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Glycosaminoglycan synthesis by normal human mammary epithelial cells in primary culture 1986

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GLYCOSAMINOGLYCAN SYNTHESIS BY NORMAL HUMAN MAMMARY EPITHELIAL CELLS IN PRIMARY CULTURE -' by NANCY JONES B . S c , The U n i v e r s i t y of A l b e r t a , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n FACULTY OF GRADUATE STUDIES Department of Anatom7 We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA June, 1986 (c) Nancy Jones I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f f^\Q jrt> fUX- j The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date »E-6 n / a n ABSTRACT The extracellular matrix (ECM) influences c e l l growth and differen- t ia t ion . Glycosaminoglycans (GAGs), alone or complexed with protein (proteoglycans), are a major component of the ECM affecting c e l l behavior. GAG synthesis has been studied extensively i n animal models and malignant c e l l s . This research centres on studying GAG production by normal human mammary epi thel ia l ce l ls i n culture. Mammary ,tissue obtained from reduction mammoplasties were dissociated to single c e l l s . The epi thel ia l c e l l population was seeded onto hydrated collagen gels at 2-2.5x10 cells/cm i n medium containing 5$ FCS and 5ug/ml of i n s u l i n . Ultrastructural studies confirmed the epi thel ia l nature of the cultures. To measure GAG synthesis, cultures were incubated with H-glucosamine for 24 hours at 3 time points; days 3-4, 9-11 and 17-18. The cultures were proliferat ing at the early time point and had reached a stationary phase at the later time points. C e l l , ECM and medium fractions were analyzed for GAGs as identif ied by enzyme degradation and cellulose acetate electrophoresis. At day 4, when ce l l s were actively growing, the majority of GAGs produced were released into the medium fraction (75-80$). The predominant GAG was the nonsulfated GAG, hyaluronic acid (HA). Of the sulfated GAGs chondroitin sulfate (CS) 4 and 6 comprised only 18$ of total GAGs; dermatan sulfate (DS) synthesis was negligible . At the later time periods, when cultures had ceased growing a higher percentage of total GAG was incorporated into an ECM (50-65$). The sulfated GAGs were preferentially incorporated into the ECM, CS 4 and 6 comprising 70$ and DS comprising 30$. The marked difference i n type and location of GAGs produced was not merely a function of time i n culture. Cultures seeded at high 5 2 densities (5x10 cells/cm ) were not proliferating when terminated at day 4. Their GAG prof i le was similar to that of lower density cultures at day 10. This data provides a b a s e l i n e from which we can determine i f c e l l - s y n t h e s i z e d GAGs, play a r o l e i n maintaining d i f f e r e n t i a t e d and malignant phenotypes. - i i i - TABLE OF CONTENTS Page ABSTRACT i i LIST OP TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS ix ABBREVIATIONS.. x CHAPTER 1 - INTRODUCTION GENERAL INTRODUCTION 1 I) The Basement Membrane • 1 1) Collagen 2 2) Fibronectin 4 3 ) Laminin 4 4) Entactin 5 II) Proteoglycans 5 1) Hyaluronic Acid 11 2) Sulfated Proteoglycans 16 III) Mammary Epithelial Cells '. 19 IV) Proteoglycans and Malignancy 2 0 V) Thesis Problem Formulation 25 CHAPTER 2 - MATERIAL AND METHODS I) Dissociation Procedures 27 II) Preparation of Collagen Gels 28 III) Cell Culture Procedures 2 9 IV) Radiolabelling Procedures 2 9 V) Resolution into Fractions 3 0 VI) Extraction of Glycosaminoglycans 3 0 - iv - Page VII) Analysis of Glycosaminoglycans by Enzyme Digestion 32 1 ) Hyaluronic Acid I d e n t i f i c a t i o n . . . . . 3 2 2 ) Chondroitin Sulfate Identification 3 4 3 ) Heparan Sulfate Identification 3 4 VIII) S c i n t i l l a t i o n Counting 35 IX) Calculation of Assay Results 3 7 X) Identification of GAG using Electrophoresis 3 8 XI) Film Developing for Autoradiography 3 8 XIII) Electron Microscopy 4 0 CHAPTER 3 - RESULTS I) Growth 4 1 II) Total GAG Synthesis 4 1 III) Hyaluronic Acid Synthesis 46 IV) Chondroitin Sulfate Synthesis 4 8 V) Dermatan Sulfate Synthesis 4 8 VI) Heparan Sulfate Synthesis 5 5 VII) Electrophoresis Results 55 VIII) Cultures Labelled with 5 5 S - S u l f a t e 5 7 IX) Autoradiography 6 1 X) Electron Microscopy 6 1 CHAPTER 4 - DISCUSSION I) Culture Status 6 4 II) Distribution of Synthesized GAGs 65 - v - Page I I I ) Types of Synthesized GAGs 68 1) Hyaluronic A c i d 68 2) S u l f a t e d GAGs 74 IV) O v e r a l l Amount of Synthesized GAGs 78 V) Future Research 81 VI) Summary 83 APPENDICES •••• 84 BIBLIOGRAPHY 87 ML LIST OF TABLES PAGE Table I The Proteoglycans 10 Table I I Di s t r i b u t i o n of -glucosamine labelled glycosaminoglycans i n normal human mammary e p i t h e l i a l c e l l s i n tissue culture 44 Table I I I ^H-glucosamine incorporation into i n d i v i d u a l glycosaminoglycans i n normal human mammary e p i t h e l i a l c e l l s i n culture 49 Table IV Electrophoresis I d e n t i f i c a t i o n of GAG {% of total) 56 Table V Percentage of Sulfated GAG i n the Medium and ECM of high and Low Density Cultures Labelled with 3 5 S-Sulfate 58 - v i i - LIST OF FIGURES Page Fig . 1 Schematic representation of the basement membrane 3 F i g . 2 Chemical Structures of HA (non-sulfated) and DS (sulfated). 7 Fi g . 3 The l i n k trisaccaride 8 Fig. 4 Schematic representation of postulated ECM interactions 12 F i g . 5 Flow chart of i d e n t i f i c a t i o n of GAG by enzymes . . 33 F i g . 6 Enzyme digestion over time 36 F i g . 7 Growth study of normal human mammary e p i t h e l i a l c e l l s seeded at low density 42 F i g . 8 Total ^H-glucosamine incorporation into GAG i n cultures of normal human mammary e p i t h e l i a l c e l l s 45 F i g . 9 ^-glucosamine incorporation into HA/cell 50 F i g . 10 ^H-giucosamine incorporation into CS/cell 51 F i g . 11 a) ^H-glucosamine incorporation into HA, CS and DS/cell i n the medium fr a c t i o n 52 b) ^H-glucosamine incorporation into HA, CS and DS/cell i n the ECM fra c t i o n 53 F i g . 12 ^H-glucosamine incorporation into DS/cell 54 F i g . 13 5 5 s _ s u l f a t e incorporation into CS and DS 59 Fig. 14 Autoradiograph of glucosamine incorporated GAG i n a l l fractions of an early stationary culture ••• 62 F i g . 15 Electron Micrograph of normal human mammary c e l l s i n culture 63 - v i i i - ACKNOWLEDGEMENT I am happy to have this opportunity to thank the people who helped me i n various ways: To Dr. Anne Adams and Theresa Lee who had the dubious honor of introducing me to the laboratory and tissue culture techniques. To Dr. Gordon Parry and co-workers at Berekely who taught me GAG analysis techniques and to Dr. Terry Crawford at UBC who taught me the electrophoretic technique and was there to answer my numerous questions. To Dr. Joanne Emerman, my advisor, for her perseverance i n teaching me the principles of c e l l biology, her continued enthusiasm and encouragement throughout my time i n her laboratory and for labouring through the numerous drafts of this thesis. To Nancy Sehindelhuer and Paul Johnson for a l l their assistance i n preparing the typed thesis. Finally, to Phil Jones who helped with the typing but mostly for a l l the moral support he gave me when the going got tough. This study was supported by a grant from the National Cancerr Institute of Canada. - ix - ABBREVIATIONS ECM - extracellular matrix PG - proteoglycan. GAG - glycosaminoglycan HA - hyaluronic acid (hyaluronate) CS - chondroitin sulphate HS - heparan sulphate , DS - dermatan sulphate BM - basement membrane INTRODUCTION H i s t o r i c a l l y , the ext r a c e l l u l a r matrix (ECM) was considered to be an iner t substrate providing support for c e l l s and not interacting i n any si g n i f i c a n t manner with these c e l l s . Over the l a s t two decades, however, information has accumulated indicating that t h i s was an over-simplification of i t s function. The interactions between c e l l s and the ECM are regarded as s i g n i f i c a n t and imperative i n such important areas of c e l l functioning as growth (Gey et a l . , 1974), migration (Greenberg et a l . , 1981), d i f f e r e n t i a t i o n (Emerman and P i t e l k a , 1977, Emerman et al.,1977, 1981; Kleinman et a l . , 1981; Parry et a l . , 1982) and determination of c e l l shape (Emerman et al.,1979 , Gospodarowicz et a l . , 1978). The ECM i s composed of macromolecules from four major classes - collagen, proteoglycans, glycoproteins and e l a s t i n (Hay, 1981). Together these components form a str u c t u r a l l y stable material that l i e s under e p i t h e l i a and surrounds connective tissue c e l l s . The Basement Membrane The term basement membrane refers to an organized complex of ECM components which include collagen, proteoglycans and glycoproteins, that are associated with the basal surfaces of e p i t h e l i a l c e l l s whenever they contact connective tissue (Vracko, 1974). To further c l a s s i f y the basement membrane, i t can be described according to i t s morphological appearance which include 3 or 4 major zones (Martin et a l . , 1982): l ) the lamina lucida externa (or lamina rara) which i s an electron lucent region 20 to 40 nm wide found just below the e p i t h e l i a l basal c e l l surface, 2) the lamina densa (or basal lamina) i s a middle layer 20 to 100 nm wide and contains a meshwork of fine filaments giving i t an electron dense appearance, 3) the JL lamina lucida interna which i s an electron lucent region of variable wide and found between the lamina densa and the underlying connective tissue (Figure l ) and the ret icular lamina. The reticular lamina i s a meshwork of fine collagen fibers between the basal lamina and connective tissue. The major collagen component present i n the ECM i s Type IV collagen (Kleinman, 1982). This has been demonstrated using various techniques including autoradiography and immunolocalization (Sano et a l . , 1981). Type IV collagen was f i r s t identif ied by Kefalides i n 1966. He was able to solubil ize a unique collagen protein after pepsin digestion from canine glomerular basement membranes. While i t was i n i t i a l l y thought that Type IV contained a single type of chain i t has been subsequently proven that this collagen has two dist inct chains designated pro a 1 (IV) and pro a 2 (IV) (Crouch et a l . , 1980; Glanville et a l . , 1979). Unlike Types I, II and III, Type IV collagen does not arise biosynthetically from precursor molecules called procollagens. Instead, the pro a I (IV) and pro a 2 (IV) chains are incorporated into the basement membrane as such (Heathecote et a l . , 1976; Karakashian et a l . , 1982; R i s t e l i et a l . , 1981). Type IV collagen has been localized to the lamina densa portion of the basement membrane by several studies (Yaoita et a l . , 1978; Laurie et a l . , 1980; R o l l et a l . , 1980). The actual function of Type IV collagen i s not known, however, i t s n o n - f i b r i l l a r structure may be lending both e l a s t i c i t y and s t a b i l i t y to the basement membrane. It has been suggested that l ike ends of the collagen type IV molecules interact with each other forming a continuous network (Timpl et a l . , 1981).' Other models have also been proposed and w i l l be discussed later i n the Introduction. Another collagen present i n basement membranes, as well as other areas, Type V collagen, was or iginal ly isolated from placenta (Bailey et a l . , - 3 - Epithelial Cells Lamina lucida Lamina dens.a Connective Tissue F i g . 1 Schematic r e p r e s e n t a t i o n of t h e basement membrane.(Martin e t a l . , 1982.) 1979; Burgeson et a l . , 1976; Chung et a l . , 1976). I t also contains two d i s t i n c t chains, a 1 (v) and a 2 (V). The role of t h i s collagen i n basement membranes i s unclear. Immunolocalization studies indicate i t s presence i n the lamina densa (R o l l et a l . , 1980) as well as emanating from there to the underlying connective tissue (Martinez-Hernandez et a l . , 1982). This l a t t e r finding has led Martinez-Hernandez et a l . to suggest that Type V collagen may act by anchoring d i s s i m i l a r tissue types together. The glycoproteins found i n the basement membrane include fibronectin (Vaheri et a l . , 1978), laminin (Timpl et al.,198l) and entactin (C a r l i n et a l . , 1981). Fibronectin i s a large glycoprotein (MW 440,000) made up of 2 i d e n t i c a l 220,000 Dalton chains linked by d i s u l f i d e bonds and found i n serum, on c e l l surfaces and i n the ECM of connective tissues (Ruoslahti et a l . , 1981). I t s function i n the ECM has been related to i s a b i l i t y to aggregate and bind to a number of other molecules (Vahe r i et a l . , 1978). I t has been localized by immunoelectron microscopy to the lamina lucida as well as throughout the basement membrane (Foidart et a l . , 1980). This glycoprotein binds various types of c e l l s to collagens Type I , I I , I I I & IV. A few of the c e l l types studied include primary fibroblasts (Murray et a l . , 1978), rat hepatocytes (Hooper et a l . , 1976), myoblasts (Ketley et a l . , 1976) and established c e l l l i n e s such as CHO and 3T3 (Gr i n n e l l , 1978). From the studies to date i t appears reasonable to conclude that fibronectin i s the glycoprotein used as an attachment mediator between other ECM components and c e l l s of mesenchymal o r i g i n (Kleinman, 1982). I t s place i n the structured basement membrane i s not known for certain but i t has been shown to bind to numerous c e l l types as well as to collagens (Type I to V) and proteoglycans (Woodley et a l . , 1984). - 5 - g Laminin i s a very l a r g e g l y c o p r o t e i n (MW 10 ) f i r s t i s o l a t e d from the Engelbreth-Holm-Swarm (EHS) tumor (Timpl et a l . , 1979) and found i n a l l basement membranes (Timpl et a l . , 1980). I t i s composed of two types of chains (200,000 and 400,000 Daltons) that are l i n k e d by d i s u l f i d e bonds i n a cross formation ( L i o t t a , 1983), w i t h 3 chains being 200,000 Daltons (sh o r t arms) and 1 chain being 400,000 Daltons (long arm). Laminin has been i m p l i c a t e d i n the adhesion mechanisms between ECM components, p a r t i c u l a r l y Type IV c o l l a g e n and PGs, i n the s e v e r a l c e l l types i n c l u d i n g breast e p i t h e l i a l c e l l s (Terranova et a l . , 1980) guinea p i g epidermal c e l l s , bovine lens e p i t h e l i a l c e l l s and monkey pigmented e p i t h e l i a l c e l l s . Laminin binds p r e f e r e n t i a l l y to Type IV c o l l a g e n and promotes the adhesion of e p i t h e l i a l and e n d o t h e l i a l c e l l s (Terranova et a l . , 1980). I t has a l s o been shown th a t l a m i n i n binds to proteogylcans w i t h the highest a f f i n i t y being to heparin and heparan s u l f a t e (Del Rosso et a l . , 1981). C e r t a i n c e l l s such as metastic T241 fibrosarcoma c e l l s (Murray et a l . , 1980) w i l l adhere to Type I c o l l a g e n v i a l a m i n i n . C e l l s that adhere v i a l a m i n i n can synthesize t h e i r own l a m i n i n (Terranova et a l . , 1980) and the same i s probably true of c e l l s r e q u i r i n g f i b r o n e c t i n f o r adhesion (Dessau et a l . , 1978). C e l l s that are capable of s y n t h e s i z i n g both p r o t e i n s are l i k e l y capable of u t i l i z i n g both f o r adhesion ( F o i d a r t et a l . , 1980). E n t a c t i n i s the most r e c e n t l y discovered g l y c o p r o t e i n and a l s o appears to be a component of many basement membranes (Bender et a l . , 1981; C a r l i n et a l . , 1981). In an u l t r a s t r u c t u r a l study of the b a s a l sufaces of e p i t h e l i a l c e l l s a f t e r exposing them to an t i b o d i e s to the p r o t e i n , Bender et a l . were able to l o c a l i z e e n t a c t i n i n c l o s e a s s o c i a t i o n w i t h these s u r f a c e s . L i t t l e i s known as yet regarding the f u n c t i o n of the g l y c o p r o t e i n i n the basement membrane. - 6 - Proteoglygans Proteoglycans are another class of molecules abundant i n the BM as well as the ECM i n general (Kleinman, 1982). Proteoglycans are long chain polymers of repeating disaccharides with either carboxyl or sulfate groups (Toole, 1982). - The disaccharide unit i s made up of either a glucuronic or iduronic acid residue, with either N-acety-D-glucosamine or galactosamine. The number of sugar residues can vary from 300, which i s a common amount with sulfated proteoglycans, to 2,000 to 3,000 residues seen i n the average HA molecule . The proteoglycan i s dist inct from glycoproteins because of the high percentage (90-95$) of carbohydrate. Glycoproteins typical ly have less than 60% carbohydrate. Structurally identif ied proteoglycans include hyaluronic acid, chondroitin sulfate 4, chondroitin sulfate 6, ^ dermatan sulfate, heparin and heparan sulfate. (Keratan " sulfate i s also an identif ied proteoglycan but, because i t i s related almost solely to cartilage and cornea, i t w i l l not be discussed further) . A representation of two proteoglycans i s presented i n Figure 2. Proteoglycans d i f f e r from each other i n several ways. A l l proteoglycans i n their native state are linked to a protein core with the exception of hyaluronic acid . An average protein core may contain 1900-2000 amino acid residues which are generally serine-r ich. More recent research indicates that the protein core can vary markedly i n size and amino acid content (Rapaeger et a l . , 1985). This difference i n protein core may i n part be responsible for the f i n a l location of synthesized proteoglycans (Rapaeger et a l . , 1985; Chang et a l . , 1985). This w i l l be discussed further i n another section of the thesis . Attached to the protein core are from 100-150 side chains of a particular carbohydrate sequence as shown i n Figure 2. The protein core i s attached covalently to the carbohydrate side HA ^ COO" GLUCURONIC ACID 0 ^ CHo / 0 H N--ACETYLGLUCOSAMINE DS S 0 3 IDUR0N1C ACID N-ACET YLGAL ACTOSAM I N E ( 4 - SUL FATE RESIDUE) F i g . 2 Chemical s t r u c t u r e s of H A ( n o n - s u l f a t e d ) and D S ( s u l f a t e d ) . - 8 - F i g . 