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

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GLYCOSAMINOGLYCAN SYNTHESIS BY NORMAL HUMAN MAMMARY E P I T H E L I A L C E L L S I N PRIMARY CULTURE -'  by NANCY JONES B.Sc,  The U n i v e r s i t y  of Alberta,  1979  A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in 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 to the required  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA J u n e , 1986  (c) N a n c y J o n e s  »E-6  In presenting  this  thesis i n partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e of B r i t i s h Columbia, I agree that it  freely  the L i b r a r y s h a l l  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 .  agree t h a t p e r m i s s i o n f o r extensive for  University  s c h o l a r l y p u r p o s e s may  for  financial  of  f^\Q  jrt> f U X - j  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3 Date  n/an  Columbia  my  It is thesis  s h a l l n o t be a l l o w e d w i t h o u t my  permission.  Department  thesis  be g r a n t e d by t h e h e a d o f  copying or p u b l i c a t i o n of t h i s  gain  further  copying of t h i s  d e p a r t m e n t 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 . understood that  I  make  written  ABSTRACT  The  extracellular  tiation.  (ECM) influences  Glycosaminoglycans  (proteoglycans), GAG synthesis cells.  matrix  This  are a major  research  mammoplasties  were  centres  complexed  of the ECM a f f e c t i n g  confirmed the e p i t h e l i a l nature  days 3-4,  9-11 and 17-18.  to  Mammary ,tissue  medium  fractions  cell  behavior.  single  obtained from reduction  cells.  collagen  The  gels  at  of the cultures.  epithelial  2-2.5x10  To measure  H-glucosamine f o r 24 hours at  cell  cells/cm  Ultrastructural  studies  GAG synthesis, 3 time  points;  The cultures were p r o l i f e r a t i n g at the early  point and had reached a stationary phase at the l a t e r and  protein  GAG production by normal human  hydrated  were incubated with  with  on studying  i n medium containing 5$ FCS and 5ug/ml of i n s u l i n .  cultures  and d i f f e r e n -  i n animal models and malignant  i n culture.  onto  or  growth  extensively  dissociated  was seeded  alone  component  has been studied  mammary e p i t h e l i a l c e l l s  population  (GAGs),  cell  were  analyzed  for  GAGs  as  degradation and c e l l u l o s e acetate electrophoresis.  time points. identified  time  C e l l , ECM by  enzyme  At day 4, when c e l l s were  a c t i v e l y growing, the majority of GAGs produced were released into the medium fraction acid  (75-80$).  The predominant GAG was the nonsulfated GAG, hyaluronic  (HA). Of the sulfated GAGs chondroitin sulfate  only 18$ of t o t a l the l a t e r of t o t a l  GAGs; dermatan  sulfate  (DS) synthesis was n e g l i g i b l e .  time periods, when cultures had ceased  At  growing a higher percentage  GAG was incorporated into an ECM (50-65$).  p r e f e r e n t i a l l y incorporated  (CS) 4 and 6 comprised  The sulfated GAGs were  into the ECM, CS 4 and 6 comprising 70$ and DS  comprising 30$.  The marked difference i n type and location of GAGs produced  was  a  not merely 5  densities 4.  (5x10  function  of time  i n culture.  Cultures  seeded  at  high  2 cells/cm ) were  not p r o l i f e r a t i n g when terminated  at day  Their GAG p r o f i l e was s i m i l a r to that of lower density cultures at day  10.  This  data  provides  a  baseline  from  which  we  can  determine  i f  c e l l - s y n t h e s i z e d GAGs, p l a y a r o l e i n m a i n t a i n i n g d i f f e r e n t i a t e d and m a l i g n a n t phenotypes.  - iii  -  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OP TABLES  vii  LIST OF FIGURES  v  ACKNOWLEDGEMENTS  i  i  i  ix  ABBREVIATIONS..  x  CHAPTER 1 - INTRODUCTION GENERAL INTRODUCTION I)  II)  1  The Basement Membrane  •  1  1)  Collagen  2  2)  Fibronectin  4  3)  Laminin  4  4)  Entactin  5 5  Proteoglycans 1)  Hyaluronic Acid  11  2)  Sulfated Proteoglycans  16 '.  III)  Mammary E p i t h e l i a l Cells  19  IV)  Proteoglycans and Malignancy  20  V)  Thesis Problem Formulation  25  CHAPTER 2 - MATERIAL AND METHODS I)  Dissociation Procedures  27  II)  Preparation of Collagen Gels  28  III)  C e l l Culture Procedures  29  IV)  Radiolabelling Procedures  29  V)  Resolution into Fractions  30  VI)  Extraction of Glycosaminoglycans  30  - 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 . . . . .  32  2)  Chondroitin Sulfate I d e n t i f i c a t i o n  34  3)  Heparan Sulfate I d e n t i f i c a t i o n  34  VIII) S c i n t i l l a t i o n Counting  35  IX)  Calculation of Assay Results  37  X)  I d e n t i f i c a t i o n of GAG using Electrophoresis  38  XI)  Film Developing for Autoradiography  38  XIII) Electron Microscopy  40  CHAPTER 3 - RESULTS I)  Growth  41  II)  Total GAG Synthesis  41  III)  Hyaluronic Acid Synthesis  46  IV)  Chondroitin Sulfate Synthesis  48  V)  Dermatan Sulfate Synthesis  48  VI)  Heparan Sulfate Synthesis  55  VII)  Electrophoresis Results  55  VIII) Cultures Labelled with  55  S-Sulfate  57  IX)  Autoradiography  61  X)  Electron Microscopy  61  CHAPTER 4 - DISCUSSION I)  Culture Status  64  II)  D i s t r i b u t i o n of Synthesized GAGs  65  - v -  Page III)  Types o f S y n t h e s i z e d GAGs  68  1)  Hyaluronic Acid  68  2)  S u l f a t e d GAGs  74  IV)  O v e r a l l Amount o f S y n t h e s i z e d GAGs  78  V)  F u t u r e Research  81  VI)  Summary  83  APPENDICES  •••• 84  BIBLIOGRAPHY  87  ML  LIST OF TABLES  PAGE Table I  The Proteoglycans  10  Table I I  D i s t r i b u t i o n of -glucosamine l a b e l l e d 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  ^H-glucosamine i n c o r p o r a t i o n i n t o 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 t o t a l )  56  Table V  Percentage of Sulfated GAG i n the Medium and ECM of high and Low Density Cultures Labelled with 3 5 - S u l f a t e  58  Table I I I  S  - vii -  LIST OF FIGURES Page Fig.  1  Schematic representation of the basement membrane  Fig.  2  Chemical Structures of HA (non-sulfated) and DS ( s u l f a t e d ) .  7 8  Fig.  3  The l i n k t r i s a c c a r i d e  Fig.  4  Schematic representation of postulated ECM 12  interactions Fig.  5  Flow chart of i d e n t i f i c a t i o n  Fig.  6  Enzyme d i g e s t i o n over time  Fig.  7  Growth study of normal human mammary e p i t h e l i a l seeded at low density T o t a l ^H-glucosamine incorporation i n t o GAG i n  Fig.  Fig.  8  9  F i g . 10 F i g . 11  3  . . 33  of GAG by enzymes  36 cells 42  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  45  ^-glucosamine incorporation i n t o HA/cell  50  ^H-giucosamine incorporation i n t o C S / c e l l a) ^H-glucosamine incorporation i n t o HA, CS and D S / c e l l i n the medium f r a c t i o n b) ^H-glucosamine incorporation i n t o HA, CS  51  and D S / c e l l i n the ECM f r a c t i o n  52 53  F i g . 12  ^H-glucosamine incorporation i n t o D S / c e l l  54  F i g . 13 F i g . 