fold increase in 350 cultures, respectively. Such changes are also observed in IMW-HA, 20.6— and 11.2—fold increases for the lst and 2nd EGTA fractions, respectively. In the LMW-species, there is a 3.7—fold increase in the 1st EGTA fraction but only a 1.31-rfold increase in the 2nd EGTA fraction. Thus it appears that major alterations occur in the ”loosely-associated" hyaluronic acid upon transforma— tion in the high and intermediate size HA. Again, this alteration may play an important role in the many changes observed in normal cell-cell and cell-substratum interactions upon transforma— tion. Finally, analysis of the trypsinate fraction of cell surface HA reveals a small and consistant decrease in the ratio of 350 to 410 HA in all molecular weight species. Hyaluronidase Digestion of Isolated Hyaluronic Acid To confirm that EGTA-released material was hyaluronic acid, the isolated GAG was monitored by digestion with Streptomyces hyaluronidase, a HA—specific endoglycosidase, followed by analysis of the digest by Sepharose CL—ZB chromotography. Conditions for digestion of HA are described in Materials and Methods. Results from typical digestions are shown in Figure 29, panels e and f and Figure 30. Figure 30 demonstrates that all molecular weight species from EGTA—released [3H]—labeled HA from 350 cultures labeled 4-8 h after temperature shift, are digested under these 143 20 1 101' 3H, dpm X 10’2 ‘16 A 22 ‘728 34 40 46 F faction (ml) Figure 30. Hyaluronidase treatment of EGTA-released [3H]—labeled hyaluronic acid. Secondary LA24GZ—infected CEF were seeded at 1x10 cells/100 mm culture dish and cultured at 410 for 20 h. 4 h after temperature shift, c ltures at 410 and3350 were labeled with lmCi [H]acetate for 4 h. [H]-—labeled hyaluronic acid was isolated from various fractions as described in Materials and Methods. [ H]—Iabeled HA from 1st EGTA- released fraction of 350 cells was analyzed for purity by treatment with the HA—specific endoglycosidase from Streptomyces hyalurolyticus as described in Materials and Methods. {THY-labeled Ist EGTA-HA was analyzed by Sepharose CL-ZB column chromatography (0.9 x 55 cm) in 0.05 M Tris-CI buffer, pH 8.0, 0.15 N NaCI and 0.02% sodium azide at room teglperature. 1 ml fractions were collected. (———), [H]—labeled lst EGTA-HA. ( ----- ), identical sample treated with hyaluronidase. 144 conditions. In addition, it has been previously determined that the method used to isolate HA (114) results in greater than 95% pure HA (Ullrich, S.J. and Hawkes, S.P., unpublished observa— tions). These results therefore confirm the earlier results and demonstrate that the isolated tritium—labeled material is indeed hyaluronic acid. DISCUSSION Increases of hyaluronic acid in transformed cells has been described in several systems. Increased HA synthesis by chicken embryo fibroblasts after viral transformation with Rous sarcoma virus was an early observation (89). Other systems exhibiting increased levels of hyaluronic acid upon transformation include chicken embryo fibroblasts infected with temperature sensitive mutant of Rous sarcoma virus, the Bryan stain and cultured at the permissive temperature (’90), RSV—transformed chicken chondrocytes (91) and chicken chondrocytes transformed with the temperature—sensitive mutant of RSV, Prague A, LA24 (92), SV40- transformed hamster embryo fibroblasts (93), SV40-transformed human fibroblasts (94), herpes simplex type 2 virus—transformed primary African green monkey kidney cells (93), human glioma cells (95), certain carcinomas (96,97,98), Morris hepatomas (99,100), Wilm's tumor (101) and fibrosarcomas (102). However, certain transformed cell systems do not possess increased levels of hyaluronic acid including mouse melanomas (103) and SV40— transformed 3T3 fibroblasts (104). It appears though, that most transformed systems tend to synthesize increased levels of hyaluronic acid. Indeed, Chiarugi _e_t_ a_|_. (96) have compared glycosaminoglycans isolated from several mammalian tumors to the 145 146 corresponding normal tissues and have observed several variations, the most notable being increased hyaluronic acid. The extracellular organization of hyaluronic acid is not well defined. It has been shown that hyaluronic acid forms high mole- cular weight proteoglycan aggregates in cartilage, glial cells and aortic tissue as a result of its interaction with the core protein of proteoglycan monomers (70). It has also been found to bind to the cell surface glycoprotein, fibronectin (27,28). In addition, there is a report describing a low molecular weight protein which is covalently bound to hyaluronic acid isolated from RSV— transformed chicken embryo fibroblasts (157). Recently, hyaluronic acid binding proteins have been described which are capable of binding to a Dowex-hyaluronic acid affinity column. Moreover, addition of these to urea—pretreated fibroblast monolayers followed by addition of hyaluronic acid specifically leads to an increase in the amount of HA which attaches to the cell glycocalyx (158). Furthermore, Underhill and Toole (114,159) have shown that SV—3T3 fibroblasts have specific cell surface receptors for HA. This information supports the idea that hyaluronic acid must in some way be specifically organized on the cell surface but does not answer how. In this chapter we have analyzed the cell surface distribution of hyaluronic acid by dividing it into several fractions, EGTA incubation (lst EGTA-HA) and wash (2nd EGTA-HA) fractions, HA remaining on the cell surface of EGTA—suspended cells and removed by trypsin (trypsinate-HA) and HA remaining 0 associated with the cell—substratum of the EGTA-removed cells 147 (SAM—HA). We have defined HA released from the cell surface and/or substratum by the two EGTA treatments as "loosely- associated" HA and that which is still attached to the cell surface (trypsinate-HA) or the cell—substratum (SAM-HA) as "tightly—associated" HA. The "loosely—associated" HA appears to interact with the cell surface and/or substratum via a cation- dependent manner as it is released by the calcium chelator, EGTA. Furthermore, we have confirmed earlier observations of two distinct molecular weight species in the cellular, cell surface and medium fractions (160) and have shown how these species are distributed among the various cell surface fractions described. The results described in this chapter demonstrate many alter- ations in the distribution and organization of hyaluronic acid in transformed and nontransformed CEF. Transforming CEF have more HA in each major fraction analyzed (cellular, cell surface and medium) than do nontransforming CEF. Analysis of the cell surface subfractions also show increased amounts of total hyalu- ronic acid from 350 