3 The common l i n k t r i s a c c a h a r i d e between t h e GAG component and t h e p r o t e i n c o r e of PG. - 9 - chain via a xylose-serine linkage (Figure 3). His tor ica l ly , the proteoglycans were named according to their carbohydrate side chains and no account was taken at that time as to the nature of the protein core (Kraemer, 1979). The proteoglycans d i f f e r from each other i n the type of monosaccharide present i n the repeating disaccharide uni t . It i s on the basis of this difference that several of the degradative enzymes differentiate between proteoglycans (Yamagata et a l . , 1968). HA, CS 4 and CS 6 a l l have glucuronic acid as one monosaccharide whereas dermatan sulfate and heparan sulfate have either glucuronic or iduronic acid . CS 4, CS 6 and dermatan sulfate a l l have N-acetyl-galactosamine as the second monosaccharide while HA and HS have N-acetyl-glucosamine. The third area where proteoglycans d i f f e r i s i n their degree of sulfat ion. The following are l i s ted according to degree of sulfation from least to greatest: CS 4, CS 6, DS HS. CS 4 i s sulfated on the carbon 4 and CS 6 on carbon 6 of the N-acetyl-galactosamine residue (Kraemer, 1979). DS i s similar to CS 4 but has epimerized 3-D-glucuruonic acid to 8-L- iduronic acid (Kraemer, 1979). However, i t appears that a large amount of heterogeneity exists i n the CS and DS proteoglycans such that copolymers of both have been observed (Kraemer, 1979). They may contain sequences of iduronic acid interspersed with sequences of CS 4 or CS 6 either non-sulfated or disulfated on the N-acetyl-galactosamine residue. Heparan sulfate i s unique i n that i t possesses an ac id- labi le sulfate group linked to the amine group of the hexosamine residue. HA i s a non-sulfated proteoglycan. A summary of the proteoglycans can be seen i n Table I . Proteoglycans have had several functions attributed to them i n their role as ECM components. They are generally believed to be involved i n - 10 - TABLE 1: PROTEOGLYCANS1 Glycosaminoglycan Molecular Repeating Disaccharide Weight A B S u l f a t e s / Disaccharide U n i t P r o t e i n Core Hyaluronic a c i d 4,000 - 8,000,000 D-glucu- r o n i c a c i d N-acetyl- D-glucos- amine NO Ch o n d r o i t i n S u l f a t e - 4 500 - 50,000 D-glucu- r o n i c a c i d N-acetyl- galactos- amine 0.2-1.0 YES Cho n d r o i t i n S u l f a t e - 6 500 - 50,000 D-glucu- r o n i c a c i d N-acetyl- galactos- amine 0.2-2.3 YES Dermatan 15,000 - D-glucu- N - a c e t y l - 1.0-2.0 YES S u l f a t e 40,000 r o n i c OR g a l a c t o s - L - i d u r o n i c amine a c i d Heparan S u l f a t e 5,000 12,000 D-glucu- r o n i c OR L - i d u r o n i c a c i d N-acetyl- glucos- amine 0.2-3.0 YES Heparin 6,000 25,000 D-glucu- r o n i c OR L - i d u r o n i c a c i d N-acetyl- glucos- amine 2.0-3.0 YES 1 A l b e r t et al.,1983- - 11 - cell-substrate adhesions (Toole, 1982; Culp, 1976; Culp et a l . , 1979). Both HA and HS are present on the surfaces of ce l ls (Culp et a l . , 1979; Rapraeger et a l . , 1985). These proteoglycans have also been shown to bind to fibronectin (Yamada et a l . , 1980) and laminin (Kleinman et a l . , 1981). Prom this information i t would appear that proteoglycans not only help l ink c e l l surface to basement membrane (cell-associated proteoglycan) but also help to s tabil ize basement membrane components (BM proteoglycan) within this structurally defined area. How do these molceules discussed thus far interact to form the structurally stable basement membrane? To summarize b r i e f l y , i n some manner the glycoprotein present, with the help of proteoglycans, enhances or enables the c e l l to adhere to a particular type of collagen. There are numerous theories on the actual structure and several are shown i n Figure 4. They mainly d i f f e r i n the arrangement of the components within the basement membrane although they a l l demonstrate one or more receptors for ECM components on the plasmalemma. More recent studies (Laurie et a l . , 1985) using rotary shadowing electron microscopy, found laminin mainly bound to Type IV collagen 81 nm from the carboxyl terminus and large heparan sulfate bound to Type IV collagen 206 nm from the carboxyl terminus. Laurie et a l . postulate a model whereby heparan sulfate binds to the collagen molecule i n such a way as to allow interaction between the free long arm of laminin and the c e l l . However, they have also suggested other possible models from their data and this area of interaction of ECM components i n the BM i s by no means conclusive. I w i l l now discuss proteoglycans i n depth as they are the basis of the thesis work. - 12 - F i g . 4 S c h e m a t i c r e p r e s e n t a t i o n s o f p o s t u l a t e d ECM i n t e r a c t i o n s . A. B a s e d on H y n e s ( 1 9 8 1 ) B. K l e i n m a n e t a l ( 1 9 8 1 ) C. T o o l e ( 1 9 8 1 ) D. S u g r u e and H a y ( 1 9 8 2 ) HA= H y a l u r o n i c a c i d PG = P r o t e o g l y c a n FN = F i b r o n e c t i n C0= C o l l a g e n LN = L a m i n i n A l t h o u g h t h e y show d i f f e r e n t i n t e r a c t i o n s b e t w e e n t h e ECM c o m p o n e n t s , a l l m o d e l s e n v i s i o n r e c e p t o r s a t t h e c e l l s u r f a c e f o r one o r more o f t h e s e c o m p o n e n t s . - 13 - Hyaluronic Acid HA has .heen implicated i n motili ty and growth of developing tissue (Toole, 1977)• Toole et a l . (1971) observed that the major proteoglycan being synthesized during chick embryo corneal migration i s HA. They also observed that the onset of HA synthesis actually occurs before the onset of migration. HA has also been shown to promote detachment of a variety of ce l ls including neural crest ce l ls and dog kidney ce l ls (Turley, 1984; Abatangelo et a l . , 1982). HA has been implicated i n the a b i l i t y of tissue to reach high degrees of hydration (McCabe, 1972). Hydrated tissues exert a hydrostatic pressure capable of opening pathways through the tissue to allow for migration of c e l l s . Coupled with this function are the weak adhesions or bonds formed by HA (Toole, 1977). HA has been noted i n footpads of motile fibroblast ce l ls (Culp, 1976; Latterra et a l . , 1982) and radiolabelled and fluorescent hyaluronate occurs i n retraction fibers (Turley, 1984). Its presence here may be related to the a b i l i t y of HA to "lubricate" an area (Culp, 1976) and allow the c e l l to slide along unimpeded by strong cell-substrate attachments., HA has also been implicated i n the prevention of precocious differentiat ion (Toole et a l . , 1972). Small amounts of hyaluronate added to high density cultures of stage 26 chick embryo somite ce l ls inhibi t the formation of car t i lage- l ike nodules that otherwise develop. Coinciding with this finding are the< results of studies on chick embryo sclerotomal ce l ls and chick embryo limb mesoderm hyaluronidase ac t iv i ty (Toole, 1972). These c e l l s , as well as the corneal c e l l s , show a large increase i n hyaluronidase ac t iv i ty at the end of the active c e l l migration stage. This enzyme removes HA and signif ies the onset of differentiat ion. The effects of HA on growth as opposed to migration are not well documented. In - 14 - studies involving embryonic tissue, growth and migration are occurring simulataneously and i t i s therefore d i f f i c u l t to separate the effect of HA on each. It has been suggested that with regard to certain c e l l types, HA does not promote locomotion. Neural crest c e l l s (Erickson et al.,1983) show l i t t l e or no locomotion when seeded on a hyaluronate-coated substratum. The adhesion a b i l i t y of HA also now appears more c e l l s p e c i f i c than o r i g i n a l l y thought. Although, as stated e a r l i e r , HA i s associated with weak cell-substratum and c e l l - c e l l interactions, for certain c e l l s HA does act as an adhesion mediator. For example, HA i s involved i n the attachment of SV40-3T3 c e l l s to a sulfated proteoglycan substratum (Toole, 1982) and the attachment of chondrocarcinoma c e l l s to tissue culture surfaces (Mikuni-Takagaki et a l . , 1980). The type of involvement of HA i n adhesions may be related not only to the c e l l type but also the type of substrate the c e l l i s adhering to, the time i n culture and the developmental sequence (Turley, 1984). This may relate to the a b i l i t y of a c e l l to synthesize HA binding s i t e s , the a v a i l a b i l i t y of HA or the number of binding s i t e s available to interact (Underhill et a l . , 1981). In general, HA has mainly been found to mediate adhesion to non-fibronectin substrates (Schubert et a l . , 1982; Brennan et a l . , 1983) as opposed to other proteoglycans, which mediate adhesion to both fibronectin and non-fibronectin substrates (Schubert et a l . , 1982). A f i n a l point about HA i s i n regard to i t s a b i l i t y to aggregate other proteoglycans. There i s a binding s i t e on the protein core of some proteoglycans containing CS 4, CS 6 or DS for HA (Hascall et al.,1981; Oegema et a l . , 1981). As a r e s u l t , HA i s able to form large aggregates of these molecules. I t may be v i a th i s mechanism that cell-associated HA - 15 - (Tur l e y , 1984) may a t t a c h to proteoglycans present i n the basement membrane thereby anchoring the c e l l to the substratum. The r o l e of HA as a b i n d i n g molecule i s not f u l l y understood and i s c u r r e n t l y under i n v e s t i g a t i o n (McCarthy et a l . , 1985; Lacey et a l . , 1985; Marks et a l . , 1985). McCarthy et a l . (1985) examined the mechanisms by which e x t r a c e l l u l a r aggregates of PGs are maintained a t the chondrocyte c e l l s u r f a c e . They were able to determine i n both normal c h i c k and Swarm r a t sarcoma chondrocytes that 50-60$ of the aggregated proteoglycans are hel d at the c e l l surface v i a i n t e r a c t i o n w t i h hyaluronate, which i s s u s c e p t i b l e to streptomyces hyaluronidase. In p r e l i m i n a r y s t u d i e s they have been able to i d e n t i f y hyaluronate-binding s i t e s a t the c e l l surface and b e l i e v e i t may be v i a these receptors that the HA-aggregates a t t a c h . HA bi n d i n g s i t e s are a l s o being i d e n t i f i e d on a number of other c e l l s i n c l u d i n g 3 T 3 c e l l s (Lacey et a l . , 1985), a d u l t c h i c k b r a i n (Marks et a l . , 1985) and c h i c k n e u r a l c r e s t c e l l s ( T u r l e y , 1984). As research continues i n t h i s area the r o l e of HA w i l l become c l e a r e r , however, at t h i s p o i n t i t would be reasonable to assume that HA i s not simply a proteoglycan that hydrates t i s s u e and allows f o r ready detachment and m o t i l i t y of c e l l s , although a s s i s t a n c e w i t h c e l l m o t i l i t y i s almost c e r t a i n l y a f u n c t i o n of proteoglycans (HA and others) as treatment w i t h DON w i l l stop c e l l movement i n v i t r o ( T u r l e y , 1980). DON ac t s by i n h i b i t i n g the sy n t h e s i s of glycosaminoglycans v i a i n h i b i t i o n of the formation of glucosamine, a precursor f o r GAG. Here again the r e s u l t s of experiments on d i f f e r e n t c e l l types r e v e a l s that the response to HA appears to be c e l l - t y p e s p e c i f i c . For example, c h i c k heart f i b r o b l a s t s w i l l migrate i n t o c o l l a g e n g e l s (Bernanke et a l . , 1979) and move over a p l a s t i c c u l t u r e d i s h when hyaluronate i s added to the medium (Tu r l e y , 1984). Conversely, i t s a d d i t i o n to 3H3 c e l l c u l t u r e s ( T u r l e y , 1984) - 16 - leucocytes ( F o r r e s t e r et a l . , 1981) and n e u r a l c r e s t c e l l s ( E r i c k s o n et a l . , 1983; Newgreen et a l . , 1982) e i t h e r does not a f f e c t m o t i l i t y , as i n the case of the 3T3 c e l l s , or i n h i b i t s m o t i l i t y , as i n the case of the l a t t e r two c e l l types. Again, i s i t a question of a v a i l a b i l i t y of HA, HA receptors or both or do these c e l l s type not respond to HA under any circumstances; r a t h e r , do they u t i l i z e another molecule or mechanism f o r m o t i l i t y ? I t i s a l s o p o s s i b l e that HA may bind c o m p e t i t i v e l y w i t h other proteoglycans causing i n h i b i t i o n of m o t i l i t y . To summarize the f u n c t i o n s of HA i n the ECM and s p e c i f i c a l l y the basement membrane i t appears i n some instance's to a i d i n c e l l detachment and f a c i l i t a t e m o t i l i t y and i n others to act as an adhesion molecule to maintain c e l l - s u b s t r a t e p r o x i m i t y . In some c e l l s HA i s found i n c e l l l a m e l l a e i n others i t i s dispersed over the e n t i r e c e l l surface (Turley, 1984). I t has been reported to be i n c l o s e a s s o c i a t i o n w i t h the a c t i n component of the cy t o s k e l e t o n of 3T3 c e l l s (Lacey et a l . , 1985). S u l f a t e d Proteoglycans S u l f a t e d proteoglycans have been l i n k e d w i t h c y t o d i f f e r e n t i a t i o n ( T r e l s t a d et a l . , 1974; Praus et a l . , 1971). In t h e i r s t u d i e s on c h i c k embryo c o r n e a l development, T r e l s t a d and co-workers found t h a t , c o i n c i d i n g w i t h the onset of d i f f e r e n t i a t i o n , the major proteoglycan present changes from HA to s u l f a t e d proteoglycan (they d i d not i d e n t i f y s p e c i f i c p r o t e o g l y c a n s ) . Toole et a l . (1977) a l s o analysed 4 and 8-day c h i c k embryo limb chondrocytes f o r c h o n d r o i t i n s u l f a t e . They determined experimentally that the CS formed a t the e a r l i e r day i s l e s s r e a c t i v e to c o l l a g e n (decreased binding) and l e s s s u l f a t e d and of a sm a l l e r molecular weight than the CS from the l a t e r day when the c e l l s are f u r t h e r d i f f e r e n t i a t e d . However, as w i t h HA, the e f f e c t s of CS on va r i o u s c e l l types are - 17 - d i s s i m i l a r . For example, when added to cultures of f i b r o b l a s t s , yolk sac or neural crest c e l l s i t i n h i b i t s spreading and causes detachment of c e l l s from the substrate. Due to the poor spreading a b i l i t y and r a p i d i t y of movement associated with CS (Turley et a l . , 1979; Erickson, 1984), t h i s proteoglycan i s thought to have weakly adhesive properties. In some c e l l s , such as human melanoma c e l l s (Reisfeld, 1984), an antibody s p e c i f i c to CS stops the i n i t i a l spreading of these c e l l s over basement membrane components. CS has been found at the c e l l surface of mammalian skin fibroblasts where i t interacts with the c e l l membrane v i a i t s carbohydrate chains (Saito et a l . , 1972) or v i a i t s protein core, which does not appear to be intercalated with the c e l l membrane but held by another "attachment" molecule (Glossyl et a l . , 1983) • I t i s not known i f there i s a s p e c i f i c binding s i t e f o r t h i s proteoglycan but s i t e s have been isolated which appear to have a high a f f i n i t y for CS and CS hybrid or dermatan sulfate molecules (Glossyl et a l . , 1983; Truppe et a l . , 1978). Heparan sulfate, unlike HA and CS, i s known for i t s a b i l i t y to form adhesions between ECM components (Hook et a l . , 1982; K e l l e r et a l . , 1982). HS binds to both fibronectin and laminin (Yamada et a l . , 1980; Hynes et a l . , 1982; Woodley et a l . , 1984) and i s present both on the surfaces of c e l l s and i n the ECM deposited by c e l l s (Culp et a l . , 1979). Reports show that for some c e l l types, including neural crest (Erickson et a l . , 1983) 3T3 c e l l l i n e s (Turley, 1984) and neuronal c e l l s (Stamatoglou et a l . , 1983), the addition of HS to the culture medium supports both attachment and spreading. Conversely, heparan reduces attachment of CHO c e l l s (Klebe et a l . , 1982) and M3A c e l l s to fibronectin-containing substrates. One reason for t h i s discrepency may be i n the method of analysing attachement - 18 - of c e l l s . L a t e r r a et a l . (1983) f i n d that i f they looked at attachment r a t h e r than c e l l - r e m o v a l assays, the a d d i t i o n of heparan to t h e i r c u l t u r e s of f i b r o b l a s t s does not i n h i b i t adhesion to f i b r o n e c t i n and treatment w i t h heparinase does not detach the c e l l s . Woodley et a l . (1984) f i n d that HS binds p r e f e r e n t i a l l y to l a m i n i n over f i b r o n e c t i n and to Type IV c o l l a g e n over Types I to I I I . They d i d not f i n d that the a d d i t i o n of HS to e i t h e r a Type IV c o l l a g e n or l a m i n i n premixed coated c u l t u r e d i s h i n c r e a s e s the bin d i n g between the two ( c o l l a g e n and l a m i n i n ) . Others have reported that HS does enhance the b i n d i n g between f i b r o n e c t i n and c o l l a g e n (Johansson et a l . , 1980). I t would appear, however, that HS i s , i n some way, r e l a t e d to adhesion as i t i s oft e n l o c a t e d i n footpads and adhesion s i t e s (Lark et a l . , 1984). HS has been shown to bind to r a t l i v e r c e l l s v i a i t s carbohydrate chains ( K j e l l e n et a l . , 1977) and to mouse mammary c e l l s v i a d i r e c t i n t e r c a l a t i o n of the p r o t e i n core i n t o the plasma membrane (Rapraeger et a l , 1985). Although i t has been shown, as s t a t e d , that HS does enchance b i n d i n g of g l y c o p r o t e i n s and c o l l a g e n to the c e l l surface i t has not been c l e a r l y demonstrated that i t ac t s as a c e l l receptor f o r these molecules (Culp et a l . , 1982). I t i s known t h a t , u n l i k e CS, which can be aggregated by HA v i a a b i n d i n g s i t e on the CS p r o t e i n , HS does not have such a b i n d i n g s i t e or, at l e a s t , does not aggregate around an HA molecule (Culp et a l . , 1982). HS has a l s o been i m p l i c a t e d i n kidney f u n c t i o n where i t i s found to be c e l l membrane as s o c i a t e d and a l s o present i n the glomerular basement membrane (Kanwar et a l . , 1979). I t i s b e l i e v e d to play an important r o l e i n p r o v i d i n g a s e l e c t i v e b a r r i e r to molecules t r y i n g to pass i n t o the kidney tubules. A HS molecule (MW 750,000) was i s o l a t e d from the basement membrane of kidney tumor t i s s u e ( H a s s e l l et a l . , 1980) and an antibody to - 19 - t h i s molecule ( t o the p r o t e i n core) demonstrated i t s presence i n EHS tumor basement membrane and i n normal kidney t i s s u e basement membranes (Kanwar et a l . , 1979). To summarize, the s u l f a t e d proteoglycans appear to p l a y a major r o l e i n c e l l - s u b s t r a t e adhesion, o r g a n i z a t i o n and c o n t i n u i n g s t a b i l i t y of the basement membrane and are present i n g r e a t e r amounts when c e l l s are beginning to d i f f e r e n t i a t e or at l e a s t to have stopped growing and / or mi g r a t i n g . The same could be s a i d of the proteoglycan HA. I t appears that proteoglycans i n v o l v e d i n f u n c t i o n s such as adhesion f o r some c e l l types are not i n v o l v e d i n the same f u n c t i o n i n other c e l l types: i e . , what one type of preteoglycan does f o r one c e l l type another proteoglycan does f o r another c e l l type or, some f u n c t i o n s may not be re q u i r e d by a l l c e l l s . I t would then seem important to analyse the f u n c t i o n s of proteoglycans w i t h i n a s p e c i f i c c e l l type and not assume that what one proteoglycan does i n one instance w i l l h o l d t r u e f o r a l l c e l l s . Mammary E p i t h e l i a l C e l l s In the area of mammary e p i t h e l i a l c e l l research i t has been reported by S i l b e r s t e i n and Dani e l s (1982) that growing mouse mammary e p i t h e l i a l c e l l s s y n t h e s i z e predominantly HA _in v i v o and i t i s found i n the basement membrane. The non-growing e p i t h e l i a l c e l l s synthesize predominantly s u l f a t e d GAG, which i s f u r t h e r i d e n t i f i e d by enzyme degradation to be CS. Parry et a l . (1985) analysed proteoglycan s y n t h e s i s by .mouse mammary e p i t h e l i a l c e l l s _in v i t r o . The c u l t u r e s , a l l nongrowing, show v a r i a t i o n s i n PG s y n t h e s i s depending on the type of sub s t r a t e on which they are maintained. They produce HA, CS 4, CS 6, DS and HS i n v a r y i n g amounts and i n d i f f e r e n t l o c a t i o n s ( c e l l , ECM and medium). Chandrasekaran et a l . (1979) f i n d that the normal human mammary c e l l l i n e , HBL-100, mantained on - 20 - a p l a s t i c s u b s t r a t e , synthesize mainly HA and i t i s present i n the medium. I t was not s t a t e d , however, whether these c e l l s are growing or at some l e v e l of d i f f e r e n t i a t i o n , t h e r e f o r e i t i s d i f f i c u l t to compare r e s u l t s of t h i s work w i t h the previous two. This e x e m p l i f i e s the problems encountered when comparisons are made between experiments. Furthermore, S i l b e r s t e i n and D a n i e l s (1982) looked at v i r g i n mouse mammary gland that had not been s t i m u l a t e d to d i f f e r e n t i a t e as were the mouse mammary c e l l s i n Parry et a l . ' s work. Whereas S i l b e r s t e i n and Daniels and Parry et a l . report proteoglycans i n an ECM, Chandrasekaran et a l . d i d not analyse an ECM f r a c t i o n . P arry et a l . f i n d that t h e i r mouse mammary e p i t h e l i a l c e l l s s y n t h e s i z e almost equal amounts of HS and CS and these are found i n an ECM while S i l b e r s t e i n and Daniels f i n d mainly CS i n the BM of the s o - c a l l e d " s t a b i l i z e d " non-growing c e l l s . However, Parry et a l . ' s c e l l s were exposed to hormones designed to f a c i l i t a t e d i f f e r e n t i a t i o n ( i n s u l i n , C o r t i s o l and p r o l a c t i n ) w h i le S i l b e r s t e i n and D a n i e l s ' mouse mammary glands were not. Another d i f f e r e n c e i n experimental design that may account f o r discrepency i n l i k e c e l l types and proteoglycan s y n t h e s i s i s whether the c e l l s are observed in_ v i t r o or i n . v i v o . I t has been demonstrated t h a t c e l l s i n c u l t u r e synthesize g r e a t e r amounts of proteoglycan and matrix components when compared to t h e i r counterparts i i i v i v o (Muir, 1977; Nevo et a l . , 1984). A v a r i a t i o n as s u b t l e as changing the feeding schedule from d a i l y to every other day has been shown to a l t e r the ECM components synthesized by c h