14  5 5 s _ l f a t e incorporation i n t o CS and DS Autoradiograph of glucosamine incorporated GAG i n a l l f r a c t i o n s of an e a r l y s t a t i o n a r y culture E l e c t r o n Micrograph of normal human mammary c e l l s i n culture  59  F i g . 15  s u  - viii -  ••• 62 63  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 techniques and to Dr. Terry Crawford at UBC  who  taught me  taught me  GAG  analysis  the electrophoretic  technique and was there to answer my numerous questions. To Dr. Joanne Emerman, my advisor, f o r her perseverance i n teaching me principles throughout  of my  cell  biology,  her  continued  time i n her laboratory and  enthusiasm  and  f o r labouring through  the  encouragement 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 P h i l Jones who  helped with the typing but mostly f o r 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 I n s t i t u t e of  Canada.  - ix -  ABBREVIATIONS ECM PG GAG HA CS HS DS BM  -  e x t r a c e l l u l a r matrix proteoglycan. glycosaminoglycan hyaluronic acid (hyaluronate) chondroitin sulphate heparan sulphate , dermatan sulphate basement membrane  INTRODUCTION  Historically, inert  the e x t r a c e l l u l a r matrix  substrate  providing  (ECM) was considered  support f o r c e l l s  s i g n i f i c a n t manner with these c e l l s .  to be an  and not i n t e r a c t i n g i n any  Over the l a s t two decades, however,  information has accumulated i n d i c a t i n g that t h i s was an o v e r - s i m p l i f i c a t i o n of i t s f u n c t i o n .  The i n t e r a c t i o n s 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 as  growth  i n such important  (Gey e t a l . , 1974),  differentiation  migration  (Emerman and P i t e l k a ,  areas of c e l l f u n c t i o n i n g  (Greenberg  1977, Emerman  et a l . , 1981),  et al.,1977, 1981;  Kleinman et a l . , 1981; Parry et a l . , 1982) and determination (Emerman et al.,1979 , Gospodarowicz et a l . , 1978). macromolecules  from  four  major  classes  glycoproteins and e l a s t i n (Hay, 1981). structurally  stable  material  that  -  of c e l l shape  The ECM i s composed of  collagen,  proteoglycans,  Together these components form a  lies  under  epithelia  and surrounds  connective t i s s u e c e l l s . The Basement Membrane The  term  basement membrane  components which include  refers  to an organized  collagen, proteoglycans  are associated with the basal surfaces  complex  of ECM  and glycoproteins,  that  of e p i t h e l i a l c e l l s whenever they  contact connective t i s s u e (Vracko, 1974).  To f u r t h e r c l a s s i f y the basement  membrane, i t can be described  to i t s morphological  according  appearance  which include 3 or 4 major zones (Martin et a l . , 1982): l ) the lamina l u c i d a externa (or lamina rara) which i s an e l e c t r o n lucent region 20 to 40 nm wide found j u s t 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 l a y e r 20 to 100 nm wide and contains a meshwork of f i n e filaments g i v i n g i t an e l e c t r o n dense appearance, 3) the  JL  lamina lucida interna which i s an electron and found between  lucent region of variable wide  the lamina densa and the underlying connective  (Figure l ) and the r e t i c u l a r lamina.  The r e t i c u l a r lamina i s a meshwork of  fine collagen f i b e r s between the basal lamina and connective The major (Kleinman,  collagen  1982).  component present  This  has been  tissue  tissue.  i n the ECM i s Type IV collagen  demonstrated  using  various  techniques  including autoradiography and immunolocalization (Sano et a l . , 1981). IV collagen was f i r s t solubilize  i d e n t i f i e d by Kefalides i n 1966.  a unique collagen protein  glomerular basement membranes.  after  He was able  pepsin digestion  While i t was i n i t i a l l y thought that Type IV  chains  designated  pro a 1  (IV) (Crouch et a l . , 1980; G l a n v i l l e et a l . , 1979). III,  Type  molecules  IV collagen  does  not  c a l l e d procollagens.  to  from canine  contained a single type of chain i t has been subsequently proven that collagen has two d i s t i n c t  Type  arise  Instead,  this  (IV) and pro a 2  Unlike Types I, II and  biosynthetically the pro a I  from  precursor  (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 l o c a l i z e d 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).  however,  The actual  function of Type IV collagen  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  of  interact  the collagen  continuous  type  network  IV molecules  (Timpl  et  i s not known,  a l . , 1981).'  with  each  Other models  that l i k e  other have  ends  forming a also  been  proposed and w i l l be discussed l a t e r i n the Introduction. Another collagen present Type V collagen,  i n basement membranes, as well as other areas,  was o r i g i n a l l y isolated  from placenta  (Bailey  et a l . ,  - 3  -  Epithelial Cells  Lamina lucida Lamina dens.a  Connective  Fig.  1  Tissue  S c h e m a t i c r e p r e s e n t a t i o n o f t h e basement m e m b r a n e . ( M a r t i n e t a l . , 1982.)  1979; Burgeson et a l . , 1976; Chung et a l . , 1976). chains, a 1 (v)  distinct  and  a 2 (V).  basement membranes i s unclear.  The  I t a l s o contains two  role  of t h i s  Immunolocalization studies  collagen i n indicate i t s  presence i n the lamina densa ( R o l l et a l . , 1980) as w e l l as emanating from there  to the underlying  1982).  connective t i s s u e  (Martinez-Hernandez  This l a t t e r f i n d i n g has l e d Martinez-Hernandez  et  al.,  et a l . to suggest  that Type V collagen may act by anchoring d i s s i m i l a r t i s s u e types together. The glycoproteins found i n the basement membrane include  fibronectin  (Vaheri et a l . , 1978), laminin (Timpl et al.,198l) and e n t a c t i n ( C a r l i n et a l . , 1981). identical  F i b r o n e c t i n i s a large glycoprotein (MW 440,000) made up of 2  220,000 Dalton chains l i n k e d  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 t i s s u e s (Ruoslahti et al.,  1981).  I t s f u n c t i o n i n the ECM has been r e l a t e d  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 l o c a l i z e d by immunoelectron microscopy to the lamina l u c i d a as w e l l 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 f i b r o b l a s t s (Murray et  al.,  1978), r a t hepatocytes (Hooper  al.,  1976) and established  1978).  cell  et a l . ,  lines  such as CHO  From the studies to date i t appears  f i b r o n e c t i n i s the glycoprotein used  1976), myoblasts and  3T3  (Ketley et (Grinnell,  reasonable to conclude  as an attachment  that  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 f o r c e r t a i n but i t has been shown to bind to numerous c e l l types as w e l l as to collagens (Type I to V) and proteoglycans (Woodley et a l . , 1984).  - 5 g  Laminin i s a very  large glycoprotein  Engelbreth-Holm-Swarm  (EHS) tumor ( T i m p l  basement membranes ( T i m p l chains a  been  10 ) f i r s t  isolated  e t a l . , 1979) and  e t a l . , 1980).  I t i s composed  found  in a l l  o f two t y p e s o f  formation  (Liotta,  arms) and 1 c h a i n implicated  in  1983),  being  the  with  3  chains  400,000 D a l t o n s  adhesion  being  (long  mechanisms  200,000  arm).  