cultures in each subfraction except the trypsinate—HA, which is actually slightly lower. The molecular size of hyaluronic acid is shifted to the high molecular weight species upon transformation. This alteration is most significant in the high molecular weight species followed by the intermediate molecular weight species. The distribution of total cell surface HA among the various cell surface fractions indicates that a much greater percentage is of the "loosely-associated" type in transforming cultures (33.5% compared to 11.3% in normal cultures). As a result, in normal 148 cultures a very high percentage of cell surface HA is of the "tightly—associated" type (88.6% compared to 66.8% in transforming cultures). Although a greater percentage of normal cell surface HA is in SAM compared to transforming cultures, there is 1.7—fold more SAM—HA in transforming cultures. Furthermore, this can be divided into high, medium and low molecular weight HA where there is greater than 2-fold more high molecular weight SAM—HA, approximately equal intermediate weight SAM—HA and greater than 2.5-fold less low molecular weight SAM—HA in transforming cultures compared to normal cultures. As nearly 85% of SAM-HA from trans— forming cultures is high molecular weight and only 3.6% of the low molecular weight type, alterations in the HMW-species would appear to be crucial to changes in. cell—substratum behavior. Such alterations in the lst and 2nd EGTA hyaluronic acid fractions are also observed. There are very large increases in high and intermediate molecular weight species in transforming cultures which results in greatly altered distributions since the change in the low molecular weight species is small in comparison (compare panels c and e to d and f in Figure 29). In contrast to the drastic changes described above, changes in both distribution and ratio of "tightly-associated" cell surface trypsinate-HA in transforming and normal cultures is only slightly altered. From the information presented, we can now describe how hyaluronic acid is organized on the nontransformed and transforming cell surfaces. In nontransformed CEF, a large percentage of cell surface hyaluronic acid is of the "tightly- associated” type and a large percentage of this is in SAM. 149 Furthermore, greater than 60% of the SAM-HA is high molecular weight whereas greater than 60% of the other normal cell surface fractions are low molecular weight HA. Thus, under normal conditions HMW-HA is localized at the cell—substratum whereas LMW-HA makes up a majority of the other cell surface fractions. Thus, normal cell surface HA appears to be specifically compartmentalized. When the normal cells begin developing transformed characteristics, nearly 85% of the SAM-HA is of the HMW-species and there is also greater than 2.2-fold more of this SAM—HA in transforming cultures. Furthermore, there is much more high and intermediate molecular weight HA in the lst and 2nd EGTA cell surface fractions. The distribution and relative levels of trypsinate—HA is relatively unaltered. This suggests that upon transformation, much more HMW—HA must be synthesized and directed to the substratum. However, under transforming conditions there is much more ”loosely-associated" HMW-HA (lst and 2nd EGTA-released HA). It seems possible that this HMW—HA has been released to the "loosely—associated" fraction from the cell- substratum which can no longer accommodate, the large increase in HMW-HA. From here it is shed into the medium. Indeed, there is a large increase in total medium-HA in 350 cultures compared to 410 cultures and it is almost exclusively of the HMW-species. In fact, there is greater than 6—fold more HMW-HA in medium of 350 cultures compared to 410 cultures. This suggests that in addition to greater synthesis of HMW-HA and greater cell—associated HMW-HA in transforming cultures, there is also a greater turnover of the 150 HMW—HA. This is supported by pulse—chase experimental analysis performed in our laboratory (160). Such alterations in organization and turnover of hyaluronic acid could greatly influence the cells normal interactions with its environment. For example, due to the hydrodynamic properties of hyaluronic acid (67), a 2-fold increase in cell-substratum HA could essentially push the cell away from the substratum as well as from other cells. This could lead to the disruption of normal cell—substratum interactions. Indeed, Toole 9.3. _a_l_. (97) have proposed that HA exerts a swelling pressure on adjacent cells thus providing avenues for cell migration. Alternatively, a large increase plus a large turnover of cell-substratum HA could destabilize other cell surface molecules resulting in their inability to function properly. Such a possible function for increased HA has been proposed by Culp and coworkers where they suggested that increased HA in mature adhesion sites could lead to their destabilization and eventual "pinching-off" during cell migration (29). The cell surface glycoprotein, fibronectin, is a good candidate for a molecule capable of being destabilized by HA as; (1) it possesses binding sites for hyaluronic acid (27,28), (2) it is involved in cell—substratum adhesion which is decreased in transformed cells (2,3), and (3) its levels on the cell surface are decreased following transformation. Interestingly, the increase in cell surface hyaluronic acid appears to precede the decrease in cell—substratum fibronectin (Chapter 2, Figure 8) and lactoperoxi- dase iodinated—fibronectin (106) upon transformation. Other possible alterations are likely to occur as a result of these 151 described changes in cell—substratum hyaluronic acid. In the next chapter we have demonstrated that hyaluronic acid is at least partially responsible for decreasing cell—substratum adhesion in transforming cells. We have also demonstrated that there is a very large in— crease in HMW—HA which is "loosely-associated" with the cell sur- face. Once again, due to its unique physical properties such as its ability to act as a molecular sieve and its large hydrodynamic size (67), it could interfere with normal cell-cell interactions thus giving the transformed cell its apparent inability to interact normally with adjacent cells. Furthermore, we have demonstrated that cell surface hyaluronic can act as a "barrier" to a nonphysiological fluorescent reagent, fluorescamine (107). Fluorescamine reacts approximately 3-fold more with normal cells than transformed cells. Pretreatment of normal and transformed cells with Streptomyces hyaluronidase, a hyaluronic acid specific endoglycosidase, results in equivalent labeling with fluorescamine of both normal and transformed cells (107). Thus transformed cell surface hyaluronic acid