i c k chondrocytes ( K a t a g i r i et a l . , 1981). So s t a n d a r d i z i n g experimental procedure i s very important. Proteoglycans and Malignancy I f proteoglycans p l a y an important r o l e i n normal c e l l f u n c t i o n i n g , as the many s t u d i e s c i t e d so f a r i n d i c a t e , might malignant c e l l s show a l t e r e d - 21 - proteoglycan s y n t h e s i s and composition? Studies began on proteoglycan s y n t h e s i s and malignancy i n the e a r l y 1970's. Since then, r e s u l t s have accumulated i n d i c a t i n g that malignant c e l l s do indeed have a l t e r e d proteoglycan s y n t h e s i s compared to normal counterparts, both i n the type and amount produced. The r e s u l t s , however, are extremely v a r i e d . In t h e i r study on human t h y r o i d t i s s u e , S h i s h i b a et a l . (1984) compared normal to adenoma and adenocarcinoma w i t h respect to proteoglycan s y n t h e s i s . Normal t i s s u e contains mainly HS ( S h i s h i b a et a l . , 1983) while malignant t i s s u e contains e i t h e r a mixture of HS (60%) and CS or DS (40$) or mainly CS and DS (90$). As w e l l , , i n the two "malignant t i s s u e samples examined one contains a 2-3 f o l d g r e a t e r amount of proteoglycans and the other 6-15 f o l d g r e a t e r when compared to the normal. Iozzo et a l . (1982) found i n normal human colon t i s s u e a l a r g e HS proteoglycan and a sm a l l e r DS proteoglycan i n about equal amounts. In colon carcinoma c e l l s (Iozzo, 1984) there i s l e s s HS synthesized and the predominant proteoglycan i s a sm a l l CS. The HS found i n the malignant c e l l s i s e i t h e r c e l l - a s s o c i a t e d or released i n t o the medium and has d i s t i n c t s t r u c t u r a l d i f f e r e n c e s from the normal t i s s u e HS, i n that the malignant HS i s of a la r g e hydrodynamic s i z e , higher bouyant d e n s i t y and has s h o r t e r GAG si d e chains than the normal synthesized HS. These d i f f e r e n c e s caused Iozzo to hypothesize that these HS macromolecules may be re s p o n s i b l e f o r a l t e r e d surface p r o p e r t i e s seen on n e o p l a s t i c c e l l s ( I o z z o , 1984). The l i s t of a l t e r e d proteoglycan s y n t h e s i s goes on - an increase i n CS i n human lung carcinomas ( H a t a l et a l . , 1977) and human hepatoma (Kojima et a l . , 1975) has been found. The a d d i t i o n of CS to i n v i t r o and i n v i v o human mammary carcinoma c e l l s (Takeuchi, 1965; O z z e l l o et a l . , I960) show growth-stimulating r e s u l t s . The a d d i t i o n of chondroitinases ( to degrade CS) can r e t a r d the growth of these c e l l s as - 22 - w e l l (Takeuchi et a l . , 1972). Toole et a l . (1979) f i n d t hat r a b b i t V 2 carcinoma c e l l s grown i n r a b b i t s have 3 to 4 times as much HA synthesized at the i n t e r f a c e between tumor mass and connective t i s s u e than do the same c e l l s i n j e c t e d i n t o nude mice. In r a b b i t s these V 2 carcinoma c e l l s are i n v a s i v e while i n nude mice they are not. These same f i n d i n g s occur regardless of the s i t e chosen f o r i n j e c t i o n of the c e l l s . They conclude that HA provides an environment conducive to i n v a s i o n of the carcinoma c e l l s i n t o surrounding t i s s u e s . They propose that the HA enables c e l l s to migrate by e x e r t i n g f o r c e v i a increased s w e l l i n g i n t i s s u e s to open pathways along c e l l - c o l l a g e n l a y e r s . Chandrasekaran et a l . (1979) analyzed proteoglycan s y n t h e s i s by two human breast carcinoma c e l l l i n e s , MCF-7 and MDA-MB-231, and i t s l o c a t i o n e i t h e r i n the medium or c e l l ( i n t r a c e l l u l a r or c e l l - a s s o c i a t e d ) . They reported t h a t the predominant proteoglycans synthesized are CS and HS w i t h HA being a very minor (0-12$) component and the m a j o r i t y of i t (HA) present i n the medium. This i s opposite to the f i n d i n g s they reported f o r the normal c e l l l i n e (HBL-100) under the same c o n d i t i o n s (Chandrasekaran et a l . , 1979). In that case, HA i s the predominant GAG (90$) w i t h very l i t t l e d e t e c t a b l e HS (3$). They d i d not f i n d a d i f f e r e n c e i n the amount of proteoglycan synthesized per c e l l between normal (HBL-100) and the malignant (MDA-MB-231) c e l l s . T his i s co n t r a r y to the other r e p o r t s ( S h i s h i b a et a l . , 1984; Angello et a l . , 1982) that do f i n d an increase above normal i n proteoglycan s y n t h e s i s i n malignant t i s s u e . The MCF-7 c e l l l i n e a c t u a l l y s y nthesizes much l e s s than the two other l i n e s (MDA-MB-231 and HBL-100) . Angello et a l . (1982) looked a t two sub-populations of a mouse mammary c e l l l i n e . The -SA c e l l s (no growth i n s o f t agar) are non-agressive i n vi v o (slow growth) while +SA c e l l s (growth i n s o f t agar) are aggressive and - 23 - grow r a p i d l y i i i v i v o w i t h a short tumor l a t e n c y p e r i o d . Both appear m o r p h o l o g i c a l l y s i m i l a r . I t i s found that +SA c e l l s i n c u l t u r e i n c o r p o r a t e 8 times more l a b e l l e d glucosamine than do the -SA c e l l s and the major i d e n t i f i e d proteoglycan i s HA. Although both sub-populations have approximately the same percentage of t o t a l PG l o c a t e d i n the 3 f r a c t i o n s analysed (medium- 12 to 15$, c e l l suface - 50-58$, c e l l - 30-34$) the +SA synthesizes 57-7$ HA and only 29.9$ HS while -SA synthesizes 40$ HA and 46.3$ HS. They conclude that e p i t h e l i a l c e l l s are able to c o n d i t i o n t h e i r environment and t h a t the c o n d i t i o n i n g w i t h increased HA encourages p r o l i f e r a t i o n . Other s t u d i e s have a l s o shown increased HA i n mammary adenocarcinomas (Palmer et a l . , 1979; Takeuchi et a l . , 1976). Takeuchi et a l . (1976) examined the proteoglycan content of eleven human breast tumors. They f i n d t h a t i n s i x of the cases HA i s the predominant GAG (between 25 and 70 yg/mg dry wt.) and HS i s the l e a s t ( l e s s than 2 ug/mg dry wt.). The c h o n d r o i t i n s u l f a t e s are next i n amount followed by DS. I n the other 5 cases HA, CS, and DS are r e l a t i v e l y equal and lower, (under 10ug/mg dry wt.). Again HS i s lowest, ( l e s s than 2 ug/mg dry wt.). Angello et a l . (1979) suggest t h a t sub-populations i n heterogenous tumors may e s t a b l i s h a matrix t h a t not only promotes t h e i r growth but a l s o the growth of other sub-populations. As w i t h normal c e l l s , the d i s c r e p a n c i e s seen i n the l i t e r a t u r e regarding PG s y n t h e s i s and malignancy may be r e l a t e d to s e v e r a l f a c t o r s . Some r e s u l t s are reported from experiments c a r r i e d out on whole t i s s u e r e c e n t l y removed from a p a t i e n t ( S h i s h i b a et a l . , 1983; Takeuchi et a l . , 1976) . Others are based on c e l l s i n c u l t u r e and the m a j o r i t y of these are c e l l l i n e s ( l o z z o 1984; Angello et a l . , 1982; Chandrasekaran et a l , 1979). As reported e a r l i e r i n the I n t r o d u c t i o n , normal proteoglycan s y n t h e s i s appears - 24 - to be s p e c i f i c to c e l l type. The same may be true f o r malignant c e l l s . The type of proteoglycan produced by a malignant c e l l may also be related to i t s degree of d i f f e r e n t i a t i o n and invasiveness or metastatic p o t e n t i a l . For example, malignant c e l l s i n the process of rapid growth may synthesize one major proteoglycan (Toole et a l . , 1979) while c e l l s that have "tr a v e l l e d " to a new s i t e may wish to adhere and " s e t t l e down" and may synthesize a different proteoglycan (lozzo, 1984). Numerous researchers (lozzo, 1984; L i o t t a et a l . , 1979; Bauer et a l . , 1979) have pointed out that i t may be the stromal tissue surrounding the tumor c e l l s that i s stimulated to synthesize various altered matrix components. Interestingly, the small CS molecule associated with the colon carcinoma c e l l was discovered by autoradiographic techniques not to be produced by the colon c e l l s but rather synthesized by the mesenchymal c e l l s i n the surrounding CT stroma (lozzo et a l . , 1982). Alternately, these stromal c e l l s may be synthesizing a factor designed to increase or decrease synthesis of matrix components by the tumor c e l l s . David et a l . (1981, 1982) discovered that the reason the transformed NMuMG c e l l s (mouse mammary e p i t h e l i a l c e l l l i n e ) they studied does not accumulate a proteoglycan-rich BL on collagen substrates i s because they are unable to decrease proteoglycan degradation. The normal NMuMG c e l l s incorporate proteoglycan into a d i s t i n c t basal lamina and have the same synthesis rate as the transformed c e l l s . However, when these same normal c e l l s are cultured on a p l a s t i c substrate, they become unable to decrease PG degradation. An obvious problem associated with studies of malignant c e l l types and proteoglycan synthesis i s the lack of data on normal tissue to be used as a comparison, coupled with the fact that proteoglycans synthesized by different c e l l s do not appear to influence the same functions i n a l l c e l l s . - 25 - Thesis Problem Formulation Due to the obvious i n f l u e n c e of PGs on c e l l f u n c t i o n , i t seemed appropriate to determine the type and l o c a t i o n of proteoglycans synthesized by normal human mammary e p i t h e l i a l c e l l s i n c u l t u r e during the exponential and s t a t i o n a r y phases of growth and how they r e l a t e to normal c e l l f u n c t i o n . This i s not only important i n understanding the normal c e l l - p r o t e o g l y c a n r e l a t i o n s h i p but a l s o necessary f o r e s t a b l i s h i n g i f a l t e r a t i o n s occur i n the malignant c e l l . Normal human mammary e p i t h e l i a l c e l l s were obtained from r e d u c t i o n mammoplasties. The t i s s u e was processed to s e l e c t f o r e p i t h e l i a l c e l l s and the c e l l s were grown on c o l l a g e n g e l s , as i t has been demonstrated that e p i t h e l i a l c e l l s grow b e t t e r on a substrate derived from components of ba s a l lamina or stromal t i s s u e than on a p l a s t i c s u b s t r a t e (Emerman and P i t e l k a , 1977; Hay, 1981; Kleinman et a l . , 1981; Michalopoulous et a l . , 1975; Richards et a l . , 1982; Wicha et . a l . 1979, 1982; Yang et a l . , 1979). 3 The proteoglycans were l a b e l l e d w i t h H-glucosamine added to the medium 24 hours p r i o r to t e r m i n a t i o n . Cultures were terminated a t . growing and nongrowing stages as determined by DNA a n a l y s i s . A n a l y s i s of proteoglycans was accomplished by i d e n t i f i c a t i o n of the glycosaminoglycan s i d e chains. The proteoglycans were t r e a t e d w i t h a n o n - s p e c i f i c protease to cleave the carbohydrate s i d e chains from the p r o t e i n core and the l a t t e r was discarded. The GAG p o r t i o n was i d e n t i f i e d by enzymes s p e c i f i c to each and by c e l l u l o s e acetate e l e c t r o p h o r e s i s (Parry et a l . , 1985; Angello et a l . , 1982; Kanwar et a l . , 1982; Crawford et a l . , 1984; Iozzo, 1984; Sh i s h i b a et a l . , 1984). The i n f o r m a t i o n gained from these experiments on normal human mammary e p i t h e l i a l c e l l s i n c u l t u r e provides a b a s e l i n e w i t h which to compare the - 26 - e f f e c t s of va r i o u s f a c t o r s on GAG synth e s i s and f u n c t i o n . Such f a c t o r s i n c l u d e a) hormones b) sub s t r a t e s c) d i f f e r e n t i a t i o n d) receptors and e) malignancy. Of p a r t i c u l a r i n t e r e s t i s the e f f e c t s of va r i o u s hormones c o n t r o l l i n g mammary growth and d i f f e r e n t i a t i o n on proteoglycan s y n t h e s i s . Very l i t t l e i n f o r m a t i o n p e r t a i n i n g to t h i s area can be found i n the l i t e r a t u r e ( K i d w e l l et a l . , 1982). The a d d i t i o n of va r i o u s proteoglycans (GAGs) e i t h e r to the medium or incorporated i n t o a substrate would render i n f o r m a t i o n regarding the e f f e c t s of s p e c i f i c GAGs on c e l l f u n c t i o n . For example, would c r e a t i n g a sub s t r a t e r i c h i n the proteoglycan found i n non-growing c e l l s cause growing c e l l s to stop growing or would growth be sti m u l a t e d i n quiescent c e l l s i f they were on a substrate r i c h i n the proteoglycan found synthesized by growing c e l l s ? Would adding the proteoglycan (GAG) to the medium be as e f f e c t i v e as i t s presence i n a substrate? Another l a r g e area open f o r i n v e s t i g a t i o n i s that of GAG re c e p t o r s . The presence of GAG i s defined i n t h i s t h e s i s as being medium, ECM ( i n c l u d e s c e l l - a s s o c i a t e d ) or c e l l ( i n t r a c e l l u l a r ) l o c a t e d . I t would help i n the understanding of GAG f u n c t i o n to know which GAGs are c e l l - a s s o c i a t e d and how ( v i a r e c e p t o r s , i n t e r c a l a t e d by the p r o t e i n core) and which are basement membrane a s s o c i a t e d . F i n a l l y the r e s u l t s of t h i s study could be used as a b a s e l i n e w i t h which to compare r e s u l t s o f malignant human mammary c e l l s grown under the same c o n d i t i o n s . I t would enable some conclusions to be drawn regarding the e f f e c t s of a l t e r e d GAG syn t h e s i s on the growth and maintenance of the a l t e r e d phenotype. MATERIALS AND METHODS Dissociation Procedure Tissue was obtained from reduction mammoplasties. A box was delivered to the operating room containing 3 to 4 250 ml cups each containing 100 ml of sterile transport medium (see Appendix l ) on ice. Samples were brought back to the culture room where, under ste r i l e conditions, excess fat was removed from the glandular portions using a scalpel and scissors. The fat was discarded and the glandular portions were minced finely using 2 scalpel blades then placed in a 250 ml flask containing 50 ml of dissociation medium (Appendix 2). The flask was then placed i n a 37°C incubator and kept sti r r i n g for approximately 22 h. Of the three samples used for this thesis, one was dissociated after 18 h, one at 21 h and one at 22 h. The dissociation was considered complete when only small aggregates of cells remained. The solution was then divided equally i n 4 15 ml centrifuge tubes and centrifuged in a c l i n i c a l centrifuge (Fisher Scientific) for 4 min at 800 rpm (80xg). This centrifugation speed was designed to preferentially pellet the epithelial cells present in the sample. The supernatant was discarded and the pellets were combined and resuspended i n 100 ml of DME. The tubes were centrifuged again using the c l i n i c a l centrifuge for 4 min at 1000 rpm (lOOxg). The supernatants were removed and the pellets were washed a second time in DME. The washes removed any remaining collagenase. After a f i n a l centrifugation as above, the supernatant was discarded, and the pellet was resuspended in 5 ml of growth medium (Appendix 3) and put through a 150um Nitex f i l t e r to decrease clumping of ce l l s . For c e l l counting, 0.1 ml of the c e l l suspension was removed and placed in a 2 ml tube. The remaining c e l l suspension was placed i n a 37°C water bath while counting took place. A minute drop of £ 7 - 28 - trypan blue (pH 1.2) was' added to the 0.1ml solution to distinguish viable ce l ls from dead c e l l s . Counting was done using a hemocytometer . If the total number of cel ls was greater than that required for the experiment, the extra ce l ls were pelleted and resuspended i n freezing medium (Appendix 4) at a concentration af approximately 1 X 10 • cel ls / ml. They were quick frozen and immediately transferred and stored i n a cryogenic tank at -70°C (Union Carbide). Preparation of Collagen Gels The collagen solution was prepared from rat t a i l s i n the following manner. The t a i l s were placed i n 9 5 $ alcohol for 15 min. The tendons were dissected out and teased apart using scalpel blades and forceps, weighed and placed i n a 60 mm Petri dish containing s ter i le d i s t i l l e d water and exposed to the ul traviolet l ight i n the tissue culture hood for 24 h. The fibers were then suspended i n a dilute acetic acid solution (1:1000) and st irred at 4°C for 48 h. They were l e f t , to s i t for another 24 h at 4°C. F inal ly , the solution was transferred into 50 ml centrifuge tubes and centrifuged i n a Sorvall centrifuge at 10,000xg for 30 min. The supernatant, the collagen solution, was bottled and stored at 4°C. To prepare the gels, a solution of medium 199 (10X concentration) was combined with 0.34N NaOH i n a ratio of 2:1 to make a total of 0.4 ml of solution. This was added to 1.6 ml of the collagen solution and stirred together i n a tube on ice to prevent premature g e l l i n g . The solution was then transferred to a 3 5 mm Petri dish. When 16 mm wells were used, the gels were prepared by combining 0.1 ml solution of the Medium 199 and 0.34N NaOH (2:l) and 0.4 ml of the collagen solution. The culture dishes were placed i n an incubator (National, Inc.) at 37°C, 5$ CO2 and 9 5 $ a i r for one - 29 - h to allow complete gell ing of the solution. After that time, 2 ml of experimental medium was added to the 3 5 mm Petri dishes and 0 .5 ml to the wells for 2 4 h prior to the experiment to allow equil ibration. C e l l Culture Procedure 5 2 I n i t i a l cultures were seeded at of 2 - 2 . 5 X 10 cells/cm onto the hydrated collagen gels i n 3 5 mm Petri dishes on wells. Identical cultures for DNA determination were grown concurrently with the cultures destined for glycosaminoglycan analysis. The cultures were incubated at 37 ° C , 5 $ CO2 and 9 5 $ a i r and observed dai ly . The medium was changed every second day at the same time during the day. With daily observation under the phase contrast microscope, i t was determined at what point the cultures were approximately 50$ confluent and 100$ confluent. The former were identif ied as growing and the lat ter as stationary cultures by DNA analysis. The f i r s t stage was attained between day 3 and 4 , the second between days 9 -11, depending on the growth rate of each particular culture. A third stage, between- days 17-18, was identif ied as late stationary. Morphologically, the cultures appeared 85$ e p i t h e l i a l . At 3 these times, the cultures was labelled with H-glucosamine i n growth medium (S.A.