between  epithelial  cells  (Terranova  et  a l . , 1980)  Daltons  L a m i n i n has  ECM  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 breast  from t h e  (200,000 and 400,000 D a l t o n s ) t h a t a r e l i n k e d by d i s u l f i d e bonds i n  cross  (short  (MW  components,  types i n c l u d i n g  guinea  p i g epidermal  c e l l s , b o v i n e l e n s 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  cells.  L a m i n i n b i n d s p r e f e r e n t i a l l y t o Type IV c o l l a g e n and promotes the a d h e s i o n o f 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 been shown t h a t being  laminin  to heparin  binds  ( T e r r a n o v a e t a l . , 1980).  to proteogylcans  and heparan s u l f a t e  c e l l s such as m e t a s t i c  the highest  affinity  ( D e l Rosso e t a l . , 1981).  Certain  T241 f i b r o s a r c o m a  adhere t o Type I c o l l a g e n v i a l a m i n i n . synthesize  their  own  probably true  of c e l l s  1978).  that  Cells  laminin  (Terranova  with  I t has a l s o  cells Cells  (Murray e t a l . , 1980)  will  t h a t adhere v i a l a m i n i n can  e t a l . , 1980)  and  the same i s  r e q u i r i n g f i b r o n e c t i n f o r a d h e s i o n (Dessau e t a l . ,  are capable  of synthesizing  both  proteins  are  likely  c a p a b l e o f u t i l i z i n g b o t h f o r a d h e s i o n ( F o i d a r t e t a l . , 1980). E n t a c t i n i s the most r e c e n t l y d i s c o v e r e d  g l y c o p r o t e i n and a l s o  appears  t o be a component o f many basement membranes (Bender e t a l . , 1981; C a r l i n et  a l . , 1981).  epithelial cells et  a l . were  surfaces.  I n an after  able Little  ultrastructural  of  e x p o s i n g them t o a n t i b o d i e s  to l o c a l i z e is  study  known  entactin  i n close  as  regarding  yet  g l y c o p r o t e i n i n t h e basement membrane.  the basal  sufaces  of  t o the p r o t e i n , Bender association the  with  function  of  these the  - 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  polymers of repeating disaccharides with either carboxyl or sulfate  chain 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, HA molecule .  to 2,000 to 3,000 residues seen i n the average  The proteoglycan i s d i s t i n c t from glycoproteins because of  the high percentage (90-95$) of carbohydrate. less than 60% carbohydrate. hyaluronic sulfate,  acid,  identified cartilage  S t r u c t u r a l l y i d e n t i f i e d proteoglycans include  chondroitin sulfate  heparin  and heparan  proteoglycan and cornea,  Glycoproteins t y p i c a l l y have  but,  4,  chondroitin sulfate  sulfate. because  6, ^ dermatan  (Keratan " sulfate it  is  related  is  almost  i t w i l l not be discussed f u r t h e r ) .  also  an  solely  to  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 t h e i r native state are linked to a protein core with the exception of hyaluronic a c i d .  An average protein core may contain 1900-2000 amino acid  residues which are generally s e r i n e - r i c h . that  the protein core  (Rapaeger  can vary markedly i n size  et a l . , 1985).  a l . , 1985;  Chang et  another  section  100-150  side  Figure 2.  and amino acid  l o c a t i o n of synthesized proteoglycans a l . , 1985).  of the t h e s i s .  chains  indicates content  This difference i n protein core may i n part be  responsible f o r the f i n a l et  More recent research  of a  This  Attached  particular  will  be discussed  to the protein  carbohydrate  (Rapaeger further i n  core  sequence  as  are from shown i n  The protein core i s attached covalently to the carbohydrate side  HA  0^ ^  CHo /  COO" 0  GLUCURONIC  ACID  H  N--ACETYLGLUCOSAMINE  DS  S0 IDUR0N1C  N-ACET YLGAL A C T O S A M I N E ( 4 - SUL F A T E RESIDUE)  ACID  Fig.  3  2  Chemical s t r u c t u r e s of HA(non-sulfated) and D S ( s u l f a t e d ) .  - 8 -  Fig.  3  The common l i n k t r i s a c c a h a r i d e b e t w e e n t h e GAG component and t h e p r o t e i n c o r e o f PG.  - 9 chain  via  a  xylose-serine  linkage  (Figure  3).  Historically,  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 u n i t .  difference  that  proteoglycans  several  (Yamagata  of the degradative et  al.,  1968).  It  i s on the basis  of t h i s  enzymes d i f f e r e n t i a t e  HA, CS 4  and CS 6  between  a l l have  glucuronic acid as one monosaccharide whereas dermatan sulfate and heparan sulfate have either glucuronic or iduronic a c i d .  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  sulfation.  area  where  proteoglycans  differ  is  i n their  degree  of  The following are l i s t e d according to degree of s u l f a t i o n 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 is  similar  to  CS 4  but has  iduronic acid (Kraemer,  1979).  epimerized  3-D-glucuruonic  acid  to  8-L-  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 iduronic  acid  (Kraemer,  interspersed  with  1979).  They may contain sequences  sequences  of  CS 4  non-sulfated or disulfated on the N-acetyl-galactosamine  or  CS 6  residue.  of  either Heparan  sulfate i s unique i n that i t possesses an a c i d - l a b i l e sulfate group linked to  the  amine  proteoglycan.  group  of  the hexosamine  residue.  HA i s  a  non-sulfated  A summary of the proteoglycans can be seen i n Table I .  Proteoglycans  have had several  role as ECM components.  functions attributed  to them i n t h e i r  They are generally believed to be  involved i n  - 10 TABLE 1: PROTEOGLYCANS  1  Sulfates/ Disaccharide Unit  Protein Core  Glycosaminoglycan  Molecular Weight  Repeating Disaccharide A B  Hyaluronic a c i d  4,000 8,000,000  D-glucuronic acid  N-acetylD-glucosamine  Chondroitin Sulfate-4  500 50,000  D-glucuronic acid  N-acetylgalactosamine  0.2-1.0  YES  Chondroitin Sulfate-6  500 50,000  D-glucuronic acid  N-acetylgalactosamine  0.2-2.3  YES  Dermatan Sulfate  15,000 40,000  D-glucur o n i c OR L-iduronic acid  N-acetylgalactosamine  1.0-2.0  YES  Heparan Sulfate  5,000 12,000  D-glucur o n i c OR L-iduronic acid  N-acetylglucosamine  0.2-3.0  YES  Heparin  6,000 25,000  D-glucur o n i c OR L-iduronic acid  N-acetylglucosamine  2.0-3.0  YES  1  A l b e r t et al.,1983-  NO  - 11 cell-substrate  adhesions  (Toole,  Both HA and HS are present Rapraeger et a l . , 1985).  1982;  Culp,  on the surfaces  1976;  Culp  of c e l l s  et  al.,  1979).  (Culp et a l . , 1979;  These proteoglycans have also been shown to bind  to f i b r o n e c t i n (Yamada et a l . , 1980) and laminin (Kleinman et a l . , 1981). Prom t h i s information i t would appear that proteoglycans not only help l i n k c e l l surface help  to  to basement membrane (cell-associated  stabilize  basement  membrane components  proteoglycan)  but also  (BM proteoglycan)  within  this s t r u c t u r a l l y defined area. How  do  structurally  these stable  molceules  discussed  basement  membrane?  manner the glycoprotein present,  thus  far  interact  To summarize  to  form  briefly,  i n some  with the help of proteoglycans,  enhances  or enables the c e l l to adhere to a p a r t i c u l a r type of collagen. numerous theories 4.  