prevents fluorescamine from reaching the cell surface components with which it can react. The possibility that such a barrier could prevent physiological components from interacting with cell surface receptors seems likely. We suggest that this barrier may be composed of the ”loosely—associated" HA. This report is the first to describe the distribution and organization of cell surface hyaluronic acid. This has been greatly facilitated by the recent description of different molecular 152 weight species of hyaluronic acid (160). Results presented here in addition to those described by Ullrich and Hawkes (160), suggest that there may be two or more mechanisms involved in the synthesis of low and high molecular weight HA and that transformation may selectively stimulate the mechanism responsible for high molecular weight HA synthesis. The observed alterations in total HA levels, especially changes in distinct molecular weight species upon transformation may provide the necessary information required for an improved understanding of the structural and functional properties of hyaluronic acid and its role in neoplastic transformation. ROLE OF HYALURONIC ACID lN CELL-SUBSTRAT UM ADHES 1 ON I have demonstrated that a 2—fold and 20-fold increase in high molecular weight hyaluronic acid is measured in SAM and "loosely-associated" compartments, respectively, of the cell surface following initiation of transformation. In this chapter I have analyzed the potential role that hyaluronic acid may play in the loss of cell—substratum adhesion during the transformation process. 153 MATERIALS AND METHODS Cell Culture Chicken embryo fibroblasts were prepared as described with the exception that secondary or tertiary CEF to be used for the adhesion assay were seeded at 5—7 x 105 cells/60 mm plastic tissue culture dish. Density is important as confluent cultures tend to detach as monolayer sheets, invalidating the adhesion assay. Adhesion (Detachment) Assay We measure the strength of cell adherence to its substratum by the ability of a solution of 5 mM EGTA in CMF-PBS, pH 7.4, containing 1 mg/ml C—amino-n-caproic acid (Sigma, St. Louis, MO) and 1 mM phenylmethyl sulfonyl fluoride (PMSF, Sigma, St. Louis, M0) to cause cell detachment. This assay has been modified from procedures previously described (161,162). A scheme for the adhesion assay is diagramed in Figure 31. Briefly, all assays were performed with exponentially growing cultures. Subconfluent secondary or tertiary LA24G2-infected CEF were prelabeled 12-24 h prior to temperature shift with 1—2 uCi [3H]thymidine/6O mm culture dish (20 Ci/mmol, New England Nuclear, Boston, MA or 72 Ci/mmol, ICN, Irvine, CA). To assay for adhesion, medium was removed from cultures at various times following temperature shift 154 155 ADHESION ASSAY Secondary or tertlary chicken embryo Ilbroblasts trimmed with Rolls sarcoma Virus (LA 24) [SHI-thymldlne —\1 12-24 hours temperature shill ./ \ 41 35° 3-15 hours r/ \A /\ medium medium f hyaluronidase medium 15 minutes 41 4th 35H 35 I I l I lresn medlum 1 hour I l I I 5mM EGTA 1n CMF-PBS stationary 10 minutes gentle agitation 5 minutes released cells collected by centrifugation cell number and/or lncorporatlon 01 3H determined [both adherent and released cells] Figure 31. Schematic diagram of adhesion assay. 156 and the monolayers washed 2—3 times with warm (390) CMF—PBS+. This was followed by addition of 3 ml of 5 mM EGTA in CMF-PBS+, incubation of monolayers at 390 for 10 min followed by agitation on a Braun Thermonix shaker set at 100 rpm for 5 min at room temperature. Following agitation, the EGTA-detached cells were removed and pelleted by centrifugation (IEC model HN-Sll) at 400 x g for 5 min. The cell pellet and cells remaining on the tissue culture dish were solubilized in 0.1% sodium dodecyl sulfate (SDS)/0.01 N NaOH, boiled, neutralized by the addition of an equal volume of 0.01 N HCl and radioactivity of the total sample (0.5 ml) determined in a Beckman LS9000 scintillation counter. The percentage of cells removed by EGTA is calculated as: dpm associated with detached cells dpm associated with detached plus remaining cells x 100' Values obtained represent the average of triplicate determinations. 3 . . . . Use of [ H]thym1dlne to count cells was validated by comparison of results to those obtained when absolute numbers of cells released and remaining were counted. Hyaluronidase Treatment To assess the affect hyaluronic acid has on the alteration of adhesion of transforming cells, a HA—specific endoglycosidase, hyaluronidase, isolated from Streptomyces hyalurolyticus (Miles Laboratories, Elkhart, IN), was used to specifically remove this glycosaminoglycan. This preparation is reported to be protease free. Prior to the adhesion assay, LA24GZ-infected CEF cultured at 350 and 410 were incubated in 2 ml medium 221+ containing 10 157 turbidity reducing units (TRU) of hyaluronidase for 15 min at 390. The presence of the protease inhibitors, PMSF (1 mM, Sigma, St. Louis, MO) or ovomucoid (5 ug/ml, Sigma, St. Louis, MO), did not alter results suggesting that no proteases which might effect our results were present in the hyaluronidase preparation. The hyaluronidase was removed and cells further incubated at their appropriate temperature for 1 h. Adhesiveness was then determined as described. Analysis of Cell Surface HA following Hyaluronidase Treatment The efficiency of removal of cell surface hyaluronic acid by hyaluronidase treatment was analyzed. [3H]-labeled hyaluronic acid was isolated from the cell surface of 410 and 350 cells which had been incubated in 3ml medium 221+ containing 5 ug/ml ovomucoid with or without Streptomyces hyaluronidase (5 TRU/ml) per 100 mm tissue culture dish for 15 min. Hyaluronic acid was isolated basically as described by Underhill and Toole (114). Six 100 mm culture dishes with subconfluent monolayers were grown at 350 and 12 at 410 for 15 h in the presence of 500 Ci/culture dish [3H]acetate (2.3 Ci/mmol, New England Nuclear, Boston, MA) to label the hyaluronic acid. Following the Pronase digestion used in the HA purification, isolated cell surface components were analyzed on a 1.4 x 118 cm Sepharose 68 column having a molecular weight exclusion for polysaccharides of 1 x 106. The presence of HA was assessed by comparing isolated material to a duplicate sample treated with Streptomyces hylauronidase (25 TRU/ml) for 24h at 39°. 