=30-60 MCi/mmol; NEN, DuPont Canada, Inc . ) . The growth medium contained only insul in (5wg/ml) and 5$ PCS (necessary for growth). No hormones were added to stimulate differentiat ion. 5 Cultures were also seeded at a higher density of 5«0 x 10 2 cells/cm . These cultures were identif ied as stationary at day 4 by DNA analysis. Radiolabelling Procedures A l l experiments were labelled with H-glucosamine at 100 yCi/ml i n 35 1.5ml of growth medium. Two experiments were labelled with S-sulphate - 30 - at 100 viCi/ml (NEN, carrier-free) instead of H-glucosamine. This was done as an alternate means of identifying synthesized sulphated GAG. A l l label l ing was done for a 24 h time period. Resolution into Fractions - Medium, Cel l and Extracellular Matrix After label l ing the medium was removed from the culture and stored on ice i n a 15 ml centrifuge tube. The gel was placed i n a 1.5 ml Eppendorf tube (VWR S c i e n t i f i c , Inc.) and centrifuged i n a mirocentrifuge (Western Sc ient i f i c Services) at 13,000xg for 5 min at 40°C. This was done to squeeze any medium out of the i n t e r s t i t i a l spaces. This medium was then removed and added to the original medium. The gel was then rinsed i n approximately 1 ml of Tris -sal ine solution, pH 7«4 (Tris lOmM, 0.85$ NaCl) for 30 min to remove additional trapped medium. This tube was also kept on ice during the time period and vortexed every few minutes. The gel was then spun down at 13,000xg for 15 min i n the microcentrifuge at 4°C. The supernatant was added to the original medium tube. To lyse the c e l l s , the gels were extracted on ice with 1 ml of detergent solution (Appendix 5) and vortexed occasionally over a 30 min period. The gels were then centrifuged a f i n a l time at 13,000xg for 15 min at 4°C. The supernatant constituted the soluble c e l l fraction while the pellet was called the extracellular matrix fract ion. They were separated and placed into 15 ml centrifuge tubes and stored on i ce . Extraction of Glycosaminoglycans To each of these three fractions was added a nonspecific protease solution (Sigma, type XIV, Streptomyces griceus) at a concentration of lOmg/ml i n lOmM T r i s , pH 7.4. One ml of protease solution was added for - 31 - every 2 ml of fraction sample. A drop of sodium azide (0.01$ w/v) was added to each tube to prevent bacterial growth. A l l fractions were then incubated i n a water bath at 38°-41°C for 48 h . Fresh protease was added after 24 h of incubation. Some samples were l e f t for 60-72 h. These were compared to those that were incubated for the 48 h time period to ensure no further enzyme action took place after that time. Digests were precipitated with 10$ trichloroacetic acid (Sigma) for 1 h at 4°C. They were then centrifuged using a tabletop centrifuge (Western Scient i f ic Services) at 200-250 Xg for 10 minutes. The supernatants were saved and the pellets discarded. The supernatants were divided into 1.5 ml Eppendorf tubes (VWR Scientif ic ) with each tube receiving 0.3 ml of the solution. The tubes were clearly marked so as not to mix the 3 fractions (medium, c e l l and ECM). A GAG mixture was prepared containing Img /ml of each of the following GAGs: hyaluronic acid (Sigma, umbilical cord), chondroitin sulfate type A (Sigma, whale cartilage) , type B (Sigma, shark cartilage) and heparin (Sigma, porcine intest inal mucosa). Twenty-five ul of this mix was added to each Eppendorf tube. This acted as a cold carrier to help "bring down" labelled GAG. Each tube was diluted with 3 volumes of 90$ ethanol/l.4$ potassium acetate to give a f i n a l volume of 1.5 ml. The ethanol/potassium acetate solution acted to help precipitate labelled GAG. Each tube was vortexed and allowed to s i t for 24 h at - 2 0 ° C . Precipitated GAGs were collected by centrifuging a l l Eppendorf tubes for 30 minutes i n the microcentrifuge at 4°C and 13,000xg. The supernatants were discarded and the pellets resuspended i n 0.3 ml of glucosamine (0.1 mM) i n water using a Pasteur pipet. ' The tubes were then allowed to s i t for 1 h at 4°C to ensure that the pellet completely dissolved. Afterwards - 32 - another 3 volumes of 90$ ethanol/l.4$ potassium acetate was added to each tube to bring the total volume to 1.5 ml. The procedure described above was repeated, that i s , a l l tubes were vortexed and stored for 24 h at - 2 0 ° C . The whole precipitation procedure was carried out 3 times i n t o t a l . After the f i n a l centrifugation, the pellets from one fraction (up to 12 Eppendorf tubes may contain pellets from one fraction) were pooled and the total sample of GAG was dissolved i n 1.5ml of d i s t i l l e d water. The samples were marked MEDIUM, CELL or ECM. A l l were stored for at least 24 h at -20°C before the identif icat ion of GAG procedure began. Analysis of GAG by Enzyme Digestion ( lhyaluronic Acid Identification Streptomyces hyaluronidase (500 units/ml, Sigma) was dissolved i n 1 ml of 50 mM sodium acetate at pH 5 « 0 . The assay was carried out i n 1.5 ml Eppendorf tubes by adding 100 y l of sample, 30 ul of enzyme and 15 Ml of NaCl (1.5 M). A l l samples were run i n t r i p l i c a t e . The samples plus enzyme were incubated at 40°C for 4 h. The enzyme was then inactivated by boi l ing i n water at 100°C for 15 min. The samples were allowed to cool completely. A 25 Ml aliquot of the GAG mix (lmg/ml of each GAG) was added to each sample followed by 1 ml of 90$ Et0H/l.4$ KAC. Each tube was then vortexed and allowed to s i t overnight at - 2 0 ° C . A l l tubes were then centrifuged (microcentrifuge, 13,000xg, 30 min, 4°C) and the supernatants and pellets separated and counted (to be described under s c i n t i l l a t i o n counting). One tube containing the pellet from each sample (MED., CELL, ECM) was kept for the next assay. - 33 - MED. Initial Sample CELL (1.5 ml) ECM HA Enzyme Assay HS Enzyme Assay divided into Divided into Pellet =undigested GAG so not HA / \ Supernatant =digested GAG identified as HA Pellet' undigested GAG so not HS Supernatant =undigested GAG identified as HS (Supernatant discarded) Pellet resuspended CS ABC Enzyme Assay CS AC ErLZ"<nne Assav Pellet undigested GAG so not CS ABC Supernatant =digested GAG so identified as CS 4,6 or DS Pellet =undigested GAG so not CS 4 or 6 Supernatant =digested GAG so identified as CS 4+6 Fig. 5 Flow Chart of Identification of GAG by Enzymes - 34 - (2) Chondroitin S u l f a t e I d e n t i f i c a t i o n Two enzymes were used to i d e n t i f y c h o n d r o i t i n s u l f a t e (Yamagata et a l . , 1968): ( i ) chondroitinase ABC (Sigma, 10 units/ml) - degrades a l l CS ( i i ) c hondroitinase AC (Sigma, 10 units/ml) - degrades CS 4 and 6 (not DS) Because chondroitinase ABC w i l l a l s o d i g e s t HA, the samples used f o r these assays must have HA removed f i r s t . Therefore, the p e l l e t from one of the t r i p l i c a t e tubes from the HA enzyme assay f o r each f r a c t i o n was resuspended i n 400 Ml of d i s t i l l e d water. These were used f o r the CS assays. Both enzymes (CS ABC, CS AC) were d i s s o l v e d s e p a r a t e l y i n d i s t i l l e d water (Sigma, 10 u n i t s / m l ) . The assay was c a r r i e d out i n 1.5 ml Eppendorf tubes by adding 60 Ml of the resuspended p e l l e t from the HA samples (MED., CELL, ECM), 60 Ml of the enzyme and 20 Ml of "enriched" T r i s (Appendix 6) at pH 8.0. A l l samples were done i n d u p l i c a t e . A l l samples were incubated at 37°C f o r 4 h a f t e r which time they were b o i l e d i n water at lOO^C f o r 15 min to i n a c t i v a t e the enzyme. • A f t e r c o o l i n g completely, 25 Ml of GAG mix (img/ml of each GAG) was added to each tube followed by 1 ml of 90$ Et0H/l.4$ KAC. A l l tubes were vortexed and allowed to s i t overnight at -20°C. The next day a l l tubes were c e n t r i f u g e d i n the m i c r o c e n t r i f u g e at 13,000xg f o r 30 min at 4°C and the supernatants and p e l l e t s separated and counted (to be described under s c i n t i l l a t i o n c o u n t i ng). (3)Heparan S u l f a t e I d e n t i f i c a t i o n One hundred Ml of n i t r o u s a c i d (prepared by adding 0.3 M sodium n i t r a t e - 35 - and 2.9 M acetic acid) was added to 50 ul of the i n i t i a l sample of MED., CELL and ECM fractions i n 1.5 ml Eppendorf tubes. A l l samples were done i n duplicate. After incubating the tubes at room temperature for 80 min, the reaction was stopped by the addition of 50 ul of ammonium sulfamate (2 M) to each tube and incubated at room temperature for another 30 min. One ml of 90$ EtOH/l.4$ KAC was added to each of the tubes which were then vortexed and stored overnight at -20°C. The tubes were then centrifuged (microcentrifuge, 13,000xg, JO min at 4°C) and the supernatant and pellets separated and counted. S c i n t i l l a t i o n Counting Supernatants were placed i n 20 ml glass s c i n t i l l a t i o n via ls (NEN Products) and 15 ml of s c i n t i l l a t i o n f l u i d (Aquasol, NEW) was added. Pellets were dissolved i n 200 ul of d i s t i l l e d water and placed i n 6 ml plastic s c i n t i l l a t i o n via ls (NEN Products) to which 5.8 ml of s c i n t i l l a t i o n f l u i d was added (Aquasol, NEN). A l l v ia ls were counted i n a Mark II S c i n t i l l a t i o n Counter for 4 min. A quench curve was constructed from six 3 known samples containing H-solutions with various amounts of quench. These samples were commercially purchased (Amersham/Searle, Ltd . and NEN, DuPont Canada, Inc . ) . A control containing only s c i n t i l l a t i o n f l u i d was read with every set of v ia ls to indicate background radioactivity. The s c i n t i l l a t i o n counter delivered counts only i n CPM (counts per minute) and as the amount of quenching was variable i n the samples, the CPMs were converted to DPMs (disintegrations per minute) to remove the variable 35 14 quench as a factor i n the results . S results were read using a C quench curve and standards commercially purchased (Amersham/Searle, L t d . ) . - 36 - j > i !_ 2 4 6 8 T ime ( h o u r s ) F i g . 6 Enzyme d i g e s t i o n o v e r t i m e . A l l enzymes were i n c u b a t e d w i t h a known sample and t h e optimum t i m e f o r maximum d e g r a d a t i o n of t h e s u b s t r a t e was chosen f o r subsequent a s s a y s . - 37 - Calculation of Assay Results The supernatant contained the amount of GAG which was degraded by the par t i c u l a r enzyme used and the pe l l e t contained the amount of GAG which was not degraded by the enzyme. The ra t i o of the degraded to non-degraded was calculated as a percentage of the t o t a l and i d e n t i f i e d as the GAG attacked by that pa r t i c u l a r enzyme. A l l ratios were compared to standard samples which were run concurrently with a l l samples and contained a l l additives except the enzyme, which was substituted by an equal amount of d i s t i l l e d water. I n i t i a l l y , enzyme assays were carried out over various incubation times (Fig. 6 ) and the times chosen for subsequent assays were those that permitted maximum degradation. To ensure that the enzymes were degrading the GAG i n question, a sample of each GAG at Img/ml was spotted on a Whatman f i l t e r paper and stained with Alcian blue i n water, pH 2.5. Enzyme was then added to each known sample and the incubation times carried out as described. Afterwards a second spot was placed on a f i l t e r paper and stained with Alcian blue. A positive result was obtained when the second spot did not s t a i n , indicating the degradation of that GAG had occurred. This procedure was also carried out between a l l known standards and a l l enzymes to determine the s p e c i f i c i t y of each enzyme. I t was noted that streptomyces hyaluronidase was s p e c i f i c to HA, chondroitinase ABC degraded HA, CS 4+6 and DS, chondroitinase AC degraded CS 4+6 and HA (s l i g h t decrease i n staining after enzyme) and nitrous acid degraded only HS. A l l enzyme assays were carried out i n duplicates or t r i p l i c a t e s . - 38 - Id e n t i f i c a t i o n of GAG Using Electrophoresis The GAG present i n each sample was also i d e n t i f i e d by cellulose acetate electrophoresis. Cellulose acetate s t r i p s (Gelman Sciences, Inc.) were soaked f o r 20 min i n 100 ml of 0.15 M zinc acetate, pH 5.8, blotted dry and marked at one end as "standard" or "sample". They were then placed i n an electrophoretic unit (Gelman Sciences, Inc.) containing 1 l i t e r of 0.15 M zinc acetate, pH 5.8, so that both ends were i n contact with the buffer. At the midpoint of the s t r i p , 5 Ml of sample and 3 Ml of standard solution containing 0.5 mg/ml of each GAG were applied widthwise across the s t r i p with a few centimeters separating the two. The output was from a constant power supply apparatus (LKB) and was set at 2 mA/strip and allowed to run for 3 h. After the a l l o t t e d time, the s t r i p s were removed and placed i n a solution containing 500 ml of 1% Alcian blue, 5% acetic acid and 10$ EtOH for 0.5 h. The s t r i p s were then rinsed overnight i n 5% acetic acid and water and dried f l a t . A l l standards were determined by running known quantities of each separate GAG and staining as outlined. The stained areas, corresponding to the known standards, were caref u l l y cut out and placed i n a 20 ml s c i n t i l l a t i o n v i a l with 10 ml of Aquasol (NEN, DuPont Canada, Ltd.) and counted i n a MARK I I S c i n t i l l a t i o n Counter. Film Developing for Autoradiography Several samples of each f r a c t i o n (MED., CELL, ECM) separated and i d e n t i f i e d using the electrophoretic and staining procedures described above where further i d e n t i f i e d by autoradiography. In the darkroom, s t r i p s and x-ray f i l m were secured by tape' and held f l a t between two pieces of glass. These were then covered i n aluminum f o i l and stored i n a l i g h t - r e s i s t e n t bag and allowed to s i t i n a cool dark place for up to 6 wk - 39 - undisturbed. The strips from the nongrowing cultures were developed after 2 wk. Leaving these longer (up to 4 wk) did not improve the autoradiographs. However, the strips from growing cultures could not be developed before 6 wk. Even at 6 wk, the autoradiographs were fa int . The stationary culture strips were found to contain 5-fold higher counts i n each band representing a GAG then growing culture s t r ips . A minimum of 1000 counts per 5 Ml sample applied was necessary to affect the H-sensitive f i l m . The fi lm was developed i n developer (Picker International) i n d i s t i l l e d water (1:4) for 4 min, stopped i n 5% acetic acid i n water and fixed (Kodak Rapid Fix) for 10 min. The f i lm was then rinsed for 3 0 min i n running water and allowed to dry overnight. DNA Determination Cultures for DNA assay were frozen prior to DNA analysis to f a c i l i t a t e membrane lys ing . Thawed gels were sonicated i n 1 ml of Na^HPO^ buffer, pH 7«4 (Appendix 7) for 3 min. Afterwards, a 300 Ml aliquot was removed, combined with 150 Ml of a Horchst dye solution (1 mg/ml of water) and vortexed to mix thoroughly (Labarca et a l . , 1980). A l l assays were done i n t r i p l i c a t e . The solutions were read immediately i n a fluorospectrophotometer (American Instrumentation Co.) with an activating wavelength of 356 nm and a fluorescence wavelength of 458 nm. The results were compared with standards ranging from 10 - 100 Mg of DNA using a calf thymus DNA stock (Sigma) at 100 Mg/ml. Calculations of c e l l number were based on the fact that 7pg = 1 mammary epi thel ia l c e l l (Kraenbuhl et a l . , 1981). - 40 - Electron Microscopy Cultures for electron microscopy were prepared by removing the medium and washing each gel 3 times with Karnovsky's solution, (Appendix 8) then l e f t for 1 h i n th i s solution. After t h i s , the gels were washed 3 times i n 0.1 M sodium cacodylate, pH 7.4, and stored i n t h i s solution at 4°C u n t i l embedding. The embedding, sectioning and photography were carried out by Mrs. Hella Prochaska. RESULTS Growth Normal human mammary e p i t h e l i a l c e l l s were seeded onto c o l l a g e n 5 2 gel-coated 35 mm P e t r i dishes a t 2-2.5 X 10 c e l l s /cm . They were maintained i n c u l t u r e f o r 17-18 days. E a r l y i n c u l t u r e , days 3 - 4 , these c e l l s formed sub-confluent monolayers w i t h l i t t l e evidence of c e l l s t r a t i f i c a t i o n . L a t e r i n c u l t u r e , at days 9 - 11, the c e l l s were confluent and patches of s t r a t i f i e d c e l l s were evident. By days 17 - 18, the co l l a g e n g e l s had r e t r a c t e d from the c u l t u r e d i s h and contracted to approximately 10-15$ of t h e i r o r i g i n a l s i z e and the c e l l s could not be v i s u a l i z e d by phase-contrast microscopy. Growth s t u d i e s (Figure 7 ) show th a t days 3 - 4 c u l t u r e s were i n an a c t i v e phase of growth. Cultures terminated on days 9 - 1 1 were i n a s t a t i o n a r y growth phase, i n d i c a t e d by those terminated on days 17 -18. Cultures v a r i e d by 2 - 3 days i n the time i t took them to reach sub-confluent (50$) and confluent s t a t e s , as a l l c u l t u r e s d i d not grow a t i d e n t i c a l r a t e s . To determine i f high d e n s i t y i n h i b i t e d growth i n c u l t u r e , s e v e r a l 5 2 c u l t u r e s were seeded at 5 X 10 c e l l s / c m . No growth had occurred i n these c u l t u r e s by day 4 and the c e l l number was comparable to that of low de n s i t y c u l t u r e s terminated a t day 9 - 11. I t was concluded that c u l t u r e s seeded a t high d e n s i t y appeared to be i n a s t a t i o n a r y growth phase. • T o t a l Glycosaminoglycans Synthesis C u l t u r e s of human mammary e p i t h e l i a l c e l l s incubated w i t h H- glucosamine were terminated at 3 time p o i n t s . Table 2 shows the Ml - 42 - 2.0 - 3-4 9-11 17-18 T ime ( d a y s ) F i g . 7 Growth study of d u p l i c a t e c u l t u r e s of normal human mammary e p i t h e l i a l c e l l s seeded a t low d e n s i t y . Day 3-4 was c o n s i d e r e d growing; day 9-11 and 17-18 were c o n s i d e r e d s t a t i o n a r y . C e l l number was determined by DNA assay. E r r o r s b a r s i n d i c a t e SEM. (N = 3) - 43 - percentages of total GAG synthesized present i n the medium, c e l l and ECM fractions during these time points. While the ce l ls were prol iferat ing (days 3 - 4 ) the majority of synthesized GAG was found i n the medium fract ion. The ECM contained approximately 1/5 of the total GAG synthesized at this stage of growth. When the cultures reached a confluent state and were no longer growing ( Table 2 - early stationary - days 9 - 11 i n culture and late stationary - days 17 - 18 i n culture ), the ECM contained approximately 1/2 of a l l the synthesized GAG. To determine i f the differences seen i n local izat ion and type of glycosaminoglycan synthesized by cultures of human mammary epi thel ia l ce l l s were due to their growth status and not a phenomenon of time i n culture, 5 2 two cultures were seeded at high density (5 X 10 cells/cm ) and terminated on day 4. The cultures, which were not growing on day 4, showed a pattern of GAG distr ibution similar to that of the nongrowing cultures (days 9 - 1 1 and later) seeded i n i t i a l l y at low density (Table 2). This suggests that GAG local izat ion was dependent on growth phase rather than time i n culture. When total GAG synthesized i n each fraction i s expressed per c e l l (Figure 8 ), the growing cultures showed the ce l ls were releasing the majority of the GAG into the medium, a 4 - fold increase over the amount found i n the ECM. By the early stationary growth phase both medium and ECM showed greater amounts of synthesized GAG / c e l l indicating that more overall GAG was synthesized at this stage. The GAG / c e l l found i n the medium was less than 1-fold greater than that found i n the medium of the growing culture.' In contrast, synthesized GAG / c e l l i n the ECM i n early stationary cultures was 8 - fold greater than that of growing cultures. Also by the stationary phase the ECM had more synthesized GAG / c e l l than did the medium. The late stationary cultures (days 17 - 18) showed the - 44 - Dis t r i bu t i on of 3H-glucosamine l abe l l ed glycosaminoglycans In normal human mammary ep i t h e l i a l c e l l s in cu l tu re . Culture Conditions Medium Ce l l ECM % incorporat ion o LOW DENSITY:^ Growing 81 + 1.50 1 3 + 0.54 16 + 1.59 Ear ly s tat ionary 39 0.76 9 + 1.22 51 + 0.96 Late s tat ionary 44 + 2.26' 6 + 0.91 46 + 2.25 High dens i ty: Stat ionary 28 + 0.70 4 + 0.70 68 + 1.00 1. Mean + S.E.M. 2. R e f e r s t o s e e d i n g F i g . 