on the actual structure  They mainly d i f f e r  i n the arrangement  on the plasmalemma.  1985)  using rotary  of the components  bound  to Type  heparan terminus. the  sulfate  IV collagen bound  to  studies  microscopy,  found  81 nm from the carboxyl Type  IV collagen  within the  one or more receptors f o r  More recent  shadowing electron  There are  and several are shown i n Figure  basement membrane although they a l l demonstrate ECM components  the  206  (Laurie  et a l . ,  laminin mainly  terminus  nm from  the  and large carboxyl  Laurie et a l . postulate a model whereby heparan sulfate binds to  collagen molecule i n such a way as to allow i n t e r a c t i o n between the  free long arm of laminin and the c e l l .  However, they have also  suggested  other possible models from t h e i r data and this area of i n t e r a c t i o n 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 -  Fig. 4  Schematic representations interactions. B a s e d on Hynes(1981) A. Kleinman etal(1981) B. Toole(1981) C. Sugrue and Hay(1982) D. HA= PG = FN = C0= LN =  of postulated  ECM  Hyaluronic acid Proteoglycan Fibronectin Collagen Laminin 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 models 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 components.  - 13 -  Hyaluronic Acid HA has .heen (Toole,  1977)•  implicated i n m o t i l i t y and growth Toole et a l . (1971) observed that  of developing the major  tissue  proteoglycan  being synthesized during chick embryo corneal migration i s HA. They also observed that the onset of HA synthesis a c t u a l l y occurs before the onset of migration. cells  HA has also been shown to promote detachment  including  neural  crest  Abatangelo et a l . , 1982).  cells  of a variety of  and dog kidney c e l l s  (Turley,  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  a hydrostatic  through  allow  pressure  capable  of opening pathways  f o r migration of c e l l s .  adhesions  or bonds  Coupled with t h i s  formed by HA (Toole,  (Turley,  and fluorescent  1977).  1984).  "lubricate"  hyaluronate  an area  (Culp,  1976)  unimpeded by strong c e l l - s u b s t r a t e HA  has  also  differentiation  been  and allow  the tissue  HA has been  occurs  Its presence here may be related  exert to  function are the weak  footpads of motile f i b r o b l a s t c e l l s (Culp, 1976; Latterra radiolabelled  1984;  noted i n  et a l . , 1982) and  in  retraction  fibers  to the a b i l i t y of HA to  the  cell  to  slide  along  attachments.,  implicated  (Toole et a l . , 1972).  in  the  prevention  Small amounts  of  precocious  of hyaluronate added  to high density cultures of stage 26 chick embryo somite c e l l s i n h i b i t the formation with  of c a r t i l a g e - l i k e  nodules  this finding are the< results  that  otherwise  of studies  develop.  on chick embryo  c e l l s and chick embryo limb mesoderm hyaluronidase a c t i v i t y These  cells,  as well  as  the corneal  cells,  show  a  Coinciding sclerotomal  (Toole,  large  1972).  increase  hyaluronidase a c t i v i t y at the end of the active c e l l migration stage. enzyme removes HA and s i g n i f i e s the onset of d i f f e r e n t i a t i o n . of  HA on growth as  opposed  to migration  are not well  in This  The effects  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 e f f e c t of HA  on each. I t has been suggested that w i t h regard to c e r t a i n c e l l types, HA does not promote locomotion. little The  Neural c r e s t  or no locomotion when seeded  adhesion  originally  ability  thought.  of HA  also  cells  (Erickson et al.,1983) show  on a hyaluronate-coated substratum.  now  appears  Although, as stated  more c e l l  earlier,  specific  than  HA i s associated with  weak cell-substratum and c e l l - c e l l i n t e r a c t i o n s , f o r c e r t a i n 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 s u l f a t e d proteoglycan substratum (Toole, 1982) and the  attachment  of chondrocarcinoma  (Mikuni-Takagaki et a l . , 1980).  cells  to  tissue  culture  surfaces  The type of involvement of HA i n adhesions  may be related not only to the c e l l type but a l s o the type of substrate the c e l l i s adhering t o , the time i n c u l t u r e and the developmental (Turley, 1984). binding  sites,  sequence  This may r e l a t e to the a b i l i t y of a c e l l to synthesize HA the a v a i l a b i l i t y  of HA  or the number of binding  a v a i l a b l e to i n t e r a c t ( U n d e r h i l l et a l . , 1981).  sites  In general, HA has mainly  been found to mediate adhesion to non-fibronectin substrates (Schubert e t 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  proteoglycans containing CS 4, Oegema et a l . , 1981). these molecules.  on the p r o t e i n  core  of some  CS 6 or DS f o r HA ( H a s c a l l et al.,1981;  As a r e s u l t , HA i s able to form large aggregates of  I t may be v i a t h i s mechanism that  cell-associated  HA  - 15 ( T u r l e y , 1984) may a t t a c h to p r o t e o g l y c a n s thereby  anchoring  molecule  present  the c e l l t o t h e s u b s t r a t u m .  i s not f u l l y  understood  i n t h e basement membrane  The r o l e o f HA as a b i n d i n g  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 e t a l . , 1985; Lacey e t a l . , 1985; Marks e t a l . , 1985).  McCarthy  e t a l . (1985) examined t h e mechanisms by which e x t r a c e l l u l a r a g g r e g a t e s o f PGs  a r e maintained  determine  i n both  a t the chondrocyte normal  50-60$ o f t h e aggregated interaction  wtih  hyaluronidase.  these r e c e p t o r s  al.,  proteoglycans  In preliminary  identified  sites  surface.  and Swarm  hyaluronate,  hyaluronate-binding  being  chick  cell  r a t sarcoma  are held  which  is  studies  they  a t the c e l l  a t the c e l l  have  surface  been  will  ( T u r l e y , 1984).  As r e s e a r c h  become c l e a r e r , however,  continues  at this  able  to  identify  and b e l i e v e  i t may be v i a  HA b i n d i n g  s i t e s are also  for  ready detachment and m o t i l i t y  i n t h i s area  point  assume t h a t HA i s n o t s i m p l y a p r o t e o g l y c a n  i t would  that hydrates  of c e l l s ,  although  (Lacey e t  t r e a t m e n t w i t h DON w i l l acts  by i n h i b i t i n g  the f o r m a t i o n  cell  movement i n v i t r o  the synthesis  on d i f f e r e n t c e l l  types  t o be c e l l - t y p e s p e c i f i c .  migrate i n t o collagen gels  plastic  culture  dish  1984).  Conversely,  f o r GAG. reveals  F o r example,  reasonable  t i s s u e and a l l o w s  (Turley,  cell  1980).  the r e s u l t s  t h e response  chick  DON  v i a i n h i b i t i o n of  Here a g a i n  that  to  (HA and o t h e r s ) as  o f glycosaminoglycans  o f glucosamine, a precursor  experiments  appears  stop  be  crest  t h e r o l e o f HA  assistance with  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 o f p r o t e o g l y c a n s  will  streptomyces  1985), a d u l t c h i c k b r a i n (Marks e t a l . , 1985) and c h i c k n e u r a l  cells  of  to  that  surface v i a  including 3 T 3 cells  cells  able t o  chondrocytes  susceptible  t h a t t h e HA-aggregates a t t a c h . on a number o f o t h e r  They were  t o HA  heart f i b r o b l a s t s  (Bernanke e t a l . , 1979) and move over a  when h y a l u r o n a t e i t s addition  to  i s added 3H3  cell  t o the medium cultures  (Turley,  (Turley,  1984)  - 16 leucocytes al., the  ( F o r r e s t e r e t a l . , 1981) and n e u r a l  1983; Newgreen e t a l . , 1982) e i t h e r does n o t a f f e c t case  o f t h e 3T3 c e l l s ,  l a t t e r two c e l l receptors  motility?  