158 Attachment Assay The attachment of LA24GZ—infected CEF to serum-coated glass scintillation vials was determined in the presence or absence of hyaluronidase. Generally, 12-16 h after temperature shift and labeling cells with 1—2 thi [3Hlthymidine/culture dish, cells were removed from their substratum by incubation with 5 mM EGTA in CMF—PBS+ (15 min at 390) and gentle pipetting. After centrifuga— tion at 400xg for 5 min to pellet the cells, they were resuspended in medium 221+ and added to 15 ml glass scintillation vials (NEN, Boston, MA), which had been pretreated by overnight incubation at 390 in medium 221+. A total of 2 x 105 cells was added to each vial in 2 ml medium 221+. The amount of radio- activity associated with this number of cells was determined by scintillation counting and represents total dpm added. At various times after addition of cells and incubation at the appropriate temperature, the vials were gently shaken and the medium removed. The attached cells were solubilized in 0.1% SOS/0.01 N NaOH and radioactivity determined. The percentage of cells attached is: dpm associated with attached cells dpm associated with total cells added per vial x 100' Growth in Agar The ability of cells to grow in an agar suspension with or without exogenously added HA was monitored as described by Roberts t l. with minor modifications (163). Briefly, 1-2 mg/ml of bovine vitreous hyaluronic acid (Sigma, St. Louis, MO) was 159 suspended in medium 221+ containing 0.3% (w/v) agar (Difco, Noble agar, Detroit, MI). Cells were suspended to a final concentration of 1—2 x 103 cells/35 mm tissue culture dish. This mixture (2 ml) was poured onto a base layer consisting of medium 221+ containing 0.5% (w/v) agar in 35 mm tissue culture dishes (Falcon, Oxnard, CA). For positive and negative controls, Prague A-infected and uninfected CEF were also cultured in agar, respectively. Cultures were grown at 390 for two weeks without feeding. At two weeks, colonies were stained by addition of 1.0 ml of a sterile aqueous solution of 0.5 mg/ml of 2—(p— iodophenyl)-3-(p-nitrophenyl)-5-—phenyl tetrazolium chloride (Sigma, St. Louis, M0) for 24 h at 390. After removal of dye, plates were scored. Only colonies larger than 0.75mm in diameter were counted. Cell Spreading SAM was prepared in 60 mm dishes from subconfluent cultures grown at 410 and 350 (14.5 h) as described previously, using 1/2 volume of buffers required for 100 mm tissue culture dishes. One half the SAM plates from each temperature were incubated in a 1 ml solution/60 mm dish of PBS, pH 7.4 containing c-amino—n—caproic acid (1 mg/ml) and Streptomyces hyaluronidase (8 TRU/ml) for 15 min at 390. The remaining half of the culture dishes at each temperature were incubated without hyaluronidase. Following the incubation, all plates were washed with ice cold sterilized water and drained. Plates were then 0 . stored at 4 until use. 160 Subconfluent CEF at 410 and 350 (15 h) were removed from their substrata with 3 ml of 5 mM EGTA in CMF-PBS+, collected by O O SAM/hyaluronidase 5 centrifugation and seeded on 35 SAM, 35 treated, 41°-SAM, and 410—SAM/hyaluronidase treated, at 5 x 10 cells/culture dish in serum—free medium. Serum-free medium was used to avoid the complicating effects of attachment and spreading factors present in serum. Photographs of representative areas were taken 20, 35, and 60 min after seeding with a Zeiss inverted microscope (200 X). Polyacrylamide Gel Electrophoresis Tritium—labeled proteins synthesized following hyaluronidase treatment were analyzed by SOS-polyacrylamide gel electrophoresis using the buffer system of Laemmli (131) with 4% stacking and 7.5% separating polyacrylamide gels. Fluorography was performed as described by Bonner and Laskey (132). RESULTS Removal of Cell Surface Hyaluronic Acid by Hyaluronidase To determine the efficiency of removal of cell surface HA by the hyaluronidase treatment described for the adhesion assay, 0 [3H]acetate—Iabeled monolayers cultured at 35 for 15 h were incubated in serum—free medium with or without Streptomyces hyaluronidase (5 TRU/ml) for 15 min as described in the legend to Figure 32. Serum—free medium was used to avoid potential complicating factors possibly present in serum. Cell surface hyaluronic acid was isolated as described in Chapter 4 except that following the protease digestion step, supernatant fractions were analyzed by Sepharose 68 column chromatography (Figure 32). Panel A represents the elution profile of cell surface material isolated from control (non—hyaluronidase treated) cultures (-—-—-—) and the same material after hyaluronidase treatment ( ----- ). These results demonstrate that most of the material eluting in the column void volume is hyaluronic acid. Panel 8 represents the elution profile of material isolated from the cell surface of cells pretreated with hyaluronidase. Note the lack of material eluting at or near the void volume. Thus it appears that pretreatment with hyal uronidase removes most cell surface hyaluronic acid. This is quantitatively confirmed in Table Vlll where total cell surface hyaluronic acid (dpm) per 119 protein was 161 Figure 32. 162 Effect of hyaluronidase treatment of cell monolayers on cell surface GAG. Secondary LA2462-infected CEF were seeded at 1 x 10 cells/100 mm culture dish cultured at 410 for 20.5 h and then trarksferred to 35 for 15 h in the presence of 0.5 mCi [H]acetate/IOO mm dish. Prior to isolation of cell surface GAG as described by Underhill and Toole (114), three 350 cultures were each incubated with 3ml medium 221+ containing 5 pg/ml ovomucoid with Streptomyces hyaluronidase (5 TRU/ml) for 15 min. Three identical cultures were treated without hyaluronidase. Following the Pronase d'gestion described for HA purification (114), isolated [ H]acetate-labeled cell surface components were analyzed by Sepharose 68 column chromatography (1.4 x 118 cm). 4 ml fractions were collected. Panel A represents the elution profile of isolated cell surface GAG from 350 cultures not pretreated with hyaluroni- dase (-——). A duplicate sample was incubated with Streptomyces hyaluronidase (25 TRU/ml) in 0.05 M sodium acetate, pH 5.5, 0.15 M NaCl for 24 h at 39° ( ----- ). Panel B represents théa elution profile of isolated cell surface GAG of 35 cultures pretreated with hyaluronidase. 163 25 35 45 55 Fraction (4ml) 15 99 x 5% .1. 164 TABLE VIII. Effect of Hyaluronidase Treatment on Cell Surface Hyaluronic Acid. Treatmenta [3H]-labeled HA dpm/pg proteinb o 35 , 200+ 325.3 35°, HYase 11.9 41°, 200+ 49.4 410, HYase 5.7 a. Represents treatment of cells cultured at 350 or 410 with serum-free medium (200+) containing 5 ug/ml ovomucoid with or without hyaluronidase (5 TRU/ml) as described in Materials and Methods. . o o b. Total protein from parallel cultures grown at 35 and 41 was determined by the method of Lowry (166). 