8 T o t a l 3 H - g l u c o s a m i n e i n c o p o r a t i o n i n t o g l y c o s a m i n o g l y c a n s i n c u l t u r e s o f n o r m a l h u m a n m a m m a r y e p i t h e l i a l c e l l s . T h e m e d i u m , c e l l a n d E C M f r a c t i o n s w e r e a n a l y s e d a t g r o w i n g ( d a y s 3 - 4 ) , e a r l y s t a t i o n a r y ( d a y s 9 - 1 1 ) a n d l a t e s t a t i o n a r y ( d a y s 1 7 - 1 8 ) p h a s e s . . E r r o r b a r s i n d i c a t e S . E . M . o f t h r e e d e t e r m i n a t i o n s o f o n e e x p e r i m e n t . - 46 - greatest v a r i a t i o n i n GAG synthesis. Of the 3 experiments, one synthesized a greater amount of GAG, one a comparable amount and one less than the amount of GAG synthesized by the early stationary cultures. Figures 8, 9 and 11 are representative of one experiment. Possible explanations for the variations i n the late stationary cultures can be found i n the Discussion. The cultures that were seeded at high density and terminated day 4 closely resemble the early stationary cultures i n both overall l o c a l i z a t i o n of synthesezed GAG ( Table 2 ) and synthesized GAG / c e l l ( Fig. 11a and b) indicating that these patterns were related to growth status and not time i n culture. Hyaluronic Acid Synthesis Hyaluronic acid (HA) was the most predominant GAG present i n the medium regardless of the growth state of the culture. In growing cultures the medium had more than a 7 - fold greater percentage of HA then sulphated GAG. In the stationary cultures the medium contained a 4 - fold greater percentage of HA ( Table 3 )• The percentage of t o t a l GAG i d e n t i f i e d as HA changed very l i t t l e from the growing to confluent periods i n the medium fr a c t i o n and i n the ECM. However, the percentage of HA i n the medium was 20$-30$ higher than the percentage found i n the ECM at a l l times . Although the t o t a l percentage of GAG i d e n t i f i e d as HA i n each f r a c t i o n did not change greatly between growing and confluent stages, the actual amount synthesized / c e l l did vary ( Figure 9 )• During growth the amount of HA was 5 - fo l d higher i n the medium than i n the ECM on a per c e l l basis. Once the stationary growth phase had been reached, while a l l fractions showed an increase i n the amount of HA synthesized / c e l l , there was 4-5 times as much of the newly synthesized HA i n the ECM as there was - 47 - i n the ECM during growth while the medium showed less than a 1-fold increase. This brought the HA / c e l l i n the medium and the ECM closer to a 1:1 r a t i o (Figure 9, 11a and b). In the growing phase, c e l l s synthesized twice as much HA / c e l l when compared to the synthesis of sulphated GAGs. By stationary growth, the ECM had approximately a 1:1 ra t i o of HA to sulphated GAGs. This pattern was quite d i s t i n c t from GAG synthesis into the medium which showed predominately synthesized HA, and had a r a t i o of nonsulphated to sulphated GAGs closer to 3:1 throughout a l l stages of growth. In the cultures seeded at high density, where no growth occurred, the amount of HA synthesized / c e l l was very s i m i l a r to the low density stationary cultures (Figure 11a and b). In the medium, the high density cultures had a 3:1 r a t i o of HA to sulphated GAGs ; i n the ECM the HA to sulphated GAG r a t i o was 1:1. Chondroitin Sulphate Synthesis The t o t a l percentage of GAG i d e n t i f i e d as CS was less than that i d e n t i f i e d as HA i n a l l fractions regardless of growth state ( Table 3 )• This difference was between 1.5 and 8 f o l d . When comparing the percentage of GAG i d e n t i f i e d as CS among the fractions analysed, the ECM had the greatest percentage of CS regardless of the growth state, between 1.5 - 3 fold higher than that found i n the medium. This i s i n contrast to synthesized HA l o c a l i z a t i o n . The greatest percentage of HA was found i n the medium regardless of growth state. Table 3 also shows the percentages of i d e n t i f i e d GAG synthesized by high density cultures. The percentage of CS i n the 2 fractions of high density cultures were si m i l a r to that of the stationary cultures. - 48 - When the amount of synthesized CS was expressed / c e l l (Figure 10) the growing c u l t u r e s showed th a t a l l f r a c t i o n s contained r e l a t i v e l y s m a ll but s i m i l a r amounts. By the s t a t i o n a r y growth phase, while the medium and c e l l f r a c t i o n s showed some increase i n the CS synthesized / c e l l (1.5 - 2 f o l d ) the ECM f r a c t i o n had the gr e a t e s t i n c r e a s e , 10 - 12 f o l d . By the time growth ceased, the ECM a l s o contained a 3 - f o l d g r e a t e r amount of CS than d i d the medium. The percentage of synthesized CS i n the ECM f r a c t i o n s of growing and e a r l y s t a t i o n a r y phases showed only a 1% increase (Table 3). This was due to the l a r g e increase i n GAGs synthesized i n the ECM f r a c t i o n by the e a r l y s t a t i o n a r y c u l t u r e s . The a c t u a l amount of CS synthesized / c e l l however, was 4 - f o l d g r e a t e r i n s t a t i o n a r y versus growing c u l t u r e s . In the high d e n s i t y c u l t u r e s terminated at day 4 ,the percentage of CS (Table 3) and the amount of CS synthesized / c e l l (Figure 11a and b) were s i m i l a r , i n both medium and ECM f r a c t i o n s , to the low d e n s i t y s t a t i o n a r y c u l t u r e s . This i n d i c a t e s again that the type and l o c a l i z a t i o n of synthesized GAG are f u n c t i o n s of growth s t a t u s and not a t i m e - i n - c u l t u r e phenomenon. The r a t i o of nonsulfated to s u l f a t e d GAGs has been described under the HA Synthesis s e c t i o n of the R e s u l t s . Dermatan S u l f a t e Synthesis In a l l c u l t u r e s , regardless of growth s t a t u s , the ECM contained the highest percentages of synthesized DS. This amount ranged from 4 .- 7 f o l d h i g h er than that found i n the medium of any c u l t u r e s (Table 3). DS accounted f o r 19 - 20 percent.of the s u l f a t e d GAGs i n the ECM regardless of growth phase while only accounting f o r 8 - 1 0 percent of the s u l f a t e d GAG i n the medium, a d i f f e r e n c e of approximately 2 f o l d . The high d e n s i t y c u l t u r e s showed percentages of DS s i m i l a r to the s t a t i o n a r y c u l t u r e s f o r - 49 - T A R I E 5, ^H-glucosamine incorporation into indiv idual glycosaminoglycans 1n normal human mammary ep i me "Hal c e l l s in cu l ture. Culture Fraction^ Hyaluronic Chondroitin Dermatan Heparan Conditions Ac id Sulfate Sulfate Sulfate % incorporation 3 Low density: Growing Medium 88+0 . 83 2 10+0.84 1+0.00 1+0.43 ECM 60+1 .97 26+0.68 7+1.59 3+1.37 Early stat ionary Medium 80+1 .42 17+1.42 2+0.68 1+0.57 ECM 50+1 .53 33+1.06 9+1.22 3+0.77 Late stat ionary Medium 78+1 •S3 18+1.33 1+0.00 2+0.57 ECM 59+1 .65 30+1.44 6+0.89 3+0.61 High density: Stationary Meaium 76+0 .92 20+0.57 1+0.00 1+0.00 ECM 46+0, .79 32+0.53 12+0.42 4+0.61 1. Cultures were resolved into 3 f rac t ions : Medium, Cel l and ECM. The d i s t r ibu t ion of alycosaminoglycan did not d i f f e r in the ce l l f ract ion (HA, 76-78%; CS, 15-19%; DS, 1-3%; HS, 4-5%) regardless of growth conditions so the data from this f ract ion is not shown. 2. Mean + S.E.M. ^ d e f e r s to seeding. - 50 - F i g . 9 3 H - g l u c o s a m i n e i n c o p o r a t i o n i n t o HA/ c e l l . The medium, c e l l a n d ECM f r a c t i o n s w e r e a n a l y s e d i n g r o w i n g ( d a y 4 ) , e a r l y s t a t i o n a r y ( d a y 10) and l a t e s t a t i o n a r y ( d a y 17) c u l t u r e s o f n o r m a l human mammary e p i t h e l i a l c e l l s . E r r o r b a r s i n d i c a t e S.E.M. o f t h r e e d e t e r m i n a t i o n s o f one e x p e r i m e n t . - 51 - * 0 4 8 12 16 20 Time (Days) F i g . 10 3 H - g l u c o s a m i n e i n c o p o r a t i o n i n t o CS/ c e l l . The medium, c e l l a n d ECM f r a c t i o n s w e r e a n a l y s e d i n g r o w i n g ( d a y 4 ) , e a r l y s t a t i o n a r y ( d a y 10) and l a t e s t a t i o n a r y ( d a y 17) c u l t u r e s o f n o r m a l human mammary e p i t h e l i a l c e l l s . E r r o r b a r s i n d i c a t e S.E.M. o f t h r e e d e t e r m i n a t i o n s o f one e x p e r i m e n t . - 52 - F i g . 11 a) 3H-glucosamine i n c o p o r a t i o n i n t o HA, CS, and DS / c e l l i n the medium f r a c t i o n of t r i p l i c a t e c u l t u r e s of normal human mammary e p i t h e l i a l c e l l s at growing, e a r l y s t a t i o n a r y and hig h d e n s i t y ( s t a t i o n a r y ) phases. E r r o r bars i n d i c a t e S.E.M. (N = 3) F i g . 11 b) 3H-glucosaraine i n c o p o r a t i o n i n t h e ECM f r a c t i o n of t r i p l i c a t e c u l t u r e s of normal human mamillary e p i t h e l i a l c e l l s a t growing, e a r l y s t a t i o n a r y and h i g h d e n s i t y ( s t a t i o n a r y ) phases. E r r o r bars i n d i c a t e S.E.M.(N = 3) - 54 - Time (days) F i g . 12 3 H - g l u c o s a m i n e i n c o p o r a t i o n i n t o DS/ c e l l . The medium, c e l l a n d ECM f r a c t i o n s w e r e a n a l y s e d i n g r o w i n g ( d a y 4 ) , e a r l y s t a t i o n a r y ( d a y 10) and l a t e s t a t i o n a r y ( d a y 17) c u l t u r e s o f n o r m a l human mammary e p i t h e l i a l c e l l s . E r r o r b a r s i n d i c a t e S.E.M. o f t h r e e d e t e r m i n a t i o n s o f one e x p e r i m e n t . - 55 - both the medium and ECM (Table 3). DS was 25 percent of the t o t a l sulphated GAG i n the ECM and only 5 of that percent i n the medium. When DS was expressed / c e l l , a l l fractions showed equally low amounts i n the growing cultures (Figure 11a and b, 12). When growth ceased, DS had increased by 10 f o l d i n the ECM fr a c t i o n while the medium fra c t i o n remained low, increasing by only 1 - 2 f o l d . Heparan Sulfate Synthesis The percentage of t o t a l GAG i d e n t i f i e d as HS does not exceed 5 percent i n any fr a c t i o n at any time point (Table 3). However, the actual amount of HS synthesized i s higher i n the ECM fr a c t i o n than the medium fr a c t i o n of stationary cultures. This i s not the case i n the growing cultures where a l l fractions had only a small amount of synthesized HS (Table 3)» The high density cultures showed si m i l a r results (Table 3); that i s , uniformly low percentages i n a l l fractions. Electrophoresis Results GAGs i n a l l fractions were i d e n t i f i e d by cellulose acetate electrophoresis (Table 4). The amount of H-glucosamine labelled material applied to the cellulose acetate s t r i p was up to 20 fo l d higher for the stationary cultures compared to growing cultures due to the greater amount of t o t a l GAG synthesized by confluent cultures. This may account for some discrepancy between the electrophoretic results and the enzyme assay results, especially i n the growth stage. Since the more labelled material applied to the s t r i p the more accurate the resu l t s , any s t r i p having less than 1000 DPM was not useable. Therefore, very low counts of - 56 - TABLE 4: E l e c t r o p h o r e s i s I d e n t i f i c a t i o n of GAG (% of T o t a l ) C u l t u r e Sample %HA 7.HS/DS 7.CS C o n d i t i o n s Low MED 100 +0.00 ND ND D e n s i t y : 1 CELL 100 +0.00 ND ND Growing ECM 79 +2.64 4 +0.96 17 +1.80 Low MED 89 +1.36 3 +1.07 8 +1.17 D e n s i t y : CELL 86 +1.79 3 +0.77 11 +1.24 E a r l y ECM 58 +1.84 9 +1.08 33 +1.55 S t a t i o n a r y Low MED 89 +1.04 2 +0.51 9 +1.22 D e n s i t y : CELL 90 +1.36 1 +0.43 9 +1.40 Late ECM 71 +1.98 8 +1.11 21 +1.67 S t a t i o n a r y High MED 85 +1.54 1 +0.23 14 +0.83 D e n s i t y : CELL 81 +1.21 ' ND 19 +0.85 S t a t i o n a r y ECM 56 +2.01 16 +0.54 28 +1.07 ND = l e v e l i s not d e t e c t a b l e Mean of 3 + SEM 1 - r e f e r s to seeding - 57 - labelled material, such as i n the case of sulfated GAGs i n the medium of growing cultures, resulted i n non-detectable quantities. However, the majority of the electrophoresis results compared within 10 - 15 percent of the enzyme assay results (Table 4). The distr ibution of each GAG i n the three fractions compared favourably between the two methods for growing and stationary cultures i n i t i a l l y seeded at low density as well as i n the high density cultures. The medium showed the largest percentage of HA regardless of growth status. The ECM showed the largest percentage of CS regardless of the growth status. The percentage of HS and DS were combined together as they co-migrate on the s t r i p . The medium and c e l l fractions contained only small percentages of HS and DS while the ECM fraction contained the largest percent i n a l l cases. Presumably the increased percentage of HS and DS i n the ECM was due to the increased percent of DS as was seen with the enzyme assay results . The high density cultures compared with the stationary cultures i n a l l GAG percentages as was seen i n the enzyme assay results . , Cultures Labelled with ^ S - Sulfate 5 2 Normal human mammary epi thel ia l cel ls seeded at 5 X 10 cells/cm 5 2 (high density) and 2 X 10 cells/cm (low density) were labelled with 35 y S-sulfate and terminated on day 4. DNA analysis of these cultues indicated that cultures seeded at high density had not grown over the four day period while cultures seeded at low density had increased from 2 X f~ 2 5 2 10 cells/cm to 4 X 10 cells/cm by day 4. These cultures provided information on the synthesis of sulfated GAGs - CS, DS and HS. Again, only the medium and ECM fractions are significant as the c e l l results varied only s l ight ly between the two densities and were negligible - 58 - TABLE 5: Percentage of S u l f a t e d GAG i n the Medium and ECM of High and Low Density Cultures l a b e l l e d w i t h 5 5 s-Sulfate. Experiment F r a c t i o n % CS % DS % HS High Med. 71 + 0.54 1 8 + 0.23 2 + 0.21 Density ECM- 72 + 0.59 18 + .0.67 2 +_ 0.37 Low Med. 77 + 0.57 7 + 0.57 Density ECM 65 + 0.96 17 + 0.59 — 1 Mean of 3 + SEM. T 59 - . 0 0 0 5 f H i g h d e n s i t y •*•*•* L o w d e n s i t y 0 0 o ° o 0 . 0 0 0 4 .0003 . 0 0 0 2 •0001 ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° ° o ° 0 ~ o —2— o o 'oo .MED ECM MED ECM CS DS F i g . 13 3 5 S - s u l f a t e i n c o p o r a t i o n i n t o CS a n d DS/ c e l l i n t h e medium a n d ECM f r a c t i o n s o f d u p l i c a t e c u l t u r e s o f n o r m a l human mammary e p i t h e l i a l c e l l s a t h i g h (5 x 1 0 s c e l l s / c m 2 ) a n d l o w (2 x 1 0 5 c e l l s / c m 2 ) . - 60 - 35 i n amounts (Table 5)- S - s u l f a t e l a b e l l e d GAGs demonstrated s i m i l a r 3 p a t t e r n s to the H-glucosamine l a b e l l e d s u l f a t e d GAGs i n growing and 35 s t a t i o n a r y c u l t u r e s . In the S - s u l f a t e l a b e l l e d high d e n s i t y c u l t u r e , CS was 9 - f o l d g r e a t e r than DS i n the medium f r a c t i o n and 4 - f o l d g r e a t e r than DS i n the ECM f r a c t i o n . This was comparable to an 8 - f o l d and 4. - f o l d g r e a t e r amount of CS over DS i n the medium and ECM r e s p e c t i v e l y i n the 3 H-glucosamine ' low d e n s i t y s t a t i o n a r y c u l t u r e s . In the low d e n s i t y 35 S - s u l f a t e l a b e l l e d c u l t u r e , CS was 10 - f o l d higher than DS i n the medium f r a c t i o n and 4 - f o l d higher than DS i n the ECM f r a c t i o n . This 3 again compared favourably w i t h the H-glucosamine l a b e l l e d low d e n s i t y growing c u l t u r e s which had a 10 and 4 f o l d g r e a t e r amount of CS over DS i n the medium and ECM f r a c t i o n s r e s p e c t i v e l y . 3 As i n the H-glucosamine l a b e l l e d c u l t u r e s , the predominant s u l f a t e d GAG / c e l l i n both 5 5 S - s u l f a t e l a b e l l e d c u l t u r e s was CS followed by DS (Figure 13). HS was very low i n the high d e n s i t y c u l t u r e and not detectable i n the low d e n s i t y c u l t u r e . Also comparable was that the amounts of CS and DS / c e l l were always gre a t e r i n the ECM f r a c t i o n than i n the medium f r a c t i o n r e g a r d l e s s of growth s t a t u s . The medium f r a c t i o n contained more s u l f a t e d GAG i n the high d e n s i t y c u l t u r e than i n the low d e n s i t y , however t h i s was not a great amount when compared to that i n the ECM (20 f o l d g r e a t e r i n the ECM). Autoradiography GAGs separated by c e l l u l o s e acetate e l e c t r o p h o r e s i s were i d e n t i f i e d by autoradiography (Figure 14). The s t a t i o n a r y c u l t u r e s showed the great e s t 3 co n c e n t r a t i o n of H - glucosamine l a b e l l e d GAGs i n the ECM f r a c t i o n . DS I - 61 - was not detectable u s i n g t h i s technique. The growing c u l t u r e s showed only the medium f r a c t i o n and the grea t e s t c o n c e n t r a t i o n of l a b e l l e d m a t e r i a l there was i d e n t i f i e d as HA. E l e c t r o n Microscopy E l e c t r o n microscopy revealed human mammary c e l l s w i t h e p i t h e l i a l c h a r a c t e r i s t i c s ( F i g . 15) i n c l u d i n g t i g h t j u n c t i o n s and m i c r o v i l l i a t the a p i c a l s u r f a c e s . These were t y p i c a l f i n d i n g s i n the predominant c e l l type throughout the c u l t u r e s , both growing and s t a t i o n a r y . The r a t i o of e p i t h e l i a l to f i b r o b l a s t c e l l s was approximately 4 to 1. - 62 - M E D I U M I ^ CELL ) E C M i r HA C S F i g . 1 4 A u t o r a d i o g r a p h y o f 3 H - g l u c o s a m i n e i n c o p o r a t e d G A G i n t h e m e d i u m , c e l l a n d a n d E C M f r a c t i o n s o n a n e a r l y s t a t i o n a r y c u l t u r e o f n o r m a l h u m a n m a m m a r y e p i t h e l i a l c e l l s . - 63 - F i g . 15 E i e c t r o n m i c r o g r a p h of s t a t i o n a r y normal human mammary c e l l s i n c u l t u r e demonstrating an e p i t h e l i a l nature. N TJ MV = nucleus = t i g h t j u n c t i o n = micro v i l l i DISCUSSION The type, amount and l o c a l i z a t i o n of glycosaminoglycans synthesized hy normal human mammary e p i t h e l i a l c e l l s i n culture were shown to vary with the growth status of the culture. The results w i l l be discussed under the following headings: 1) culture status 2) d i s t r i b u t i o n of synthesized GAGs 3) type of synthesized GAG 4) overall amount of synthesized GAG The discussion w i l l be completed with a b r i e f outline of questions generated by. the research presented i n this thesis and the direction of future work. l ) Culture Status The cultures of normal human mammary e p i t h e l i a l c e l l s described i n t h i s thesis are growing or nongrowing (stationary). B r i e f l y , growing cultures 5 are 3 - 4 day old cultures seeded at low density (2.0-2.5 X 10 2 cells/cm ). Stationary cultures are 9 -11 day old cultures seeded at low 5 2 density (2.0-2.5 X 10 cells/cm ) and 4 day old cultures seeded at high 5 2 density (5«0 X 10 cells/cm ). The GAG p r o f i l e of stationary cultures i s the same regardless of time i n culture suggesting that the type, amount and l o c a l i z a t i o n of synthesized GAG depends on the growth status of a culture and not time spent i n culture. This finding supports the work of Cohn et a l . (1976). The 17 - 18 day old cultures i n i t i a l l y seeded at low density (2.0-2.5 X 10 ) (late stationary cultures) have varied GAG patterns and w i l l be - 65 - discussed separately from the other nongrowing cultures. 