or i n h i b i t s  Again,  o r do  motility,  these  cells  type  utilize  I t i s also possible  o f the  o f HA, HA  t o HA  another molecule  t h a t HA may b i n d  as i n  as i n the case  n o t respond  the functions  o f HA  under any  o r mechanism f o r  competitively with  i n t h e ECM  basement membrane i t appears i n some instance's facilitate  motility  and i n o t h e r s  cell-substrate proximity.  lamellae i n others 1984).  motility,  other  causing i n h i b i t i o n of m o t i l i t y .  summarize  maintain  (Erickson et  i s i t a question of a v a i l a b i l i t y  r a t h e r , do they  proteoglycans To  types.  or both  circumstances;  and  crest cells  I t has been r e p o r t e d  specifically  to a i d i n c e l l  the  detachment  t o a c t as an a d h e s i o n m o l e c u l e t o  I n some  i t i s dispersed  and  cells  HA  i s found  i n cell  surface  (Turley,  over t h e e n t i r e c e l l  t o 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 o f t h e c y t o s k e l e t o n o f 3T3 c e l l s (Lacey e t a l . , 1985). Sulfated  Proteoglycans  Sulfated (Trelstad  proteoglycans  have  been  linked  e t a l . , 1974; P r a u s e t a l . , 1971).  with  cytodifferentiation  In their  studies  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 with from  t h e onset o f d i f f e r e n t i a t i o n , HA  to  sulfated  proteoglycans).  the major p r o t e o g l y c a n  proteoglycan  (they  T o o l e e t a l . (1977) a l s o a n a l y s e d  limb chondrocytes f o r c h o n d r o i t i n s u l f a t e . that  t h e CS  (decreased  did  formed  binding)  a t the e a r l i e r  as  with  identify  specific  day i s l e s s  experimentally  reactive  to  and l e s s s u l f a t e d and o f a s m a l l e r m o l e c u l a r  HA,  the e f f e c t s  of  CS  changes  4 and 8-day c h i c k embryo  They determined  t h a n t h e CS from t h e l a t e r day when t h e c e l l s However,  not  present  collagen weight  are further differentiated. on  various  cell  types  are  - 17 dissimilar.  For example, when added to c u l t u r e s 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 from the substrate.  Due  to the poor spreading a b i l i t y  movement associated with CS  (Turley et a l . ,  and  of c e l l s  rapidity  1979; Erickson, 1984),  proteoglycan i s thought to have weakly adhesive p r o p e r t i e s .  of  this  In some c e l l s ,  such as human melanoma c e l l s ( R e i s f e l d , 1984), an antibody s p e c i f i c to CS stops  the  initial  spreading  of  these  cells  over  basement  membrane  components. CS has been found at the c e l l where i t i n t e r a c t s  with  surface of mammalian s k i n  the c e l l  fibroblasts  membrane v i a i t s carbohydrate chains  (Saito et a l . , 1972) or v i a i t s p r o t e i n core, which does not appear to be intercalated  with  the  cell  membrane but  molecule ( G l o s s y l et a l . , 1983) • binding  site  for this  held  by  another  "attachment"  I t i s not known i f there i s a s p e c i f i c  proteoglycan but  sites  have been i s o l a t e d  appear to have a high a f f i n i t y f o r CS and CS hybrid or dermatan  which sulfate  molecules ( G l o s s y l et a l . , 1983; Truppe et a l . , 1978). Heparan s u l f a t e , u n l i k e HA and CS, i s known f o r 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 f i b r o n e c t i n 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 f o r some c e l l types, i n c l u d i n g neural crest (Erickson et a l . , 3T3  cell  lines  (Turley,  1984)  and  neuronal c e l l s  1983)  (Stamatoglou et a l . ,  1983), the a d d i t i o n of HS to the c u l t u r e medium supports both attachment and spreading. Conversely, heparan reduces attachment of CHO et  al.,  1982)  and  M3A  cells  reason f o r t h i s discrepency may  to f i b r o n e c t i n - c o n t a i n i n g  c e l l s (Klebe  substrates.  One  be i n the method of analysing attachement  - 18 of  cells.  L a t e r r a e t a l . (1983) f i n d  that  i f they l o o k e d a t  r a t h e r t h a n c e l l - r e m o v a l a s s a y s , t h e a d d i t i o n o f heparan  attachment  to t h e i r cultures  o f f i b r o b l a s t s does n o t i n h i b i t a d h e s i o n t o f i b r o n e c t i n and t r e a t m e n t h e p a r i n a s e does n o t d e t a c h t h e c e l l s . binds p r e f e r e n t i a l l y  to laminin  Woodley e t a l . (1984) f i n d  over f i b r o n e c t i n and t o Type  with  that  HS  IV c o l l a g e n  over Types I t o I I I . They d i d n o t f i n d t h a t t h e a d d i t i o n o f HS t o e i t h e r a Type  IV c o l l a g e n  or laminin  premixed  coated  culture  b i n d i n g between t h e two ( c o l l a g e n and l a m i n i n ) .  dish  increases  Others have r e p o r t e d  the 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 e t a l . , 1980).  I t would appear, however, t h a t HS i s , i n some way, r e l a t e d t o  adhesion as i t i s o f t e n l o c a t e d al.,  1984).  HS  has been  i n f o o t p a d s and a d h e s i o n  shown  to bind  to r a t l i v e r  sites  (Lark et  cells  via  its  c a r b o h y d r a t e c h a i n s ( K j e l l e n e t a l . , 1977) and t o mouse mammary c e l l s v i a direct  intercalation  (Rapraeger  of  e t a l , 1985).  the  protein  core  into  the  plasma  membrane  A l t h o u g h i t has been shown, as s t a t e d , t h a t  HS  does enchance b i n d i n g o f g l y c o p r o t e i n s and c o l l a g e n t o t h e c e l l s u r f a c e i t has n o t been c l e a r l y demonstrated  t h a t i t a c t s as a c e l l r e c e p t o r f o r these  m o l e c u l e s ( C u l p e t a l . , 1982).  I t i s known t h a t , u n l i k e CS, which  aggregated  site  by HA v i a a b i n d i n g  c a n be  on the CS p r o t e i n , HS  does n o t have  such a b i n d i n g s i t e o r , a t l e a s t , does n o t aggregate around  an HA m o l e c u l e  (Culp et a l . ,  1982).  HS has a l s o been i m p l i c a t e d i n k i d n e y f u n c t i o n where i t i s found cell  membrane  associated  and  membrane (Kanwar e t a l . , 1979). in  providing  also  present  i n the glomerular  I t i s believed  a s e l e c t i v e b a r r i e r to molecules  kidney tubules.  A HS m o l e c u l e  t o be  basement  t o p l a y an i m p o r t a n t r o l e trying  (MW 750,000) was i s o l a t e d  t o pass  i n t o the  from t h e basement  membrane o f k i d n e y tumor t i s s u e ( H a s s e l l e t a l . , 1980) and an a n t i b o d y t o  - 19 t h i s molecule  ( t o t h e p r o t e i n c o r e ) demonstrated  i t s presence  i n EHS tumor  basement membrane and i n normal k i d n e y t i s s u e basement membranes (Kanwar e t al.,  1979).  