165 determined for 350 and 410 cultures treated with or without hyaluronidase. These results demonstrate that cell surface HA from 350 cultures incorporate greater than 6.5-fold more tritium than 410 cultures after 15 h of labeling at'the appropriate tempera- ture. Furthermore, greater than 96% of 350 cell surface hyaluronic acid was removed by hyaluronidase treatment and greater than 88% of 410 cell surface HA was removed by the same treatment. Thus it appears that this hyaluronidase treatment efficiently removes almost all cell surface HA. The fact that only 4 to 12% of the cell surface HA remains may be due to its location at the cell-substratum, for example, beneath the cells and possibly inaccessible to hyaluronidase. Thus even though this represents only a small amount of the total HA, its location makes it very important in the adhesion process. Changes in Cell-Substratum Adhesion Upon Transformation The strength of adhesion between cells and their substratum was measured by the ability of cells to withstand removal from the substratum when gently agitated in CMF—PBS+ containing 5 mM EGTA, pH 7.4, as described in Materials and Methods. A decreased adhesiveness results in an increased percentage of cells released, as monitored by this assay. Figure 33 shows that as early as 4 to 8 h following temperature shift, transforming cells begin to become less adhesive. This change occurs before any morpholo— gical characteristics of transformed cells are observed by light microscopy. By 12 h following temperature shift, approximately 48% of the transforming cells were released by this assay whereas Figure 33. dpm associated with detached plus remaining cells PERCENTAGE OF CELLS RELEASED 166 40 r q 30- "‘ 20 -' 10 r l-P—I--I A 8 12 TIME AFTER TEMPERATURE SHIFT (HOURS) Effect of transformation on adhesion of cells to substratum Secondary LA24GZ-infected CEF were seeded at 5 x 10 cells/60 mm culture dish and cultured at the nonpermissive temperature (410) for 23 h. 12 h prior to temperature shif all cultures were labeled with 1 ilCi/60 mm plate [H]thymidine. After transfer of one half the culture dishes to the permissive o . temperature (35 ), cultures were assayed for adheSlon as described in Figure 31 at 0, 4, 8 and 12 h. (O——O), cultures transferred to the permissive temperature. (I ----- I), cultures grown at the nonper- missive temperature. Percentage of cells released is equal to: dpm assoc1ated w1th detached cells x 100. 167 approximately 6% were removed from cells cultured continuously at . . o the nonpermisswe temperature (41). In this experiment, this represents approximately an 8-fold difference. From experiment to experiment, variation in the actual percentages of cells released by EGTA treatment varied, however, the final result was always the same, _i_._e_;, cells cultured at the permissive temperature (35°) rapidly become less adhesive than cells remaining at the nonpermissive temperature (410). The reason for the variability is unknown at this time but may be at least partially due to inconsistant density of cells over the tissue culture dish. The results described here confirm observations reported earlier (164) and in addition confirm the validity of this adhesion assay. Effect of Hyaluronidase on the Change in Cell-Substratum Adhesion Following Initiation of Transformation To determine if removal of hyaluronic acid from the cell surface by hyaluronidase treatment could restore adhesive properties lost upon transformation, CEF cultured at 41C) or transferred to 350 were pretreated with hyaluronidase and adhesion monitored as described at various times after temperature shift. As shown in Figure 34, removal of HA .by hyaluronidase restores the adhesive properties of a significant proportion of the cells. This effect is most evident between 4 and 16 h after temperature shift to 350. A similar though smaller effect is observed for cells at 410. Although the effect of hyaluronidase is not large at the 16 h time point in this experiment, several subsequent experiments demonstrated a consistant and significant 60 (IO 20 PERCENTAGE Of CELLS RELEASED Figure 34. 168 TIME AFTER TEMPERATURE SHIFT (HOURS) Effect of hyaluronidase on adhesion of LA24GZ-infected CEF. econdary LA24GZ-infected CEF were seeded at 5 x 10 cells/60 mm tissue culture dish and cultured at the nonpernfissive temperature (410) for 23 h. Cultures were labeled with 1 ilCi [H]thymidine 12 h prior to temperature shift. At various times after transfer of one half the cultures to the permissive temperature (350), cells were incubated with or without hyaluronidase (Streptomyces hyalurolyticus) at a concentration of 5 turbidity reducing units/ml of medium (2 ml) containing 1 mM phenyl methyl sulfonyl fluoride and assayed for the ability to adhere to the culture dishes as described in Figure 31. (O—éOg; 350. (O ----- O); 350, hyaluronidase. (I——-'); 41. (I ----- I); 41 , hyaluronidase. 169 effect at this time (data not shown). To determine if the increase in adhesion after hyaluronidase treatment was due to the synthesis of adhesive proteins, we analyzed proteins labeled with 3H--labeled amino acids for 1 h and 4 h following hyaluronidase treatment, by gel electrophoresis of total cellular proteins. The results suggest that hyaluronidase treatment does not stimulate the synthesis of any major cellular proteins which may be involved in the increased adhesion (data not shown). Effect of Hyaluronidase Treatment on Cell Attachment To determine if hyaluronic acid plays a role in the initial rate of cell attachment, [3H]thymidine—labeled, EGTA-released cells, cultured at 410 or 350 for 13 h, were seeded on serum— coated glass scintillation vials at the appropriate temperature in 2 ml media with or without Streptomyces hyaluronidase (4 TRU/ml) as described in Materials and Methods and in the legend to Figure 35. At various times following seeding, cells remaining attached to the substratum following gentle aggitation were solubilized and radioactivity determined. The results shown here demonstrate that 350 cells (panel A) attach at a slightly slower rate than 410 cells (panel B) and that hyaluronidase treatment does not significantly influence the rate of attachment of the EGTA-removed cells to serum—coated substrata. This suggests that the processes of attachment and detachment may involve different mechanisms. As we have demonstrated that EGTA treatment of cells removes a significant amount of cell surface HA, especially in Figure 35. 