2) D i s t r i b u t i o n of Synthesized GAGs The cultures were separated into 3 fractions, the medium, the c e l l (soluble c e l l f r a c t i o n only) and the ECM (including the c e l l membrane) as previously described by Parry et a l . (1985) and Nevo et a l . (1984). However, i t should be noted that c e l l surface and ECM GAG are not equivalent. Recent studies have shown that certain types of GAGs are c e l l surface s p e c i f i c and are not found i n the ECM and vice versa (Rapraeger and Bemfield, 1985). Many studies do not define an ECM component (Cohn et a l . , 1979; Angello et a l . , 1982; Chandrasekaran et a l . , 1979) but instead use one, two, or a l l of the following - c e l l , c e l l surface and medium. Most of the synthesized GAGs i n t h i s study were either i n the medium or the ECM fraction with very l i t t l e i n the c e l l (Table 2) indicating that these molecules were not accumulating there during any stage of growth. Parry et a l . (1984) also found l i t t l e accumulation of synthesized GAG i n mouse mammary e p i t h e l i a l c e l l s cultured on collagen gels. Conversely, Cohn et a l . (1976) found a large amount (up to 90 percent) of synthesized GAG present ' i n t h e i r 3T3 c e l l s cultured on p l a s t i c and Parry et a l . (1985) found equal amounts of GAG i n the c e l l and medium synthesized by mouse mammary e p i t h i l i a l c e l l s cultured on p l a s t i c . In growing tissue _in vivo, i t has been c l e a r l y demonstrated that c e l l s migrate toward or create t h e i r own suitable e x t r a c e l l u l a r environment conducive to continued growth. In-embryonic tissues release of GAGs allow migratory pathways to form (Toole et a l . , 1971). Growing c e l l s have been observed moving along these pathways u n t i l t h e i r f i n a l destination i s reached (Hay, 1982). No further release of the migration-stimulating GAG - 66 - occurs and the cel ls begin to differentiate and accumulate an ECM. In culture, the cel ls may also be releasing synthesized GAG into the medium to create a suitable environment for growth. During growth the cultures accumulated most of the synthesized GAG i n the medium (Table 2). With cessation of growth, the local izat ion of GAG changed so that the ECM contained a substantial increase i n the percentage of synthesized GAG. There s t i l l remained a portion of the synthesized GAG i n the medium, however this was half of what i t was during growth. Nevo et a l . (1984) suggest i n their study on bovine corneal endothelial ce l ls i n culture that the ECM does not play a role i n ' e a r l y growth. They grew cel ls on plastic and on ECM and noted that the growth rate for both i s the same over the f i r s t 3 days i n culture. However, by the f i f t h day, the f i n a l c e l l count for cultures on the ECM i s 18 percent higher than cultures on plas t i c . They conclude that the major function of the ECM occurs i n a confluent, culture. An ECM may permit higher c e l l densities, however, as a confluent state i s not attained u n t i l day 4 and greater growth was noted for the cultures on the ECM than on plastic for the 24 hours prior to the confluent state. Therefore, an ECM may not be necessary for growth and cel ls i n culture need not incorporate synthesized GAG into an ECM during this stage. From their experiments on a normal human breast c e l l l ine (HBL-100) and two malignant human breast c e l l lines (MDA-MB-231 and MCF-7), Chandrasekaran et a l . (1979) show that the majority of synthesized GAG i s found i n the medium. Their cultures were labelled when 80 percent confluent and terminated 48 h later . Although i t i s not stated i n their' paper, the cultures appear to be growing at the time of l a b e l l i n g . These results compare favourably with the results of growing human mammary - 67 - e p i t h e l i a l c e l l s described i n t h i s t h e s i s . . Cohn et a l . (1976) found that the medium contains l e s s than 20 percent 4 of the synthesized GAGs when t h e i r mouse 3T3 c e l l s are 3« 5 X 10 2 c e l l s / c m (not confluent)and a higher percentage (35 percent) when the c e l l s are 7.0 X 10^ c e l l s / c m ^ ( c o n f l u e n t ) . Although the increase i n percentage of GAGs i n the medium i s not l a r g e , they conclude that the i n h i b i t i o n of the growth of c e l l s a t higher d e n s i t y may be r e l a t e d to the increased presence of GAG i n the medium. Although t h e i r methodology and r e s u l t s d i f f e r from those reported i n t h i s t h e s i s , i t seems p o s s i b l e t h a t c e l l d e n s i t y and density-dependent growth i n h i b i t i o n a l t e r the type and d i s t r i b u t i o n of synthesized GAGs i n t h e i r work as i t appeared to do i n t h i s work. A strong r e l a t i o n s h i p e x i s t s between c e l l d e n s i t y and growth such t h a t the d e n s i t y changes as growth occurrs. Because of t h i s , i t i s d i f f i c u l t t o assess which of the f a c t o r s plays the major r o l e i n a l t e r i n g GAG s y n t h e s i s . Cohn et a l . (1976) r e l a t e s many of the a l t e r e d s y n t h e s i s p a t t e r n s seen i n the c u l t u r e d 3T3 c e l l s to d i f f e r e n c e s i n c e l l d e n s i t y . I t would indeed seem reasonable to assume th a t d e n s i t y does play a r o l e i n GAG s y n t h e s i s . High c e l l d e n s i t y i n c u l t u r e s s t u d i e d f o r t h i s research i s compatible w i t h nongrowth. Nongrowing c u l t u r e s show an a l t e r e d GAG syn t h e s i s compared to growing. The growing c u l t u r e s have a much lower c e l l d e n s i t y . Although low and high d e n s i t y c u l t u r e s are e i t h e r growing or nongrowing, there are other explanations f o r changes i n GAG l o c a l i z a t i o n due to d e n s i t y besides growth. C e l l s t h a t are growing are g e n e r a l l y m i g r a t i n g across a sur f a c e . Therefore the l o c a t i o n of GAGs may d i f f e r between migrating and non m i g r a t i n g c e l l s - . C e l l s i n contact w i t h other c e l l s a l t e r t h e i r shape - 68 - resulting i n altered receptor shape or d i s t r i b u t i o n . Both of these factors may affect synthesis and d i s t r i b u t i o n of different GAGs. Factors i n addition to growth and density may affect the location of synthesized GAGs. For example, Parry et a l . (1985) looked at confluent cultures of mouse mammary e p i t h e l i a l c e l l s on three different substrates and found that the type of . substrate influences l o c a l i z a t i o n of newly synthesized GAG. Cells on p l a s t i c release the majority of t h e i r GAGs into the medium while the c e l l s on collagen gels release the majority of t h e i r GAGs into the ECM. Cells grown on p l a s t i c may not be able to incorporate synthesized GAGs into an ECM despite the fact that they are no longer growing. The human mammary e p i t h e l i a l c e l l s cultured on collagen gels incorporate GAG into an ECM at a stationary growth state (Table 2), just as mouse mammary c e l l s cultured on collagen gels do. In the la t e stationary cultures of normal human mammary e p i t h e l i a l c e l l s (cultures seeded at low density and terminated day 17-18) the d i s t r i b u t i o n of GAGs do not d i f f e r from the e a r l i e r stationary cultures. However, the a t o t a l GAG sythesis varies from the early stationary and between the 3 la t e stationary cultures, such that 1 culture has a greater amount of GAG, 1 has an equal amount and 1 culture has less than the amount of GAG found i n early stationary cultures (data not shown). Several factors could account fo r t h i s difference. A possible explanation i s that the c e l l s are i n a crowded condition; i n some cases they contract the collagen gel to approximately 10 percent of i t s o r i g i n a l s i z e . This may have made i t d i f f i c u l t for normal nutrient and gas exchange to occur resulting i n c e l l dying and death. Varied GAG synthesis appears to be related somehow to the physical state of the substrate. - 69 - Types of Synthesized GAGs Hyaluronic Acid As discussed i n the Introduction, various GAGs are associated with certain b i o l o g i c a l functions on the basis of where they are found before, during and after such functions and by the types of c e l l responsible for t h e i r synthesis ( i e . growing c e l l s ) . The term "growth" i n t h i s thesis refers to an increase i n c e l l number not an increase i n i n d i v i u a l c e l l s i z e . Normal human mammary e p i t h e l i a l c e l l s i n culture synthesize predominantly HA during growth and release i t to the medium (Table 3) • I t i s possible that these c e l l s are synthesizing HA and releasing i t into the medium because they are growing or migrating. In embryological growth and migraton of tissue, HA i s the most abundant GAG being synthesized by both the e p i t h e l i a l c e l l s i n the process of migrating and the surrounding non-migrating tissue (Toole, 1982; 1979). HA can regulate c e l l m o t i l i t y i n •a number of ways: a) by promoting c e l l surface protrusions responsible for locomotion, as seen i n rapidly moving fibroblasts where i t i s concentrated i n the extending lamella and retraction fibers (Turley et a l . , 1984), b) by maintaining only weak adhesions allowing c e l l s to detach from underlying substrates e a s i l y , as seen i n a variety of c e l l s which detach readily i n the presence of HA (Abatangelo et a l . , 1982), c) by hydrating large-areas of tissue, creating open spaces which c e l l s can move through, as seen i n neural crest c e l l s (Tosney, 1978) and d) by preventing early d i f f e r e n t i a t i o n (Toole, 1977). Toole (1972) shows that addition of HA into chick embryo somite c e l l s beginning to d i f f e r e n t i a t e stop further cartilage formation. HA synthesis i s not only associated with embryological growth. S i l b e r s t e i n and Daniels (1982, 1984) demonstrate by histochemical techniques that mouse mammary gland i n vivo synthesize HA. This HA i s - 70 - mainly associated with the cap region of the growing gland where c e l l s are shown to be a c t i v e l y p r o l i f e r a t i n g and penetrating tissue. They also show that t h i s s p e c i f i c GAG synthesis and deposition i s present i n s e r i a l l y aged glands and i s thus related to growth status and not tissue age. In v i t r o , the addition of HA promotes locomotion of various c e l l types (SV40-3T3, chick heart fibroblasts) across and into various substrates, including collagen and fibronectin (Turley, 1984; Bernanke et a l . , 1979)• Other c e l l s do not respond to the addition of HA, such as 3T3 c e l l s (Turley, 1984), leucocytes (Forrester et a l . , 1981) and neural crest c e l l s (Newgreen et a l . , 1982). This has led to the conclusion that the response to HA i s c e l l type s p e c i f i c and/or c e l l s must have available receptors to HA (Turley, 1984; Underhill et a l . , 1981). HA binding receptors may only be present at certain times during a c e l l ' s development and addition of HA during inappropriate times may account for the lack of response i n these c e l l s . Normal human mammary e p i t h e l i a l c e l l s synthesize t h e i r own HA and presumably responded to i t s presence by growing and / or migrating. HA has l i t t l e a f f i n i t y for laminin, a glycoprotein found i n the ECM (Del Rosso et a l . , 1981). Laminin, as discussed i n the Introduction, i s known to have a major role, by binding with collagen and proteoglycans, i n securing c e l l s to a basement membrane. The lack of a f f i n i t y between HA and laminin creates an environment conducive for detachment for c e l l s associated with excess HA thereby f a c i l i t a t i n g growth and / or m o t i l i t y . For growing c e l l s i n tissue i t would appear b e n e f i c i a l , i f not obligatory, to have an abundance* of HA present. This would ensure growth and / or m o t i l i t y up to the point where a f i n a l destination i s reached and prevent any p o s s i b i l i t y of early adhesion and precocious d i f f e r e n t i a t i o n . I t i s - 71 - l i k e l y that normal human mammary epi thel ia l ce l ls are able to create an environment conducive to their growth, i e . HA synthesis (Table 3). Stationary cultures, however, also synthesize HA which is found both i n the medium and the ECM. There are several explanations for t h i s . F i r s t , once the cultures are stationary there i s most l i k e l y an ongoing cycle of c e l l death and proliferat ion that may continually be causing the synthesis of HA and i t s release into the medium. Secondly, i t appears from recent studies (discussed i n the Introduction) that HA has several functions. What HA may be doing for the growing c e l l may be quite different from i t s function i n a stationary or differentiated c e l l , part icularly i f i t i s found i n a different location, i e . HA present i n the ECM of stationary cultures (Figure 9) may have a very different function from the HA present i n the medium of growing cultures. Growing cultures of normal human mammary epi thel ia l ce l l s have only approximately 20 percent of total synthesized HA i n the ECM while the stationary cultures have 50 percent of total HA incorporated there. Thirdly, synthesis of HA and i t s release into the medium may be a culture phenomenon such that some HA w i l l be found there regardless of the growth status bf the culture. It i s interesting to note that HA i s the only GAG of any abundance i n the medium of a l l growing and nongrowing cultures, 3 to 5 times the amount of CS, the next most abundant GAG. Parry et a l . (1985) demonstrate that for confluent mouse mammary epi thel ia l ce l ls cultured on plastic the medium i s r ich i n HA. This i s also true of the ce l ls on attached collagen gels but these cultures show approximately equal amounts of HA i n the medium and ECM. This finding i s identical to the findings of the stationary normal human mammary epi thel ia l cultures on attached collagen gels. Other studies also show a predominance of HA i n the medium, including studies of cloned pigmented - 72 - r e t i n a l e p i t h e l i a l c e l l s maintained on p l a s t i c substrates (Crawford and Crawford, 1984) and the normal human breast c e l l l i n e (HBL-lOO) maintained on p l a s t i c (Chandresekaran and Davidson,1979)• Cohn et a l . (1976) find that regardless of c e l l density and growth status of t h e i r 3T3 c e l l s on p l a s t i c , the medium always has mainly HA ( 60-70 percent ). They find that the percentage of HA i n the medium actually increases with a high density or nongrowth s i t u a t i o n . I t appears that c e l l s i n culture p r e f e r e n t i a l l y release HA into the medium over other GAGs being synthesized. This phenomenon appears i n cultures of various c e l l types both growing and nongrowing. Cohn et a l . (1976) f i n d that although the medium contains mainly HA i n high (stationary) and low (growing) density cultures, the overall amount of synthesized GAG (particularly. HA) i s less i n the stationary cultures so, although the percentage of HA i n the medium i s higher, the actual amount i s le s s . Parry et a l . (1985) also find that the mouse mammary e p i t h e l i a l c e l l s synthesize less HA i n the medium when they are grown on collagen gels than on p l a s t i c and are approximately equal to the sum of the other GAGs present i n the medium (CS, HS). When the collagen gels are allowed to f l o a t early i n culture they synthesize even less HA into the medium and ECM when compared to the c e l l s on attached gels. Parry et a l . (1985) point out that c e l l s on f l o a t i n g gels are more differentiated than c e l l s on either p l a s t i c or attached collagen gels, as analysed by milk protein synthesis. I t may be that c e l l s that are f u l l y d ifferentiated do not require as much HA to function or as much of any GAG, resulting i n less synthesis and turnover of GAGs. The normal human mammary e p i t h e l i a l c e l l s studied i n t h i s research are not differentiated although they do stop growing when they reached a confluent state. - 73 -. The c e l l fractions of the cultures described i n this thesis do not vary with growth and stationary phases with regard to the type and amount of GAG (Table 2). This suggested that most of the GAG synthesized i s either released to the medium, incorporated into an ECM or rapidly degraded. Parry et a l . (1985) also find that the c e l l fraction contains the least amount of GAG i n confluent mouse mammary epithelial cultures on collagen gels. This i s 2 - and 5 - fold less than the amount of GAG i n the medium and ECM respectively. The work reported here and other work (Parry et al.,1984; Nevo et a l . , 1984) also indicates that the presence of a collagen based substrate prevents intracellular GAG build-up. Both Cohn et a l . (1976), looking at 3T3 cells cultured on plastic, and Parry et a l . (1985), looking at mouse mammary cells on plastic, find greater or equal amounts of total synthesized GAG in the c e l l compared to that in the medium regardless of their growth status. The reasons for these differences may be related to the components of the substrate, the physical nature of the substrate and c e l l - c e l l interactions. The ECM of growing human mammary epithelial c e l l cultures contain l i t t l e of the total amount of synthesized HA, most of which i s found in the medium. As the cultures become stationary the amount of HA increases in the ECM by almost 4 - fold, resulting in approximately equal amounts of HA in the ECM and medium (Figure 11a and b). The major function of HA seems to be associated with growth and / or migration (Toole, 1977; 1979; 1982; Underhill, 1982). Therefore, why is the HA found in the ECM and why is HA found i n cultures of nongrowing cells? In light of recent developments on binding sites for various GAGs, HA may play a much more varied role in c e l l function than was i n i t i a l l y appreciated (Turley, 1984). HA i s known to aggregate other GAGs (except heparan sulfate) and has binding sites on c e l l - 74 - surfaces such as fibroblasts (Underhill, 1982). HA has also been shown to bind to fibronectin (Yamanda et a l . , 1980). Specific proteins which are thought to represent HA binding sites have been isolated (Turley, 1982). The binding properties of HA may relate to available receptor sites, the dependency of the particular c e l l type on HA mediated adhesion, the nature of the growth surface and the stage of adhesion to the underlying substrate (Turley, 1984). Turley suggests that HA may be involved in early adhesion formation and be replaced over time by other GAGs or possibly glycoproteins. In the normal human mammary epithelial cells cultured for this study, very l i t t l e HS i s synthesized. As pointed out in the Introduction, HS i s associated with strong cell-matrix adhesion properties (Hook et a l . , 1982). It may very well be that HA, acting as an aggregate or intermediate binding molecule, holds the collagen-bound sulfated GAGs (mainly CS and DS) and c e l l surfaces together. The mouse mammary epithelial c e l l line NMuMG has recently been shown to have distinct cell-associated HS acting as an anchoring mechanism between the c e l l and underlying ECM (Rapraeger et a l . , 1985). It may be that, in the absence of HS, cell-associated HA (found in the ECM fraction) may perform the same service for the c e l l . During growth, as opposed to nongrowth, cells require the a b i l i t y to move because of the close association between growth and motility. Thus, they may lack substantial amounts of HA in the ECM at that time. Parry et a l . (1985) find the same amount of HA present i n the medium and ECM of stationary mouse mammary epithelial cells on attached collagen gels, although on floating collagen gels these same ce l l s , which are more differentiated, have a third of the amount of synthesized HA in the ECM (6 percent as compared to 18 percent in the attached gels) and 30 percent more sulfated - 75 - GAG. Cohn et a l . (1976) find i n their 3T3 c e l l cultures that HA i s the major cell-surface GAG i n both the growing and stationary cultures although i t does decrease by 20 percent when the culture stop growing. Gordon and Bernfield (1979), while studying midpregnant mouse mammary e p i t h e l i a l c e l l s , f i n d that 60 percent of the GAG i n the ECM (including c e l l membrane) i s HA. Crawford and Crawford (1984) find that for cloned pigmented chick r e t i n a l e p i t h e l i a l c e l l s i n culture HA actually increases r e l a t i v e to sulfated GAGs during d i f f e r e n t i a t i o n indicating that the function of HA may be different i n different c e l l types. Sulfated GAGs The small amounts of sulfated GAG compared to HA (Figure 11a) released into the medium has been observed by others (Cohn et a l . , 1976; Chandrasekaran et a l . , 1979; Crawford et a l . , 1984). In contrast, Parry et a l . (1985) find that confluent cultures of mouse mammary e p i t h e l i a l c e l l s cultured on p l a s t i c release equal amounts of HA and CS into the medium, although Cohn et a l . (1976) find mainly nonsulfated GAG i n the medium of mouse 3T3 c e l l s . When these c e l l s enter a stationary growth phase they release an undersulfated CS that accounted for approximately 40 percent of the GAG found i n the medium. They also find that less than 5 percent of the GAG i n the medium i s HS regardless of growth status. This i s very s i m i l a r to the findings of t h i s study (Table 3). Although the functions of HA are s t i l l not widely understood, even less i s known of the functions of CS. I t has been shown that t h e i r adhesive properties are greater than those of HA for certain c e l l types such as neural crest c e l l s and leucocytes (Hook et a l . , 1982; Turley, 1984), although they do not appear as strong as the more highly sulfated DS and - 76 - HS. CS has also been shown to have a stronger a f f i n i t y than HA for the ECM glycoprotein laminin but not as strong as the a f f i n i t y of DS and HS (Del Rosso, et a l . , 1981). I t would appear that CS may act as an intermediate GAG between the functions of HA and the more highly sulfated GAGs. When monocytes change morphologically and express more macrophage-like characteristics (a process equivalent to dif f e r e n t i a t i o n ) the t r a n s i t i o n i s also accompanied by a switch i n synthesis of CS-4 to an over-sulfated galactosamine (Kolset et a l . , 1983, 1984). S i l b e r s t e i n and Daniels (1982) find that the ECM of mouse mammary e p i t h e l i a l c e l l s i n the flank region of developing mammary gland incorporates synthesized CS