To summarize, t h e s u l f a t e d p r o t e o g l y c a n s appear t o p l a y a major r o l e i n cell-substrate basement  adhesion,  membrane  beginning  and  o r g a n i z a t i o n and  are present  to differentiate  migrating.  continuing  i n greater  or at least  stability  amounts  t o have stopped  when  p r o t e o g l y c a n s i n v o l v e d i n f u n c t i o n s such  as a d h e s i o n  a r e n o t i n v o l v e d i n t h e same f u n c t i o n i n o t h e r c e l l type o f p r e t e o g l y c a n does f o r one c e l l another c e l l  cells  growing  The same c o u l d be s a i d o f the p r o t e o g l y c a n HA.  o f the are  and / o r  I t appears  that  f o r some c e l l  types  t y p e s : i e . , what one  type a n o t h e r p r o t e o g l y c a n does f o r  type o r , some f u n c t i o n s may n o t be r e q u i r e d by a l l c e l l s .  would t h e n seem i m p o r t a n t a specific cell  It  to a n a l y s e t h e f u n c t i o n s o f p r o t e o g l y c a n s w i t h i n  type and n o t assume t h a t what one p r o t e o g l y c a n does i n one  instance w i l l hold true f o r a l l c e l l s . Mammary E p i t h e l i a l In  Cells  the a r e a o f mammary e p i t h e l i a l c e l l  r e s e a r c h i t has been r e p o r t e d by  S i l b e r s t e i n and D a n i e l s (1982) t h a t growing mouse mammary e p i t h e l i a l synthesize  predominantly  membrane.  The  non-growing  s u l f a t e d GAG, which Parry  in  PG  in  s y n t h e s i s depending  different  (1979) f i n d  epithelial  analysed  c e l l s _in v i t r o .  maintained.  and i t i s found  i s further identified  e t a l . (1985)  epithelial  HA _ i n v i v o  cells  The c u l t u r e s , on t h e t y p e  i n the basement  synthesize  predominantly  by enzyme d e g r a d a t i o n  proteoglycan  synthesis  cells  to be CS.  by .mouse  mammary  a l l nongrowing, show v a r i a t i o n s o f s u b s t r a t e on which  they are  They produce HA, CS 4, CS 6, DS and HS i n v a r y i n g amounts and locations  (cell,  ECM  and medium).  t h a t t h e normal human mammary c e l l  Chandrasekaran  et  l i n e , HBL-100, mantained  a l . on  - 20 a p l a s t i c s u b s t r a t e , s y n t h e s i z e m a i n l y HA It  was  level  not  s t a t e d , however, whether  these  i t i s p r e s e n t i n the medium. c e l l s are  of d i f f e r e n t i a t i o n , therefore i t i s d i f f i c u l t  t h i s work w i t h the p r e v i o u s two. when comparisons a r e and  and  growing  T h i s e x e m p l i f i e s the problems  made between  experiments.  al.'s  to d i f f e r e n t i a t e  work.  Whereas  proteoglycans fraction.  i n an  Parry  were the  Silberstein ECM,  et  as  and  a l . find  that  et  their  and  a l . did  e q u a l amounts o f HS and CS and  while  and  "stabilized"  Daniels f i n d  non-growing hormones  cells.  However, to  CS  i n Parry  et  analyse  Parry  facilitate  et  of  et  a l . report  these a r e found  i n the BM  been  an  cells  i n an  the  al.'s  ECM  ECM  so-called  cells  to  Cortisol  and p r o l a c t i n ) w h i l e S i l b e r s t e i n and D a n i e l s ' mouse mammary g l a n d s Another d i f f e r e n c e i n experimental  discrepency  in like observed  cell  types  cells  are  in_ v i t r o  cells  i n culture synthesize  or  and  differentiation  were  exposed  were n o t .  designed  mainly  not  not  mouse mammary e p i t h e l i a l  s y n t h e s i z e almost Silberstein  Parry  of  Silberstein  t h a t had  mouse mammary c e l l s  Chandrasekaran  some  encountered  Furthermore,  Daniels  at  t o compare r e s u l t s  D a n i e l s (1982) l o o k e d a t v i r g i n mouse mammary g l a n d  stimulated  or  d e s i g n t h a t may  proteoglycan  in. vivo.  greater  account  for  s y n t h e s i s i s whether  the  I t has  amounts  of  (insulin,  been demonstrated proteoglycan  and  components when compared t o t h e i r c o u n t e r p a r t s i i i v i v o ( M u i r , 1977; al., daily  1984). to  A variation  every  synthesized  by  other chick  as  day  s u b t l e as has  been  shown  chondrocytes  s t a n d a r d i z i n g e x p e r i m e n t a l procedure P r o t e o g l y c a n s and  changing to  (Katagiri  the  matrix Nevo e t  feeding schedule  alter et  the  ECM  al.,  that  from  components 1981).  So  i s very important.  Malignancy  I f proteoglycans  p l a y an  the many s t u d i e s c i t e d  important  so f a r i n d i c a t e ,  role  i n normal c e l l  might m a l i g n a n t  f u n c t i o n i n g , as  c e l l s show  altered  - 21 proteoglycan synthesis  synthesis  and c o m p o s i t i o n ?  and m a l i g n a n c y  i n t h e e a r l y 1970's.  accumulated  indicating  proteoglycan  s y n t h e s i s compared  and amount produced. study  that  malignant  mainly  (90$).  cells  do  Shishiba  then,  proteoglycan results  indeed  have  both  have  altered  i n the  varied.  type  In their  e t a l . (1984) compared normal to t o proteoglycan  synthesis.  Normal  malignant  tissue  o f HS (60%) and CS o r DS (40$) o r m a i n l y  CS and  As w e l l , , i n t h e two " m a l i g n a n t  tissue  c o n t a i n s a 2-3 f o l d g r e a t e r amount o f p r o t e o g l y c a n s g r e a t e r when compared  on  HS ( S h i s h i b a e t a l . , 1983) w h i l e  contains e i t h e r a mixture DS  Since  to normal c o u n t e r p a r t s ,  adenoma and adenocarcinoma w i t h r e s p e c t contains  began  The r e s u l t s , however, a r e e x t r e m e l y  on human t h y r o i d t i s s u e ,  tissue  Studies  to t h e normal.  Iozzo  samples  examined  one  and the o t h e r 6-15 f o l d  e t a l . (1982) found i n normal  human c o l o n t i s s u e a l a r g e HS p r o t e o g l y c a n and a s m a l l e r DS p r o t e o g l y c a n i n about e q u a l amounts. HS s y n t h e s i z e d  I n c o l o n carcinoma c e l l s  ( I o z z o , 1984) there i s l e s s  and t h e predominant p r o t e o g l y c a n  i s a small  CS.  The HS  found i n t h e m a l i g n a n t 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 o r r e l e a s e d i n t o the medium and has d i s t i n c t in  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,  t h a t t h e m a l i g n a n t HS i s o f a l a r g e hydrodynamic s i z e ,  d e n s i t y and has s h o r t e r GAG s i d e These d i f f e r e n c e s caused I o z z o  chains  than  t o hypothesize  the normal that  these  higher  bouyant  synthesized  HS.  HS macromolecules  may be r e s p o n s i b l e f o r a l t e r e d s u r f a c e 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 (Iozzo,  1984).  increase  The l i s t  of a l t e r e d proteoglycan  i n CS i n human l u n g  hepatoma (Kojima  carcinomas  synthesis  I960)  e t a l . , 1975) has been found.  chondroitinases  show  growth-stimulating  on - an  ( H a t a l e t a l . , 1977) and human The a d d i t i o n o f CS to i n  v i t r o and i n v i v o human mammary carcinoma c e l l s ( T a k e u c h i , al.,  goes  results.  ( t o degrade CS) can r e t a r d t h e growth  The  1965; O z z e l l o e t addition  o f these  cells  of as  - 22 w e l l ( T a k e u c h i e t a l . , 1972). Toole  e t a l . (1979)  find  that  rabbit  V  2  carcinoma  cells  grown i n  r a b b i t s have 3 t o 4 t i m e s a s much HA s y n t h e s i z e d a t t h e i n t e r f a c e  between  tumor mass and c o n n e c t i v e t i s s u e than do t h e same c e l l s i n j e c t e d i n t o nude mice.  I n r a b b i t s these  mice they a r e n o t .  V  cells  are invasive while  i n nude  These same f i n d i n g s o c c u r r e g a r d l e s s o f t h e s i t e  f o r i n j e c t i o n of the c e l l s . conducive  carcinoma  2  They c o n c l u d e  t o i n v a s i o n o f t h e carcinoma  They propose t h a t t h e HA e n a b l e s  cells  t h a t HA p r o v i d e s an environment cells  into  to migrate  surrounding  tissues.  by e x e r t i n g f o r c e v i a  i n c r e a s e d s w e l l i n g i n t i s s u e s t o open pathways a l o n g c e l l - c o l l a g e n Chandrasekaran e t a l . (1979) a n a l y z e d b r e a s t carcinoma in  cell  lines,  t h e medium o r c e l l  t h a t t h e predominant p r o t e o g l y c a n s  p r o t e o g l y c a n s y n t h e s i s by two human  or c e l l - a s s o c i a t e d ) .  They  line  s y n t h e s i z e d a r e CS and HS w i t h HA b e i n g  (HBL-100) under t h e same c o n d i t i o n s (Chandrasekaran  They  synthesized  d i d not f i n d per  (MDA-MB-231) c e l l s . al.,  i n the  T h i s i s o p p o s i t e t o the f i n d i n g s they r e p o r t e d f o r t h e normal  cell  a  d i f f e r e n c e i n t h e amount  between  This  normal  (HBL-100)  i s contrary to the other  1984; A n g e l l o e t a l . , 1982) t h a t do f i n d  proteoglycan s y n t h e s i s i n malignant  tissue.  cell  e t a l . , 1979).  t h a t c a s e , HA i s t h e predominant GAG (90$) w i t h v e r y l i t t l e (3$).  either  reported  a v e r y minor (0-12$) component and t h e m a j o r i t y o f i t (HA) p r e s e n t medium.  layers.  MCF-7 and MDA-MB-231, and i t s l o c a t i o n  (intracellular  chosen  d e t e c t a b l e HS  of  and  In  proteoglycan  the  reports  malignant  (Shishiba et  an i n c r e a s e above normal i n The MCF-7 c e l l  line  actually  s y n t h e s i z e s much l e s s than t h e two o t h e r l i n e s (MDA-MB-231 and HBL-100) . A n g e l l o e t a l . (1982) l o o k e d a t two s u b - p o p u l a t i o n s cell  line.  The -SA c e l l s  (no growth i n s o f t  v i v o ( s l o w growth) w h i l e +SA c e l l s  o f a mouse mammary  agar) are non-agressive  in  (growth i n s o f t a g a r ) a r e a g g r e s s i v e and  - 23 grow  rapidly i i i vivo  with  morphologically similar. 8  times  more  identified  labelled  analysed  glucosamine is  46.3$ HS.  tumor  57-7$ HA  than  HA.  and  proliferation.  and  cell  only  suface  29.9$ HS  (1976)  that  the  Other  They f i n d  (between  25  and  ug/mg d r y w t . ) . the  the  that  have  -SA  cells  both  wt.).  and  -SA  the  major have  3 fractions  l o c a t e d i n the  while  - 30-34$) the  synthesizes  with  increased  HA  shown  increased  HA  40$  proteoglycan  content  the  yg/mg d r y  The  c h o n d r o i t i n s u l f a t e s are  other  5  cases  HA,  wt.).  et  tumors may  wt.)  cases  al.  and  CS,  HS  and  (1979)  eleven  is  is  HA  and  the  the  and  malignancy  r e s u l t s are reported  human  based  l i n e s ( l o z z o 1984;  on  predominant  least  (less  Angello  GAG  than  relatively  equal  lowest,  (less  2  that  that  not  than  promotes  by and  ug/mg  sub-populations only  2  in their  sub-populations.  be  related  to out  ( S h i s h i b a e t a l . , 1983;  cells  breast  are  suggest  from e x p e r i m e n t s c a r r i e d  removed from a p a t i e n t Others a r e  may  mammary  Takeuchi et  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 synthesis  their  n e x t i n amount f o l l o w e d  is  e s t a b l i s h a matrix  growth but a l s o the growth of o t h e r  HA  DS  A g a i n HS  of  +SA  encourages in  T a k e u c h i e t a l . , 1976).  70  Angello  heterogenous  PG  appear  sub-populations  - 50-58$, c e l l  also  i n s i x of  l o w e r , (under 10ug/mg d r y dry  the  PG  conditioning  studies  examined  tumors.  In  Both  c e l l s i n culture incorporate  Although  adenocarcinomas (Palmer e t a l . , 1979;  DS.  do  period.  They c o n c l u d e t h a t e p i t h e l i a l c e l l s a r e a b l e t o c o n d i t i o n  environment  al.  latency  the same p e r c e n t a g e o f t o t a l  (medium- 12 t o 15$,  synthesizes  short  I t i s found t h a t +SA  proteoglycan  approximately  a  i n c u l t u r e and et a l . , 1982;  the  several  regarding  factors.  Some  on whole t i s s u e r e c e n t l y T a k e u c h i et a l . , 1976)  majority  are  cell  Chandrasekaran e t a l , 1979).  As  r e p o r t e d e a r l i e r i n the I n t r o d u c t i o n , normal p r o t e o g l y c a n  o f these  .  s y n t h e s i s appears  - 24 to be s p e c i f i c t o c e l l The  type.  The same may be true f o r malignant  cells.  type of proteoglycan produced by a malignant c e l l may also be r e l a t e d  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 one  major  proteoglycan  (Toole  of rapid growth may synthesize  et a l . , 1979) while  cells  that  have  " t r 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 d i f f e r e n t proteoglycan  ( l o z z o , 1984).  Numerous researchers  ( l o z z o , 1984; L i o t t a et a l . , 1979; Bauer e t a l . , 1979) have pointed out that i t may be the stromal  tissue  surrounding  the tumor c e l l s  stimulated to synthesize various a l t e r e d matrix components. the  small  CS molecule  associated  discovered by autoradiographic  with  techniques  the colon  that i s  Interestingly,  carcinoma  cell  was  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  e t a l . , 1982).  synthesizing a f a c t o r designed  Alternately,  they  studied  substrates degradation.  does  David  cells  may be  et a l . (1981, 1982) discovered  that  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 )  not accumulate  i s because  stromal  to increase or decrease synthesis of matrix  components by the tumor c e l l s . the reason the transformed  these  they  The normal  NMuMG  a proteoglycan-rich  are unable cells  to  incorporate  BL on collagen  decrease  proteoglycan  proteoglycan  into  a  d i s t i n c t basal lamina and have the same synthesis rate as the transformed cells.  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 t o 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 t i s s u e to be used as a comparison,  coupled  with  the f a c t  that  proteoglycans  synthesized by  d i f f e r e n t c e l l s do not appear to influence the same functions i n a l l c e l l s .  - 25 T h e s i s Problem Due  Formulation  t o the obvious  influence  o f PGs  on  cell  function,  i t seemed  a p p r o p r i a t e t o d e t e r m i n e t h e type and l o c a t i o n o f p r o t e o g l y c a n s  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 d u r i n g t h e e x p o n e n t i a l and  stationary  function.  