170 Effect of hyaluronidase on attachment. Tertiary cultures of LA24GZ—infected CEF were seeded at 1.5 x10 cells/100 mm culture dish and cultured at the nonpermissive temperature (410) for 22 h. One half the cultures were then switched to the permissive temperature 50) for 13 h. All cultures were labeled with 2 uc1 [ H]thymidine/100 mm culture dish. At 13 h after temperature shift 350 cultures (panel A) and 410 cultures (panel B) were removejj+ fror‘g+ the culture substratum with 5 mM EGTA in Ca , Mg —free PBS pH 7.4 and seeded in serum-coated scintillation vials in 2 ml media with or without Streptomyces hyaluronidase (4 TRU/ml). At various times after addition of cells, vials wer removed, gently shaken and unattached cells removed. Remaining cells were solubilized directly in the vials and radioactivity determined. Percentage of cells attached was then determined (dpms associated with attached cells/dpm associated with total cells added per vial x 100) and plotted versus time after seeding. Panel A: O(O———O) 350 cells with hyaluroni- dase; (O ----- O) .35 cells without hyaluronidase. Panel B: (O————-—O) 41 cells with hyaluronidase; (O ----- O) 41 cells without hyaluronidase. Percentage of calls attuned 171 ,4' 40? I 20): A A A 20 40 60 80 150 Tliiiu utter seeding (minutes) 120 172 transforming cells (see Chapter 4), we are not able to unambiguously rule out the role of hyaluronic acid in attachment. Role of Hyaluronic Acid in Cell Spreading To assess the potential role that hyaluronic acid plays in the ability or inability of cells to spread, EGTA-released CEF cultured at 410 or 350 for 15 h were seeded upon 350 (14.5 h)—SAM treated or not treated with Streptomyces hyaluronidase as described in Materials and Methods and Figure 36. SAM was prepared as described in Chapter 2, 14.5 hours after temperature shift, except that 60 mm tissue culture dishes were used instead of 100 mm culture dishes and therefore the volumes of buffers were adjusted accordingly. Cell spreading was monitored visually by randomly photo— graphing tissue culture dishes at various times following seeding of cells as illustrated in Figures 36 and 37. The degree of spreading ranged from rounded cells to round cells with few extended Iamellipodia to spreading cells possessing very refractive or dark edges and finally to well spread cells with little refractivity. Hyaluronidase treatment of 35°—SAM resulted in . . . . o stimulation of cell spreading. Figure 36 demonstrates that 41 cells seeded upon 35°—SAM treated with hyaluronidase (41O CEF on 35° SAM 8 HYase) spread more rapidly (i.e., are more well spread) by 60 min than 410 cells seeded upon 350-SAM not treated with hyaluronidase (41O CEF on 350 SAM — HYase). Identical results were obtained with 350 cells seeded upon 35°-SAM treated or not treated with hyaluronidase (Figure 37). It is apparent Figure 36. 173 Effect of hyaluronidase on cell spreading. 35°—SAM was prepared as described in Chapter 2 from secondary LA24GZ—infected CEF seeded at 5 x 100 cells/6O mm tissue culture dish and cultured at 41 for 30.5 h followed by 14.5 h at 35°. One half of the 35°—5AM were each incubated with lml of hyaluronidase in PBS, pH 7.4 (8 TRU/ml) containing 1 mg/ml c—amino-n— caproic acid for 15 min at 390. The remaining half were treated identically but without hyaluronidase. Tertiary LA24GZ-infected CEF were seeded at 9 x105 cells/100 mm tissue culture dish and grown at 410 for 19.5 h and then half the cultures switched to 350 for 15 h. Cells were removed from the substratum as described in Figure 1. 410 EGTA—released cells were seeded on untreated 350-SAM (41O CEF on 350 SAM— HYOase) and hyaluronidase treated 350-SAM (41o CEF 013 35 SAM 8 HYase) in serum—free medium at 5.4 x 10 cells/60 mm dish. Photographs of representative areas were taken at 20, 35 and 60 min following addition of cells. - ._-._ ~..v- . _x-— 174 $3,: a _2- “.1 Lu. 02 O < mm 0') 0 ID a) 176 20 min. 35" SAM & HYase 177 however that HA may only affect the initial spreading of cells as RSV-infected, fully transformed CEF cultured for long periods of time in the presence of Streptomyces hyaluronidase show no alterations in morphology as well as LA24GZ—infected CEF cultured for greater than 24 h at 350 with or without hyaluronidase. Apparently, once cell attachment is established, hyaluronic acid no longer influences the degree of cell spreading. The Effect of Exogenously added Hyaluronic Acid of Growth on Chicken Embryo Fibroblasts in Agar We have demonstrated that the cell—substratum or SAM-HA and the "loosely-associated" EGTA-HA are greatly elevated upon transformation. The large increase is made up almost entirely of the high molecular weight species of HA (Chapter 4). In addition, Streptomyces hyaluronidase treatment removes approximately 90% of the cell surface—HA which is the sum total of SAM—HA, EGTA—HA and trypsinate—HA described in Chapter 4. Finally, we have demon- strated that cell surface—HA in some manner decreases adhesion of transformed cells as Streptomyces hyaluronidase treatment par— tially restores the adhesion of transformed cells. It is also known that. transformed CEF secrete or shed much more HA into the growth medium than normal CEF and we have confirmed these findings (Chapter 4). We were therefore interested in determining if the mere presence of extracellular, non- cell—associated hyaluronic acid might influence cell growth. Our approach was to determine if high concentrations of exogenously added hyaluronic acid would allow normal cells to grow in semi-solid medium as 178 transformed cells do. The ability of cells to grow in agar suggests that cell—substratum adhesion is no longer required and is a characteristic of transformed cells. Growth of normal CEF in 0.6% (w/v) agar containing 1 mg/ml and 2 mg/ml HA was monitored as described (163). The results are shown in Table IX. Under these conditions, hyaluronic acid had absolutely no effect on the ability of normal CEF to grow in agar. Note that many PrA-infected CEF formed colonies equal to or larger than the minimum size counted thus confirming the validity of this assay. These results suggest that hyaluronic acid must be specifically associated with the cell surface and/or extracellular matrix as opposed to simply present in the agar gel to exert its effects on adhesion and growth control. However,’ we do not rule out the possibility that HA endogenously shed from the transformed CEF does not subsequently bind to the extracellular matrix or back to CEF where its effects are exerted or that isolated CEF—HA added to agar (as opposed to bovine—HA) might influence growth in agar. 179 TABLE IX. Effect of Exogenous Hyaluronic Acid on Growth in Agar.a Number of Coloniesb Normal c151:C 0 Normal CEF + 1 mg/ml exogenous HA 0 Normal CEF + 2 mg/ml exogenous HA 0 Prague A infected CEF 56 a. Growth in agar for 2 weeks as described (163). b. Colonies 0.75 mm and larger counted for two 60 mm tissue cul- ture dishes. c. Normal + Prague A—CEF seeded @ 1 x 103 cells/60 mm plate HA—treated CEF seeded @ 2 x 103 cells/60 mm plate. DISCUSSION The role of increased hyaluronic acid in the development and maintenance of the transformed