as the major GAG. The c e l l s i n these region are nongrowing and considered " s t a b i l i z e d " tissue. The nongrowing and differentiated mouse mammary e p i t h e l i a l c e l l s i n cultures studied by Parry et a l . (1985) have predominantly S-GAG i n the ECM, ranging from a 4 - fo l d greater amount than HA for c e l l s on p l a s t i c to a 12 - fo l d greater amount than HA for c e l l s on f l o a t i n g collagen gels. Of the amount of S-GAG, approximately 55 percent i s CS (includes DS) and 45 percent i s HS. The c e l l s on the attached collagen gel are si m i l a r to those on the fl o a t i n g collagen gel although they do not synthesize as much HS. I t appears that mouse mammary e p i t h e l i a l c e l l s synthesize mainly CS when they are i n a stationary growth state (but not differentiated) and CS and HS when they are di f f e r e n t i a t e d . These results compare favourably with the finding of th i s work. Normal human mammary e p i t h e l i a l c e l l s i n culture that are nongrowing have a substantial amount of CS incorporated into the ECM, almost 5 - fo l d higher than the amount of CS i n the ECM of growing cultures (Figure 10). Also interesting to note i s the v i r t u a l lack of DS i n any fr a c t i o n but the ECM (Figure l l ) i n th i s study. Parry et a l . (1985) also find that DS - 77 - i s only present in appreciable amounts i n the ECM of the mouse mammary epithelial c e l l s . They find that the more differentiated the cultures are the greater i s the amount of DS i n the ECM. This amount ranges from 1 to 21 percent of the total CS pool i n the cells on plastic to 42 percent for the cells on floating collagen gels (most differentiated) with the attached collagen gel cultures being between the two. In the other fractions, medium and c e l l , the amount i s much lower (between 5 and 8 percent of total CS pool) and i n several cases i s too low to be detected. The same pattern emerges i n the human mammary epithelial cells cultured i n this study. The medium and c e l l fractions often have nondetectable levels while the ECM always has most of the DS (Figure 10). DS i s most pronounced i n the ECM of nongrowing cultures, 4 - fold greater than the amount of DS i n the ECM of growing cultures (Figure l l ) . DS contains predominately L-iduronic acid residues which are epimers of the carbon-5-glucuronic acid. The presence of iduronic acid has been linked with the function of the orderly arranging of collagen f i b r i l s (lozzo, 1985). Its presence in the ECM may be essential for proper construction or orientation of the matrix components. Heparan sulfate i s associated with having strong binding properties to c e l l surfaces and other ECM components (Hook et a l . , 1982). Increasingly, research i s demonstrating that different species of heparan sulfate exist and are either c e l l surface or ECM associated (Rapraeger et a l . , 1985). Parry et a l . (1985), as mentioned, find almost equal amounts of HS and CS in the ECM of confluent mouse mammary c e l l cultures on collagen gels. A l l cultures i n their study have a hormonal milieu designed to stimulate differentiation. Nevo et a l . (1984) also find 50 percent of the synthesized GAG i n the ECM of their differentiated bovine endothelial cells - 78 - to be HS. An i n t e r e s t i n g finding of both Parry et a l . (1984) and Cohn et a l . (1976) i s that the c e l l f r a c t i o n of their c e l l s grown on p l a s t i c have a s i g n i f i c a n t amount of HS, much higher than the other fractions contain. Presumably the c e l l s are able to synthesize HS but are unable to release i t to the medium or incorporate i t into an ECM. Only small percentages of HS are detected i n the human mammary e p i t h e l i a l c e l l cultures studied for t h i s thesis (Table 3). One explanation for t h i s may be that, i f HS i s related to the process or maintance of d i f f e r e n t i a t i o n , the c e l l s described are not differentiated and therefore are not able to synthesize or do not require HS. They are obviously able to adhere to the substrate either by the use of other proteoglycans or glycoproteins or possibly by u t i l i z i n g any HS present i n the collagen gel. Although the evidence for the importance of HS i n c e l l u l a r d i f f e r e n t i a t i o n i s not conclusive, several studies have shown that i t i s not conducive to growth. Clark et a l . (1975) f i n d that the addition of dextran sulfate (an a r t i f i a l S-GAG) to cultures of BHK c e l l s i n doses as low as 1 Mg/ml caused a nontoxic Ĝ  arrest. I t was discovered that t h i s surrogate GAG binds to c e l l surfaces and i s able- to change the morphology of the c e l l s as well as causing growth changes (Goto et a l . , 1973). Majacek and Bornstein (1984) conclude that components of an ECM can control the biosynthetic a c t i v i t y of c e l l s . They observed that addition of exogenous soluble HS or DS can increase the production of 2 noncollagenous proteins i n cultured rat smooth muscle c e l l s . Other proteoglycans do not have th i s a f f e c t . They postulate that these two proteins played a role i n growth i n h i b i t i o n . The mode by which the HS molecule acts upon the c e l l may include any or a l l of the following: l ) a l t e r a t i o n of the c e l l shape v i a the cytoskeleton, i n turn a l t e r i n g biosynthetic a b i l i t i e s , 2) v i a - 79 - surface receptor and a response evoked by a "second messenger" system or 3) endocytosis of the proteoglycan and direct delivery to i t s s i t e of action (Majacek et a l . , 1984). The f i r s t method stated would appear to be the most l o g i c a l way for HS incorporated into an ECM, including the c e l l surface, to exert i t s effect on the c e l l although the second method would work equally w e l l . To summarize, human mammary e p i t h e l i a l c e l l s do not appear to require the presence of synthesized S-GAG or an ECM to grow. On the other hand, i n nongrowing cultures an ECM i s produced and approximately one half of the synthesized GAG found there i s sulfated with the majority of that being CS (Figure l i b ) . DS i s also present i n the ECM of nongrowing cultures (Figure 10). 3) Overall Amount of Synthesized GAG The overall amount of GAG synthesized by c e l l s seems related to several factors including l ) growth and d i f f e r e n t i a t i o n , 2) i i i v i t r o vs. i i i vivo conditions, 3) normal vs. malignant, 4) substrate and 5) age of c e l l s . Parry et a l . (1984) f i n d that mouse mammary e p i t h e l i a l c e l l s on p l a s t i c , which are neither growing nor dif f e r e n t i a t e d , synthesize much larger quantities of GAG than those cultures on collagen gels (a 4 - fo l d greater amount). Cohn et a l . (1976) also find that t h e i r mouse 3T3 cells,, i n a nongrowing phase, synthesize less GAG when compared to the growing cultures, a decrease of approximatley 20 percent. The results i n th i s study show an increase i n the amount of GAG synthesized as the c e l l s go from a growing to nongrowing state (Figure 7). However, preliminary studies underway i n t h i s laboratory indicate that these c e l l s , when stimulated to d i f f e r e n t i a t e v i a addition of hormones, decrease t h e i r - 80 - overall GAG synthesis, a phenomenon found by other researchers (Parry et a l . , 1984). It has been stated that _in v i t r o c e l l s synthesize excessive amounts of matrix when compared to t h e i r i n vivo counterparts (Muir, 1977). Nevo et a l . (1984) find t h i s to be true i n thei r study on cultured bovine endothelial c e l l s . They compared t h e i r results with reports on proteoglycan content of basement-membranes isolated from other endothelial tissue and discovered that cultured ' c e l l s are synthesizing greater amounts. In t h i s ; s t u d y no analysis of GAG from normal breast tissue i s done as a comparison and there are no reports of t h i s information i n the l i t e r a t u r e . I t would be interesting to see i f th i s tissue, l i k e others, showed increase GAG synthesis i n culture. Much work has been done i n the area of malignancy and GAG synthesis. As t h i s was discussed at length i n the Introduction only differences i n t o t a l GAG synthesis w i l l be included here. Generally, i t has been found that malignant c e l l s , when they are compared to t h e i r nonmalignant counterparts, synthesize greater amounts of GAG (Shishiba et a l . , 1984; Angello et a l . , 1982). In some instances these studies compared malignant and normal c e l l s of the same c e l l type. Shishiba et a l . (1984) looked at normal human thyroid tissue and human thyroid adenocarcinoma tissue and compared them for t o t a l GAG synthesis. They find a 6 to 15 fold greater amount of GAG i n the adenocarcinoma tissue. However, some studies compared malignant to nonmalignant c e l l s with the assumption that nonmalignant and normal are synomymous. For example, Angello et a l . (1982) compared two subpopulations of a mouse mammary tumor c e l l l i n e (WAX-2T), one which i s fast growing and can grow i n soft agar and one which i s slow growing and does not grow i n soft agar. They f i n d that the more aggressive tumor subpopulation - 81 - synthesizes 8 times more GAG than the less aggressive one. Chandrasekaran et a l . (1979), i n studying GAG synthesis i n c e l l l i n e s , compared the normal human breast c e l l l i n e (HBL-100) to two human breast carcinoma c e l l l i n e s (MDA-MB-231 and MCF-7). They f i n d that the HBL-100 and MDA-MB-231 synthesize equal amount of GAG and the MCF-7 considerably l e s s . C e l l l i n e s , especially so-called "normal" ones, should be compared with normal primary c e l l s with some reservation. The c e l l s used i n the experiments presented i n th i s thesis are from three different women (de t a i l s i n Appendix 9). Two of the experiments produced approximately equal amounts o f t o t a l GAG at a l l stages of growth. One experiment produced greater amounts of GAG, up to 5 - fold higher. However, the percentages of GAG i n each fr a c t i o n and the r a t i o of one GAG to another are very s i m i l a r . Why did this occur? When dealing with normal human mammary e p i t h e l i a l c e l l s several factors must be taken into account. The age of the donor may be s i g n i f i c a n t . The two experiments with less overall GAG synthesis were from two women i n th e i r mid to late t h i r t i e s . The t h i r d experiment i s from a 19 year old woman. I t may be that c e l l s from younger women are more metabolically active. The overall potential for growth ( c e l l p r o l i f e r a t i o n and GAG synthesis) may be decreasing i n older women. S i l b e r s t e i n and Daniels (1984) looked at s e r i a l l y aged mouse mammary ducts and compared GAG synthesis and l o c a l i z a t i o n using autoradiographic and histochemcial techniques. Interestingly, they find that GAG type and l o c a l i z a t i o n i s s i m i l a r between young and old ducts but that older ducts appear to synthesize less overall GAG, i d e n t i f i e d by their decreased a b i l i t y to concentrate radiolabelled material. - 82 - Future Research The r e s u l t s of t h i s study provide the framework f o r two major areas of research. F i r s t , experiments designed to achieve a d i f f e r e n t i a t e d phenotype i n the normal human mammary e p i t h e l i a l c e l l _in v i t r o would permit a n a l y s i s of GAG sy n t h e s i s under t h i s c o n d i t i o n . A change i n GAG synth e s i s i n d i f f e r e n t i a t i o n as opposed to growth/nongrowth would a l l o w f o r f u r t h e r p o s t u l a t i o n on the f u n c t i o n of va r i o u s GAGs as they r e l a t e to c e l l u l a r development. Second, malignant mammary e p i t h e l i a l c e l l s can now be compared to a normal b a s e l i n e i n c u l t u r e regarding GAG s y n t h e s i s and l o c a l i z a t i o n . Expanding f u r t h e r on the o v e r a l l f u n c t i o n of GAGs i t would appear, given the s t r u c t u r a l heterogeneity of GAGs and proteoglycans, that they are not l i m i t e d to p r o v i d i n g h y d r a t i o n or adhesion molecules to the ECM. Enhancing the substrate w i t h v a r i o u s amounts and types of GAG may a l t e r the f u n c t i o n i n g of the c e l l s on that substrate and a i d i n e l u c i d a t i n g the r o l e of GAGs i n c e l l behaviour. For example, growing c e l l s may stop growth and d i f f e r e n t i a t e i f placed on a substrate designed to s t i m u l a t e d i f f e r e n t i a t i o n . This should increase understanding of c e l l - m a t r i x i n t e r a c t i o n s . I n d i r e c t l y r e l a t e d but c l o s e l y t i e d to GAG research i s the r o l e of the p r o t e i n core i n a proteoglycan. Recent research i n d i c a t e s that the p r o t e i n core may i n part be re s p o n s i b l e f o r the f i n a l d e s t i n a t i o n of a proteoglycan (Nevo et a l . 1984). A d d i t i o n of a B-xyloside to c u l t u r e s changes the p a t t e r n of GAG s y n t h e s i s . B-xyloside a c t s as a "pseudo" core p r o t e i n which the c e l l uses to l i n k synthesized GAG si d e chains. The study by Nevo et a l . (1984) demonstrates that the a d d i t i o n of B-xyloside causes the c e l l s to re l e a s e most of t h e i r synthesized PG i n t o the medium whereas the same - 83 - c e l l s without B-xyloside i n c o r p o r a t e d the PG i n t o an ECM. The c u l t u r e s t r e a t e d w i t h B-xyloside have 72 percent of the f i n a l c e l l count of the c u l t u r e s without B - x y l o s i d e . The importance of the p r o t e i n core i s j u s t •beginning to be examined and the system used i n t h i s study would lend i t s e l f to s t u d i e s aimed at examining p r o t e i n core f u n c t i o n . A c o r o l l a r y to t h i s would be to examine the GAGs th a t are c e l l - a s s o c i a t e d as opposed to those that are d e f i n i t e ECM c o n s t i t u e n t s . I t may be, as discussed i n the HA s e c t i o n , that h y a l u r o n i c a c i d i s mainly c e l l membrane a s s o c i a t e d . At the present time i t i s not c l e a r how these molecules are attached to the plasma membrane and what t h e i r f u n c t i o n i s there as opposed to the ECM. The system used i n t h i s study would have to be modified to separate the ECM i n t o a c e l l membrane f r a c t i o n and an ECM f r a c t i o n . The techniques and i n f o r m a t i o n presented should lend themselves t o f u r t h e r i n t e r e s t i n g and valu a b l e experiments i n the co n t i n u i n g search f o r cell-ECM i n t e r a c t i o n s as they r e l a t e to c e l l f u n c t i o n s . - 84 - SUMMARY This t h e s i s research was designed to i n v e s t i g a t e the syn t h e s i s and d i s t r i b u t i o n of GAGs by normal human mammary e p i t h e l i a l c e l l s i n t i s s u e c u l t u r e . The medium f r a c t i o n of a l l c u l t u r e s contained HA regardless of the growth s t a t u s . The ECM f r a c t i o n v a r i e d i n the type and amount of GAG depending on growth s t a t u s - the growing c u l t u r e s had only a sm a l l percentage of t o t a l synthesized GAG while the s t a t i o n a r y c u l t u r e s had approximately 50-60 percent. Of the percentage of GAG i n the ECM of s t a t i o n a r y c u l t u r e s , 50 percent were s u l f a t e d ; the s u l f a t e d GAG inc l u d e d CS (.10%) and DS (30%). HS d i d not comprise more than 5-6 percent i n any c u l t u r e . To remove the. l e n g t h of time spent i n c u l t u r e as a p o s s i b l e explanation f o r the changes seen i n GAG sy n t h e s i s between growing and s t a t i o n a r y c u l t u r e s , normal human mammary e p i t h e l i a l c e l l s were seeded a t high d e n s i t y and terminated on day 4. No growth occurred. The GAG p r o f i l e of these c u l t u r e s c l o s e l y resembled the s t a t i o n a r y c u l t u r e s seeded a t low d e n s i t y and terminated a t days 9 - 1 1 - The type and l o c a t i o n of synthesized GAG was found to be dependent on the growth s t a t u s of the c u l t u r e . S u l f a t e d GAGs appeared to be more r e l a t e d to s t a t i o n a r y growth and were l o c a t e d i n the ECM. HA appeared to be present i n a l l stages of growth and were the predominant GAG i n the medium. - 85 - Appendix 1 Transport Medium F12:DME - ( l : l ) Hepes buffer - lOmM Calf serum - 5$ I n s u l i n - 5Mg/ml DMe - Delbecco's Modified Eagles Medium Appendix 2 Dissociation Medium F12 - (1:1) Hepes buffer - lOmM BSA - 2$ Insul i n - 5Mg/l Collagenase - 300U/ml Hyaluronidase - lOOU/ml Appendix 3 Growth Medium F12:DME - (1:1) Hepes buffer - lOmM Fet a l Calf Serum - 5$ Insulin - 5ug/ml Appendix 4 Freezing Medium DME - 50$ DMSO - 6% (dimethysulfoxide) CS ' - 44$ - 86 - Appendix 5 Detergent Solution Tris pH 7.2 - lOmM Triton X-lOO - 1% Deoxycholate - 1% Appendix 6 Enriched Tris T r i s base - 3»Og Na Acetate - 2.4g NaCl - 1.46g BSA - 50yg HCl - 0.13M In 100 ml of d i s t i l l e d water. pH to 8.0 Appendix 7 a) DNA buffer Na2H0P4 - 50mM NaCl - 2mM EDTA - 2mM (tetrasodium s a l t ) adjst to pH 7.4 b) Hoechst Dye (Calbiochem) stock = 20 yg/ml i n H20 c) DNA ( c a l f thymus) stock = 100 yg/ml i n H2PO4 buffer Appendix 8 Karnovsky's solution 0.5 g paraformaldehyde 7-5 ml d i s t i l l e d water 1 to 2 drops NaOH 2.5 ml 25$ glutaraldehyde 12.5 ml 0.2M Na-cacodylate pH to 7.3 - 87 - Appendix 9 Patient Age Reason for Surgery 1) M.A.S. 34 accessory breast tissue 2) J.K. 37 reduction mammoplasty 3) J.H. 19 reduction mammoplasty BIBLIOGRAPHY Abatangelo, C , Cortivo, E., M a r t i n e l l i , M. and Vecchia, P. C e l l detachment mediated hy hyaluronic acid. Exp. C e l l Res., 1982; 137:73-78. Albert, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J. D. Molecular Biology of the C e l l . Garland Pub., Inc., New York, 1983- Angello, J., Danielson, K., Anderson, L. and Hosick, H. Glycosaminoglycan synthesis by subpopulations of e p i t h e l i a l c e l l s from mammary adenocarcinoma. Can. Res., 1982; 42:2207-2210. Bailey, A. J., Sims, T. J., Duance, V. C. and Light, N. D. P a r t i a l characteri- zation of a second basement membrane collagen i n human placenta. FEBS Sett., 1979; 99:838-839. Bender, B. L., Jaffe, R., Car l i n , B. and Chung, A. E. Immunolocalization of entactin, a sulphated basement membrane component, i n rodent tissues, and comparison with GP-2 (laminin). Am. J. Pathol., 1981; 103:419-426. Bernanke, D. H. and Markwald, R. R. Effect of hyaluronic acid on cardiac cushion tissue c e l l s i n collagen matrix cultures. Texas Rep. B i o l . Med., 1979; 39:271-285. B i s s e l l , M., H a l l , G. and Parry, G. How does the extr a c e l l u l a r matrix direct gene expression? J. of Theor. B i o l . , 1982; 99:31-68. $2 - 89 - Brennan, M. J., Oldberg, A., Hayman, E. G. and Ruoslahti, E. Effect of proteoglycan produced by rat tumor c e l l s on t h e i r adhesion to fibronectin - collagen substrata. Cancer Res., 1983; 43:4302-4307. Burgeson, R. E., E l A l d i , F. A.. K a i t i l a , H. and H o l l i s t e r , D. W. Fetal membrane collagens: I d e n t i f i c a t i o n of two collagen alpha chains. Proc. Natl. Acad. S c i . , USA 1976; 73:2579-2583. C a r l i n , B., Jaffe, R., Bender, B. and Chung, A. E. Entactin - a novel basal- lamina associated sulphated glycoprotein. J. B i o l . Chem., 1981; 256:5209- 5214. Chakrabarti, B. and Park, J. ¥. Glycosaminoglycans: Structure and interaction. CRC Crib. Rev. Biochem., 1980; 8:225-212. Chandrasedaran, E. and Davidson, E. Glycosaminoglycans of normal and malignant cultured human mammary c e l l s . Can. Res., 1979; 39:870-880. Chang, R. J. and B e r l i n , R. D. Polarized secretion of glycosaminoglycans (GAGS) by MDCK monolayers. Abstract, J. of C e l l B i o l . , 1985; 101:144A Chung, E., Rhodes, R. K. and M i l l e r , E. J. Isolation of three collagenous com- ponents of probable basement membrane o r i g i n from several tissues. Bio- , chem. Biophys. Res. Commun., 1976; 71:1167-1174. C i f o n e l l i , J. Reaction of heparin sulfate with nitrous acid. Carbohydr. Res.., 1968; 8:233-242. - 90 - Cohn, R., Cassiman, J. J., Bernfield, M. Relationship of transformation, c e l l 1 density and growth control to the c e l l u l a r d i s t r i b u t i o n of newly synthesized glycosaminoglycans. J. of C e l l B i o l . , 1976; 71:280-294. Crawford, B. and Crawford, T. Type, location and role of glycosaminoglycans i n cloned differentiated chick r e t i n a l pigmented epithelium. Tissue and C e l l , 1984; 16:885-908. Crouch, E., Sage, H. and Bornstein, P. Structure basis for apparent heteroge- neity of collagens i n human basement membranes; Type IV procollagen contains 2 d i s t i n c t chains. Proc. Natl. Acad. S c i . USA, 1980; 77:745-749. Culp, L. A. Molecular composition and o r i g i n of substrate attached material from normal and virus-transformed c e l l s . J. Supramol. Struct., 1976; 5:239-255* Culp, L. A., Murry, B. A. and R o l l i n s , B. J. Fibronectin and proteoglycans as determinants of cell-substratum adhesion. J. Supramol. Struct., 1979; 11:401-427. David, G. and Bernfield, M. Defective basal lamina formation by transformed mammary e p i t h e l i a l c e l l s : A reduced effect of collagen on basal lamina (HS-rich) proteoglycan degradation. J. C e l l Physiol., 1982; 110:56-62. David, G., Van der Schueren, B. and Bernfield, M. Basal lamina formation by normal and transformed mouse mammary e p i t h e l i a l c e l l s duplicated i n v i t r o . JNCL, 1981; 67:719-723- - 91 - Del Rosso, M. Binding of the basement membrane glycoprotein laminin to glycos- aminoglycans. Biochem. J., 1981; 199:689-699. Dessau, W., Sasse, F., Timpl, R., J i l e k , F. and vonderMark, K. Synthesis and extr a c e l l u l a r deposition of fibronectin i n chondrocyte cultures. J. C e l l B i o l . , 1978; 79:342-355. Ekblom, P. K., A l i t a l o , A., Vaheri, R., Timpl, R. and Saxon, L. Induction of a basement membrane glycoprotein i n embryonic kidney: possible role of laminin i n morphogenesis. Proc. Natl. Acad. S c i . , 1980; 77:485-489. Emerman, J.T., P i t e l k a , D.R. Maintenance and induction of morphological d i f f e r e n t i a t i o n i n dissociated mammary epithelium on f l o a t i n g collagen membranes. In V i t r o , 1977; 13:316-328. Emerman, J.T., Bartley, J.C. and B i s s e l l , M. Glucose metabolite patterns as markers of functional d i f f e r e n t i a t i o n i n freshly isolated and cultured mouse mammary e p i t h e l i a l c e l l s . Exp. C e l l Res. 1981; 134:241-250. Emerman, J.T., Burwen, S.J., P i t e l k a , D.R. Substrate properties influencing u l t r a s t r u c t u r a l d i f f e r e n t i a t i o n of mammary e p i t h e l i a l c e l l s i n culture. Tissue C e l l , 1979; 11:109-119- Emerman, J.T., Enami, J., P i t e l k a , D.R., Nandi, S. Hormonal effects on i n t r a c e l l u l a r and secreted casein i n cultures of mouse mammary e p i t h e l i a l c e l l s on f l o a t i n g collagen membranes. Proc. Natl. Acad. S c i . 