state has not been fully inves— tigated. However, there is now increasing evidence that hyaluronic acid may be involved in cell-substratum adhesion, cell—cell recognition and invasiveness of tumor cells. For example, hyaluronic acid may prevent interactions between cells by presenting a "barrier" around the transformed cell which prevents it from interacting in a normal fashion with a neighboring cell. We have presented evidence that such a "barrier" in transformed CEF does exist to the nonphysiological compound fluorescamine (107). In the previous chapter, we presented evidence that approximately 20-fold more high molecular weight hyaluronic acid is "loosely—associated" in a cation—dependent manner with the cell surface of transforming cells than with normal cells. Such an increase in cell surface hyaluronic acid is a likely candidate for this barrier. Furthermore, Burger and Martin (108) have observed that RSV transformed CEF required much less wheat germ agglutinin or concanavalin A for maximal agglutination if the transformed cells were first treated with Streptomyces hyaluroni— dase. To explain these observations they proposed that HA might simply cover up the agglutinin binding sites. McBride and Bard (109) demonstrated that the presence of 'halos' around adherent 180 181 fibrosarcoma cells appeared to protect them from lymphocyte- mediated cytolysis. Hyaluronidase treatment destroyed the 'halo' and allowed immune lymphocytes to approach the tumor cell membrane thus enhancing cytotoxic action. There is also evidence suggesting hyaluronic acid plays a role in cell-substratum adhesion. Kramer and coworkers (110,111) observed that sublines of chinese hamster ovary (CHO) cells that were slow to detach with trypsin or (EGTA had little or no surface hyaluronic acid, while sublines that detached rapidly contained much more hyaluronic acid than their parental cells. Culp and coworkers (29) have suggested that elevated levels of HA in older or mature SAM may be related to increased detachability (mobility) in these cells since SAM from growing cultures contains more HA than SAM from newly attached cultures. Schubert and LaCorbiere (112) have demonstrated the involvement of secreted glycoprotein/glycosaminoglycan complexes in the attachment of normal and anchorage—independent muscle myoblast cell lines to various substrata. Furthermore, the secreted complexes from the less adhesive variants contained more HA than complexes from parental cells. Finally, addition of exogenous hyaluronic acid to 3T3 cell cultures has been shown to lead to detachment of these cells from their substratum (115). In addition to the potential ability of hyaluronic acid to modify cell—cell and cell-substratum interactions, HA has been demonstrated to influence the process of tumor cell metastasis. The invasive carcinoma, V2, grown in rabbits contained 3-4 times more HA than the same tumor grown in the nude mouse, where it 182 is noninvasive (97). It has been proposed that HA exerts a swelling pressure which can give rise to separation of cell and collagen layers and thus provide avenues for cell migration. Fur— thermore, hyaluronic acid has been shown to play a key role in migration and proliferation of many undifferentiated cells during morphogenesis and differentiation (83). In Chapter 4 we demonstrated that [3H]glucosamine—labeled cell surface hyaluronic acid was increased by 4 h following the initiation of the transformation process in LA24GZ—infected CEF. It has also been shown that HA synthetase activity is increased by 4 h following temperature shift in LA24-infected CEF (165). Furthermore, there is greater than a 2—fold increase in high molecular weight hyaluronic acid in SAM of LA24G2—infected CEF cultured at the permissive temperature. The change in surface HA levels correlates with the decrease in adhesion following tempera- ture shift. The change in adhesion of transforming CEF following temperature shift is modulated by pretreatment of cells with a HA—specific endoglycosidase from Streptomyces hyalurolyticus. This treatment efficiently removes cell surface HA (approximately 90%). The remaining HA which is inaccessible to hyaluronidase, may be located deep within the cell-substratum compartment. Therefore, the inability to remove this HA may be partially responsible for the incomplete restoration of adhesion observed in hyaluronidase- treated, transforming CEF. However, it is more likely that many components which are involved in normal adhesion may be altered upon transformation and increased HA is only partially responsible for loss of adhesion in fully transformed cells. The 183 ability of hyaluronidase pretreatment to partially restore adhesion has also been observed in Prague A—infected, fully transformed CEF (data not shown). Removal of HA does not preferentially stimulate the synthesis of proteins which might be involved in adhesion as radioactive pulses with 3H—amino acids for 1 and 4 h following hyaluronidase treatment, followed by analysis of radioactively-labeled polypeptides by gel electrophoresis and fluorography, does not reveal any differences in protein profiles obtained with untreated controls (data not shown). This result suggests that adhesive proteins are always present but somehow prevented from functioning. Since Prague A-infected cells possess very low levels of fibronectin, this result suggests that other adhesive components can mediate cell—substratum adhesion. Furthermore, we have evidence that removal of HA followed immediately by the adhesion assay also detects increased adhesion, again suggesting the presence of adhesive components. The most reproducible results however are obtained when the adhesion assay is begun I h after enzyme treatment, presumably allowing the necessary reorganization and/or interactions to occur. These findings plus those of the previous chapter tend to support the idea that increased synthesis, presence and turnover of HA may be destabilizing and/or covering up adhesive components on the cell surface, especially at the cell substratum. Removal of excess HA (in the case of transformed CEF) may lead to the increased ability of the transformed cells to reorganize and restore adhesive components already present to a functional state. Although hyaluronidase treatment results in the strengthening of 184 the adhesive bond between transformed cells and their substratum, it does not appear to significantly influence the rate of attachment of EGTA—released CEF to a serum—coated substratum. However, we have demonstrated that EGTA treatment releases high molecular weight HA from the surface of transforming cells (Chapter 4) and this species of HA may be particularly important in the attachment process. As demonstrated in Chapter 4, SAM from transforming CEF contains greater than 2-fold more high molecular weight HA than SAM from nontransformed CEF. The influence of this HA in transforming SAM on attachment and spreading of 410 and 350 CEF was investigated by seeding cells on 350 SAM treated or not treated with hyaluronidase. The presence of HA in SAM did not significantly alter cell-attachment rates (data not shown) as was observed in the attachment of cells to serum-coated glass scintillation vials in the presence or absence of HA (Figure 35). However, the rate of cell spreading of 0 both 41 and 350 cells appears to be retarded on 350 SAM compared to spreading on 350 SAM pretreated with Streptomyces hyaluronidase (Figures 36 and 37). Although the initial rate of cell spreading is influenced by HA in SAM, after two hours the cells are equally spread on treated or untreated SAM. In addition, long term culturing of transformed cells in the presence of hyaluronidase does not influence their morphology by causing them to become more flattened (data not shown). Finally, we have demonstrated that addition of exogenous hyaluronic acid to normal cells cultured in agar does not influence their ability to form colonies like those observed with Prague A-infected CEF. As 185 hyaluronic acid has been demonstrated to be involved in cell proliferation and movement during morphogenesis and differentia- tion (83) and invasiveness of the V2 carcinoma (97) it is likely that the organization of endogenously synthesized HA is crucial to these processes and not the mere presence of the polymer. CONCLUDING STATEMENT CONCLUDING STATEMENT It is well established that addition of transformed cells to a normal ECM, or fibronectin to transformed cells in culture leads to reversion of the transformed phenotype towards that of a normal cell. How then does a new tumor cell or a small number of tumor cells overcome or escape the influence of the normal ECM? It has been shown that, in addition to decreased synthesis and/or increased turnover of cell surface fibronectin, some transformed cells have lost the ability to bind extracellular fibronectin. This property would allow the transformed cell to grow and spread without the. constraints of a normal matrix. Other transformation- sensitive alterations of ECM macromolecules, such as in the amounts of collagen and hyaluronic acid, may also give the transformed cell the ability to ignore the normal ECM. In addition, transformed cells in general are known to secrete larger quantities of proteolytic enzymes. These proteases are capable of degrading extracellular matrix components. Thus, these type of changes would free the tumor cell from the constraints of the normal matrix. We have previously reported that RSV—transformed CEF possess a "barrier" of hyaluronic acid capable of preventing the reaction of the compound, fluorescamine, with cell surface amines. Removal of HA with Streptomyces hyaluronidase resulted in an 186 187 equal extent of fluorescamine labeling with normal and transformed cells. The difference monitored by fluorescamine was not due to loss of cell surface fibronectin as CEF infected with a temperature—sensitive mutant of RSV and cultured at the permissive temperature do not show alterations in fibronectin levels until greater than 24 h after temperature shift and differences in synthesis of extracellular fibronectin are not observed until after 36 h. However, an increase in cell surface HA upon transformation occurs by 4 h and decrease in adhesion occurs soon thereafter. In addition, high molecular weight HA on the cell surface is greatly altered upon transformation, with greater than a two—fold increase in SAM and approximately 20-fold increase in the " Ioosel y—associated" cell surface 9 l ycosami noglycan . Furthermore, turnover of cell surface HA in transformed cells is greatly elevated. Thus, these alterations in HA upon transformation may provide the cell with the ability to ignore the normal ECM. Finally, we are particularly intrigued by the fact that loss of cell—substratum adhesion, mediated by hyaluronic acid (Chapter 5), correlates temporally with the deposition of the 21K protein in SAM during the early stages of the transformation process. Moreover, our results suggest that the 21K protein may be a component of a glycoprotein/GAG complex similar to that described by Schubert and LaCorbiere. Interestingly, the complex made by adhesion—deficient mutants also ‘contains elevated amounts of HA. Therefore, the 21K protein and HA may be associated in this complex and deposited together on SAM. 188 The information we have provided, in addition to that provided by others, has led us to propose the following model. We believe this model is consistent with properties of transformed cells during the development and maintenance of the cancer phenotype and is not intended to suggest that this is the only mechanism used by cancerous cells to grow and spread. Upon the development of the transformed state cells begin to produce elevated levels of cell surface and extracellular hyaluronic acid. As mentioned, HA can act as a barrier to penetration by the fluorescent probe fluorescamine. Such a barrier could also act to prevent the interaction of exogenous components with cell surface receptors. Thus cell surface receptors for extracellular matrix fibronectin, exogenous growth inhibitors or other cells would be inaccessible. Such a barrier could also act to slow the release of certain molecules such as transforming growth factors and proteases, known to be produced by many transformed cells. This might result in a concentration effect and allow these components to preferentially exert their effects on the transformed cell. Finally the rapid turnover of cell surface hyaluronic acid, especially at the cell-substratum, could result in the destabilization of large surface molecules such as fibronectin, especially as fibronectin possesses HA binding sites. HA would thus allow the transformed cell to overcome the constraints of the ECM and in fact "protect it" from the normal ECM. This might also release the cell from the requirement of the ECM for active proliferation that has been demonstrated in other systems. Furthermore, we have proposed that transforming growth factors 189 would be especially accessible to the transformed cell whereas normal growth inhibitors would not reach the transformed cell. Once the developing tumor has reached a critical size, HA would no longer be required for its primary roles, but accumulated extracellular HA could now serve as a means for migrating tumor cells to metastasize, much as in systems described for embryogenesishnorphogenesis. 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