1977; 74:4466-4470. - 92 - Erickson, C. A. and Turley, E. A. Substrata formed by combinations of extra- c e l l u l a r matix components a l t e r neural crest m o t i l i t y i n v i t r o . J. C e l l S c i . 1983; 61:299-323. Foidart, J. M., Bere, E. ¥., Yaar, M., Rennard, S. I., G u i l l i n b , M., Martin, G. R. and Katz, S. I. Dis t r i b u t i o n and immunoelectron microscopic loca- l i z a t i o n of laminin, a noncollagenous basement membrane glycoprotein. Lab. Invest., 1980; 42:336-342. Foidart, J. M., Berman, J. J., Paglia, L., Rennard, S. I. Abe, A., Perantoni, A and Martin, G. R. Synthesis of fibronectin, laminin and several collagens by a liver-derived e p i t h e l i a l c e l l l i n e . Lab. Invest., 1980; 42:525-532. Forrester, J. V. and Wilkinson, P. C. In h i b i t i o n of leucocyte locomotion by hyaluronic acid. J. C e l l S c i . , 1981; 48:315-331. Fransson, L. A., Coster, L., Malmstrom, A. and Sheehan, J. K. Self-association of s c l e r a l proteodermatan sulphate. Evidence for interaction v i a the dermatan sulphate side chains. J. B i o l . Chem., 1982; 257:6333-6338. Gey, G. 0., Svotelis, M. Foard, M. and Bareg, F. B. Long-term growth of chicken fibroblasts on a collagen substrate. Exp. C e l l Res., 1974; 84;63-71. G l a n v i l l e , R. W., Rauter, A. and Fietzek, P. P. Isolation and characterization of a native placental basement membrane collagen and i t s component chains. Eur. J. Biochem., 1979; 95:383-389. - 93 - Gordon, J. and Bernfield, M. The basal lamina of the postnatal mammary epithe- lium contains glycosaminoglycans i n a precise u l t r a s t r u c t u r a l organization. Dev. B i o l . , 1980; 74:118-135. Gospordarowicz, D., Delgado, D. and Vlodavsky, I. Permissive effect of the ext r a c e l l u l a r matrix on c e l l p r o l i f e r a t i o n i n v i t r o . Proc. Natl. Acad. S c i . USA, 1980; 77:4094-4098. Greenberg, J. H., Seppa, S., Seppa, H. and Hewitt, A. T. Role of collagen and fibronectin i n neural crest c e l l adhesion and migration. Develop. B i o l . , 1981; 87:259-266. N G r i n n e l l , P. C e l l u l a r adhesiveness and ex t r a c e l l u l a r substrata. Int. Rev. Cytol., 1978; 53:65-144. Hascall, V. C. and Hascall, G. K. Proteoglycans. _In: C e l l Biology of Extra- c e l l u l a r Matrix. E. Hay (ed.) Plenum Press, New York, 1981, pp. 39-63. Hassell, J. R., Gehron Robey, P., Barrach, H-J., Wilczek, J., Rennard, S. I. and Martin, G. R. Is o l a t i o n of a heparan sulphate containing proteoglycan from basement membrane. Proc. Natl. Acad. S c i . , 1980; 77:4494-4498. Hay, E. (ed.) C e l l Biology of the Ex t r a c e l l u l a r Matrix. Plenum Press, New York, 1981. Heathcote, J. G., Sear, C. H. J. and Grant, M. E. Studies on the assembly of rat lens capsule. Biosynthesis and p a r t i a l characterization of the c o l - lagenous components. Biochem. J., 1978; 176:283-294. - 94 - Hook, M., Robinson, J., K j e l l e n , J . , Johansson, S. and Woods, A. Heparan s u l - fate: On the structure and function of the cell-associated proteoglycan. In: Ext r a c e l l u l a r Matrix. S. Hawkes, J.C. Wang (eds.)' Academic Press, New York, 1982, pp. 15-24- Hooper, K. E., Adelmann, B. C, Gentner, G. and Gay, S. Recognition by guinea pig peritoneal exudate c e l l s of conformationally different states of the collagen molecule. Immunology, 1976; 30:249-259- Iozzo, R. Neoplastic regulation of ex t r a c e l l u l a r matrix. J. of B i o l . Chem., 1985; 260:7464-7473- Iozzo,R. Proteoglycans and neoplastic - mesenchymal c e l l interactions. Human Pathology, 1984; 15:2-10. '' Iozzo, R. Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma c e l l s and i t s l o c a l i z a t i o n at the c e l l surface. J. of C e l l B i o l . , 1984; 99:403-417- Iozzo, R. V., Bolender, R. P. and Wight, T. N. Proteoglycan changes i n the / i n t e r c e l l u l a r matrix of human colon carcinoma: an integrated biochemical and sterologic analysis. Lab. Invest., 1982; 47:124-138. Iozzo, R. V. and Wight, T. N. Isolation and characterization' of proteoglycans synthesized' by human colon and colon carcinoma. J. Biochem., 1982; 257:11135-11144- - 95 - Johansson, S, and Hook, M. Heparin enhances the binding of fibronectin to collagen. Biochem. Biophys. Acta, 1980; 631:350-358. Kanwar, Y. S. and Farquhar, M. G. Isolation of glycosaminoglycans (heparan s u l - phate) from glomerular basement membranes. Proc. Natl. Acad. S c i . , 1979; 76:4493-4497. Kanwar, Y. S. and Farquhar, M. G. Presence of heparan sulphate i n the glome- ru l a r basement membrane. Proc. Natl. Acad. SCi., 1979; 76:1303-1307. Karakashian, M.W., Dehm, P., Groomling, T. S. and Leroy, E. C. Precursor size components are the basic collagenous subunits of murine tumor basement membrane. Collagen Related Res. C l i n . Exp., 1982; 2:3-17. K a t a g i r i , Y. U. and Yamagata, T. The persistence i n the syntheisis of type H proteoglycan and type I I collagen by chondrocytes cultured i n the presence of chick embryo extract. Devi. Growth D i f f . , 1981; 23:335-348. Kefalides, N. A. A collagen of unusual composition and a glycoprotein isolated from canine glomerular basement membrane. Biochem. Biophys. Res. Commun., 1966; 22:26-32. Kefalides, N. A. Isolation of a collagen from basement membranes containing 3 i d e n t i c a l -chains. Biochem. Biophys. Res. Commun., 1971; 45:226-234. Ketley, J. N., Orkin, R. ¥. and Martin, G. R. Collagen i n developing chick muscle i n vivo and i n v i t r o . Exp. C e l l Res., 1976; 99:261-268. - 96 - Kidwell, R. J. and Mock, P. J. Effects of glycosaminoglycans on fibronectin mediated c e l l attachment. J. C e l l Physiol., 1982: 112:5-9. Kleinman, H. R. Role of c e l l attachment proteins i n defining cell-matrix interactions. In.: Tumor Invasion and Metastasis, L. L i o t t a and I. R. Hart (eds.) Martinus Nijhoff Pub., 1982, pp. 291-308. Kleinman, H. K. Klebe, R. and Martin, G. Role of collagenous matrices i n the adhesion and growth of c e l l s . J. of C e l l B i o l . , 1981; 88:473-485. Kojima, J., Nadamura, N., Kanatani, M. The glycosaminoglycans i n human hepatic cancer. Cancer Res., 1976; 36:3122-3127. Kolset, S., S e l j e l i d , R. and Lindahl, U. Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes induced by culture substrates. Biochem. J., 1984; 219:793-799- Kolset, S. K j e l l e n , L., S e l j e l i d , R. and Lindahl, U. Changes i n glycos- aminoglycan biosynthesis during d i f f e r e n t i a i o n i n v i t r o of human monocytes. Biochem. J., 1983; 210:661-667. Kraemer, I. Mucopolysaccharides: C e l l Biology and Malignancy. In.: Surfaces of Normal and Malignant C e l l s , R. Hynes (ed.) John Wiley & Sons, Chichester, 1979, pp. 149-198. Kuettner, K. and Kimura, J. Proteoglycans: An overview. J. of C e l l Biochem., 1985; 27:327-336. - 97 - Lacy, B. E. and Underhill, C. B. Association of the hyaluronate-binding s i t e with the cytoskeleton of 3T3 c e l l s . Abstract, J. C e l l B i o l . , 1985; 101:334A. Lark, M. ¥. and Culp, L. A. Multiple classes of heparin sulphate proteoglycan from fib r o b l a s t substrate adhesion s i t e s . J. B i o l . Chem., 1984; 259:6773-6782. Laterra, J., Lark, M. ¥. and Culp, C. A. Functions for fibronectin, hyal- uronate and heparan proteoglycans i n substrate adhesion of f i b r o b l a s t s . In: Ext r a c e l l u l a r Matrix. Hawkes, S. and ¥ang, J. L. (eds.) Academic Press, New York, 1982, pp. 197-209. Laurie, G. ¥., Kleinman, H. K., Hassall, J. R., Martin, G. R. and Feldman, R. J Basement membrane organizations suggested by combination of laminin and heparan suphate proteoglycan binding s i t e s with "open network" and "polygonal" models of type IV collagen. Abstract, J. of C e l l B i o l . , 1985; 101:259A. Laurie, G. ¥., Leblond, C. P., Cournil, I. and Martin, G. R. Immunohistochemi- c a l evidence for i n t r a c e l l u l a r formation of basement membrane collagen (type IV) i n developing tissues. J. Histochem. Cytochem., 1980; 26:1267-1274. Lindahl, U. and Hook, M. Glycosaminoglycans and t h e i r binding to b i o l o g i c a l macromolecules. Ann. Rev. Biochem., 1978; 47:385-417. - - 98 - Majacek, R. and Bornstein, P. Heparin and related glycosaminoglycans modulate the secretory phenotype of vascular smooth c e l l s . J. of C e l l B i o l . , 1984; 99:1688-1695. Marks, M. S. and Toole, B. P. Hyaluronate binding protein of chick and mouse brain. Abstract. J. C e l l B i o l . , 1985; 101;334A. Martin, G., Rohrabach, H., Terranova, V. and L o i t t a , L. Structure, Function and Pathology of Basement Membranes. Ch. 3 In: Connective tissue Diseases. B.M. Wagner, R. Fleischmajer and N. Kaufman (eds.) Raven Press, N.Y., 1982, pp. 16-31. Martinez-Hernandez, A. Gay, S. and M i l l e r , E. J. U l t r a s t r u c t u r a l l o c a l i z a t i o n of Type V collagen i n rat kidney. J. C e l l B i o l . , 1982; 92:343-349- McCarthy, M. T. and Toole, B. P. Association of hyaluronate and proteoglycan with the surface of rat chondrosarcoma chondrocytes. Abstract. J. C e l l B i o l / , 1985; 101:334A Mikuni-Takagaki, Y. and Toole, B. P. Cell-substrate attachment and c e l l surface hyaluronate of Rous sarcoma virus-transformed chondrocytes. J. C e l l B i o l . , 1980; 85:481-488. Muir, H. The E x t r a c e l l u l a r Matrix JEn: C e l l and Tissue Interactions. J. W. Lash and M. M. Berger (eds.) Raven Press, New York, 1977, pp. 87-99. - 99 - Murray, J. C, S t i n g l , G., Kleinman, H. K., Martin, G. R. and Katz, S. I. Epidermal c e l l s adhere p r e f e r e n t i a l l y to Type IV (basement membrane) collagen. J. C e l l B i o l . , 1978;'86:197-201. Nevo, Z., Gonzalez, R. and Gospordarowicz, D. Ex t r a c e l l u l a r matrix (ECM) pro- teoglycans produced by cultured bovine corneal endothelial c e l l s . Connect. Tissue Res., 1984; 13:45-57. Newgreen, D. F. Adhesion to ex t r a c e l l u l a r materials by neural crest c e l l s at the stage of i n i t i a l migration. C e l l Tissue Res., 1982; 227-317. Oegema, T. R., J r . and Thompson, R. C, J r . Characterization of a hyaluronic acid-dermatan sulphate proteoglycan complex from dedifferentiated human chondrocyte cultures. J. B i o l . Chem., 1981; 256:1015-1022. Ozzello, L., Lasfarques, E. Y. and Murray, M. R. Growth promoting a c t i v i t y of acid mucopolysaccharides on a s t r a i n of human mammary carcinoma c e l l s . Cancer Res., I960; 20:600-605. Parry, G., Lee, E. and B i s s e l l , M. Modulation of the differentiated phenotype of cultured mouse mammary e p i t h e l i a l c e l l s by collagen substrata. In: Ex t r a c e l l u l a r Matrix, S. Hawkes and J. Wang (eds.) Academic Press. 1982, pp. 303-309- Parry, G., Lee, E. Y-H., Farson, D., Koval, M. and B i s s e l l , M. Collagenous substrate regulates the nature and d i s t r i b u t i o n of glycosaminoglycans produced by differentiated cultures of mouse mammary e p i t h e l i a l c e l l s . Exp. C e l l Res., 1985; 156:487-499- - 100 - Rapraeger, A. and Bernfield, M. C e l l surface proteoglycan of mammary epithe- l i a l c e l l s . J. B i o l . Chem., 1985; 260:4103-4109. Richards, J. and Nandi, S. Primary culture of rat mammary e p i t h e l i a l c e l l s . I. Effect of plating density, hormones and serum on DNA sythesis. J. Natl. Cancer Inst., 1978; 61:765-771. R i s t e l i , L. and R i s t e l i , J. Basement membrane research. Med. B i o l . , 1981; 59:185-189- R o l l , F. J., Madri, J. A., Albert, J. and Furthmayr, H. Codistribution of c o l - lagen types IV and AB^ i n basement membranes and mesangium of the kidney. J. C e l l B i o l . , 1980; 85:597-616. Ruoslahti, E., Engvall, E. and Hayman, E. G. Fibronectin: current concepts of i t s structure and functions. C o l l . Res., 1981; 1:95-128. Saito, H. Yamagata, T. and Suzuki, S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. B i o l . Chem., 1968; 243:15365-1542. Sano, J., Fujiwara, S., Sato, S., I s h i z a k i , M.., Sugisaki, Y., Yajima, G. and Nagai, Y. AB (type V) and basement membrane (type IV) collagens i n the bovine lung parenchyma: Electron microscopic l o c a l i z a t i o n by the peroxidase-labelled antibody method. Biomed. Res., 1981; 2:20-29. - 101 - Schubert, D., Lacorhiere, M. Adherons and c e l l u l a r adhesion. In: E x t r a c e l l u l a r Matrix. S. Hawkes and J. C. Wang (eds.) Academic Press, New York, 1982, pp. 109-114. Shishiba, Y. and Yanagishita, M. Presence of heparan sulphate proteoglycan i n thyroid tissue. Endocrinol, Japan., 1983; 30:637-641. Shishiba, Y., Yanagishita, M., Tanaka, T., Ozawa, Y. and Kadowaki, N. Abnormal accumulation of proteoglycan i n human thyroid adenocarcinoma tissue. Endo- c r i n o l . Japan., 1984; 31:501-507. S i l b e r s t e i n , G. and Daniels, C. Glycosaminoglycans i n the basal lamina and ext r a c e l l u l a r matrix of s e r i a l l y aged mouse mammary ducts. Mechanisms of Aging and Development, 1984; 24:151-162. S i l b e r s t e i n , G. and Daniels, C. Glycosaminoglycans i n the basal lamina and ext r a c e l l u l a r matrix of developing mouse mammary duct. Dev. B i o l . , 1982; 90:215-222. Stamatogloer, S. C. and K e l l e r , J. M. Correlation between c e l l substrate attachment i n v i t r o and c e l l surface heparan sulphate a f f i n i t y f o r fibronectin and collagen. J. C e l l B i o l . , 1983; 96:1820-1823. Takeuchi, J. Growth promoting effect of chondroitin sulfate on s o l i d E h r l i c h ascites tumor. Nature, 1965; 207:537-541. - 102 - Takeuchi, J. Effect of chondroitinases on the growth of s o l i d Ehrlich ascites tumor. Br. J. Cancer, 1972; 26:115-119. Takeuchi, J., Sobue, M., Sato, E. Shamoto, M., Miura, K. and Nakagaki, S. Variations i n glycosaminoglycan components of breast tumors. Can. Res., 1976; 36:2133-2139. Terranova, V. P., Rohrbach, D. H. and Martin, G. R. Role of laminin i n the attachment of PAM12 ( e p i t h e l i a l ) c e l l s to basement mambrane collagen. C e l l , 1980; 22:719-728. Timpl, R., Rhode, H., Robey, G., Rennard, S. I., Poidart, J. M. and Martin, G. R. Laminin - a glycoprotein from basement membranes. J. B i o l . Chem., 1979; 254:9933-9937. Timpl, R. Wiedemann, H., Van Delden, V., Mayr, H. and Kuhn, K. A network model for the organization of type IV collagen i n basement membranes. Eur. J. Biochem., 1981; 120:203-211. Toole, B. Developmental role of hyaluronate. Connect. Tissue Res., 1982; 10:93-100. Toole, B.P. Glycosaminoglycans i n Morphogenesis. _In: C e l l Biology of the Ext r a c e l l u l a r Matrix, E. Hay (ed.) Plenum Press, New York, 1981; pp. 259-294. Toole, B., Biswas, C. and Gross, J. Hyaluronate and invasiveness of the rabbit V carcinoma. Proc. N a t l . Acad. S c i . , 1979; 76:6299-6303. - 103 - Toole, B., Jackson, G. and Gross, J. Hyaluronate i n morphogenesis: I n h i b i t i o n of chondrogenesis i n v i t r o . Proc. Natl. Acad. S c i . , 1972; 69:1384-1386. Toole, B., Okayama, M., Orkin, R. , Yoshimura, M., Muto, M. and K a j i , A. Developmental roles of hyaluronate and chondroitin sulfate proteoglycans. In: C e l l and Tissue Interactions. J. ¥. Lash and M. M. Burger (eds.) Raven Press; New York, 1977, pp. 139-154. Toole, B., and Trelstad, R. L. Hyaluronate production and removal during corneal development i n the chick. Dev. B i o l . , 1971; 26:28-35* Tosney, K.W. The early migration of neural crest c e l l s i n the trunk region of avian embryo: an electron microscope study. Dev. B i o l . , 1978; 62:317-333. Turley, E. A. Proteoglycans and c e l l adhesion. Cancer Metastasis Reviews, 1984; 3:325-339. Turley, E. A. P u r i f i c a t i o n of hyaluronate binding protein f r a c t i o n that modifies c e l l s o c i a l behavior. Biochem. Biophys. Res. Commun., 1982; 108:1016-1024* Turley, E. A. The control of adrenocortical cytodifferentiation by extra- c e l l u l a r matrix. D i f f e r e n t i a t i o n , 1980; 17:93-103. Turley, E. A. and Roth, S. Spontaneous glycosylation of glycosaminoglycan substrates by adherent f i b r o b l a s t s . C e l l , 1979; 17:109-115. - 104 - Underhill, C. Interaction of hyaluronate with the surface of simian virus 40-transformed 3T3 c e l l s . J. of B i o l . Chem., 1982; 56:177-189. Underhill, C. and Toole, B. Receptors for hyaluronate on the surface of parent and virus transformed c e l l l i n e s . Binding and aggregation studies. Exp. C e l l Res., 1981; 131:419-423. Vaheri, A., Ruoslahti, E. and Mosher, D. P. Fibroblast surface protein. Ann. N. Y. Acad. S c i . , 1978; 312:1-456 Vracko, R. Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure. Am. J. Pathol., 1974; 77:314-338. Wicha, M., L i o t t a , L. A., Garbisa, S. and Kidwell, W.R. Basement membrane collagen requirements f o r attachment and growth of mammary epithelium. Exp. C e l l Res., 1979; 124:181-190. Wicha, M., Lowrie, G., Kohn, E., Bagarandoss, P. and Mann, T. Ext r a c e l l u l a r matrix promotes mammary e p i t h e l i a l growth and d i f f e r e n t i a t i o n i n v i t r o . Proc. Natl. Acad. S c i . USA, 1982; 79:3213-3217. Woodley, D. T., Rao, C. N., Hassell, J. R., L i o t t a , L. A., Martin, G. R. and Kleinman, H. K. Interactions of basement membrane components. Biochim. Biophys. Acta., 1983; 761:278-283. - 105 - Yamagata, T., S a i t o , H., Habuchi, 0. adn Suzuki, S. P u r i f i c a t i o n and p r o p e r t i e s of b a c t e r i a l chondroitinases and c h o n d r o i t i n s u l f a t a s e s . J . B i o l . Chem., 1968; 243:1523-1535- Yamanda, K., Kennedy, D., Kimata, K. and P r a t t , R. C h a r a c t e r i z a t i o n of f i b r o n e c t i n i n t e r a c t i o n s w i t h glycosaminoglycans and i d e n t i f i c a t i o n of a c t i v e p r o t e o l y t i c fragments. J . B i o l . Chem., 1980; 255:6055-6063. Y a o i t a , H., F o i d a r t , J . M. and Katz, S. I . L o c a l i z a t i o n of the collagenous component i n s k i n basement membrane. J . Invest. Dermatol., 1978; 70;191-193«

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