phases  This  o f growth  i s not  cell-proteoglycan  only  and how  important  r e l a t i o n s h i p but a l s o  they  relate  t o normal  i n understanding necessary  the  cell normal  f o r establishing i f  a l t e r a t i o n s occur i n t h e m a l i g n a n t c e l l . Normal  human  mammoplasties. the  cells  cells  The t i s s u e was processed  cells  b a s a l lamina  1975;  epithelial  were  obtained  from  reduction  t o 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  were grown on c o l l a g e n g e l s , as i t has been demonstrated  epithelial  Pitelka,  mammary  grow  better  or stromal  on a s u b s t r a t e  t i s s u e than  1977; Hay, 1981; K l e i n m a n  Richards  derived  on a p l a s t i c  from  that  components o f  substrate  (Emerman and  e t a l . , 1981; M i c h a l o p o u l o u s  et a l . ,  e t a l . , 1982; Wicha e t . a l . 1979, 1982; Yang e t a l . , 1979).  3 The  proteoglycans  24 hours  prior  were  labelled  to termination.  with  H-glucosamine  Cultures  were  added  terminated  t o t h e medium a t . g r o w i n g and  nongrowing s t a g e s a s determined by DNA a n a l y s i s . Analysis  of proteoglycans  glycosaminoglycan non-specific  side  protease  was a c c o m p l i s h e d  by i d e n t i f i c a t i o n  chains.  The p r o t e o g l y c a n s  to cleave  the carbohydrate  p r o t e i n core and t h e l a t t e r was d i s c a r d e d .  were side  treated chains  of the with  a  from t h e  The G A G 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 t o each and by c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s ( P a r r y e t a l . , 1985; A n g e l l o 1984;  e t a l . , 1982; Kanwar e t a l . , 1982; Crawford e t a l . ,  I o z z o , 1984; S h i s h i b a e t a l . , 1984).  The i n f o r m a t i o n g a i n e d epithelial  from t h e s e  c e l l s i n c u l t u r e provides  experiments on normal human mammary a b a s e l i n e w i t h which t o compare t h e  - 26 effects  of various  f a c t o r s on GAG  i n c l u d e a ) hormones b) s u b s t r a t e s malignancy.  Of p a r t i c u l a r  synthesis  and f u n c t i o n .  c) d i f f e r e n t i a t i o n  interest  little  literature  information  pertaining  ( K i d w e l l e t a l . , 1982).  example,  would  stimulated  a  found  proteoglycan  substrate  cells  rich  by  GAGs on c e l l  area  were  growing on a  growing  open  hormones synthesis.  found  i n the  proteoglycans  For  found i n  o r would growth be  substrate  cells?  render  function.  i n the p r o t e o g l y c a n  (GAG) t o t h e medium be as e f f e c t i v e large  and e)  i n t o a s u b s t r a t e would  to stop  i f they  synthesized  c a n be  The a d d i t i o n o f v a r i o u s  cause growing c e l l s  i n quiescent  proteoglycan  area  the e f f e c t s of s p e c i f i c  creating  non-growing c e l l s  on p r o t e o g l y c a n  to t h i s  (GAGs) e i t h e r t o t h e medium o r i n c o r p o r a t e d information regarding  d) r e c e p t o r s  i s the e f f e c t s of various  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 Very  Such f a c t o r s  rich  Would  i n the  adding  as i t s presence  in a  substrate?  Another  receptors.  The presence o f GAG i s d e f i n e d i n t h i s t h e s i s as b e i n g medium,  ECM ( i n c l u d e s c e l l - a s s o c i a t e d ) o r c e l l help  i n the  understanding  of  GAG  f o r i n v e s t i g a t i o n i s that  the  (intracellular) function  to  located.  know  of  GAG  I t would  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 t h e p r o t e i n c o r e ) and  which a r e basement membrane a s s o c i a t e d .  study  could  be  used  as  a  baseline  m a l i g n a n t human mammary c e l l s e n a b l e some c o n c l u s i o n s synthesis  on  the  with  Finally which  compare  and  maintenance  of t h i s  results  grown under t h e same c o n d i t i o n s .  t o be drawn r e g a r d i n g  growth  to  the r e s u l t s  of  I t would  t h e e f f e c t s o f a l t e r e d GAG of  the  altered  phenotype.  M A T E R I A L S 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 s t e r i l e transport medium (see Appendix l ) on i c e . back to the culture room where, under s t e r i l e  Samples were brought  conditions, excess f a t was  removed from the glandular portions using a scalpel and scissors.  The f a t  was discarded and the glandular portions were minced f i n e l y using 2 scalpel blades  then placed  i n a 250 ml flask  medium (Appendix 2).  containing  i n a 37°C incubator and  The f l a s k was then placed  kept s t i r r i n g for approximately  22 h.  50 ml of d i s s o c i a t i o n  Of the three samples used for this  thesis, one was dissociated a f t e r 18 h, one at 21 h and one at 22 h. The d i s s o c i a t i o n was considered remained.  complete when only  The s o l u t i o n was then divided equally  at 800 rpm  preferentially supernatant 100  (80xg).  pellet  This  centrifugation  the e p i t h e l i a l  cells  ml of DME.  supernatant medium  speed  present  The tubes  were  centrifuged  was  designed  i n the sample.  again  to The  using  the c l i n i c a l  The supernatants  were removed  the p e l l e t s were washed a second time i n DME.  remaining  i n 4 15 ml centrifuge  was discarded and the p e l l e t s were combined and resuspended i n  centrifuge for 4 min at 1000 rpm (lOOxg). and  aggregates of c e l l s  centrifuge (Fisher S c i e n t i f i c ) for 4  tubes and centrifuged i n a c l i n i c a l min  small  collagenase.  After  a  final  The washes removed any  centrifugation  as  above, the  was discarded, and the p e l l e t was resuspended i n 5 ml of growth  (Appendix  3)  and put through  a 150um Nitex  filter  to decrease  clumping of c e l l s .  For c e l l counting,  0.1 ml of the c e l l  suspension was  removed and placed  i n a 2 ml tube.  The remaining  suspension  placed i n a 37°C water bath while counting  £  7  cell  took place.  was  A minute drop of  - 28 trypan blue (pH 1.2) was' added to the 0.1ml solution to distinguish viable c e l l s from dead c e l l s . If  the  total  experiment,  number of  the  extra  medium (Appendix 4 ) at ml.  Counting was done using a hemocytometer cells  cells  was greater  were  pelleted  a concentration  af  than that and  required for  resuspended  approximately  They were quick frozen and immediately  .  transferred  in  the  freezing  1 X 10 • c e l l s and stored  /  in a  cryogenic tank at -70°C (Union Carbide).  Preparation of Collagen Gels The  collagen  solution was  prepared  from rat  tails  in  the  following  manner. The t a i l s were placed i n 9 5 $ alcohol for 15 min. The tendons were dissected and  out and teased apart using scalpel blades and forceps,  placed i n a 60 mm P e t r i  dish containing s t e r i l e  exposed to the u l t r a v i o l e t l i g h t i n the tissue  weighed  d i s t i l l e d water and  culture hood for 24 h . The  f i b e r s were then suspended i n a d i l u t e acetic acid solution (1:1000) and stirred 4°C. and  at  4°C for  Finally,  the  centrifuged  supernatant,  in  48  h.  They were  solution was a  Sorvall  transferred  centrifuge  sit  into  at  for  another  24  h  50 ml centrifuge  10,000xg  for  30  at  tubes  min.  The  the collagen s o l u t i o n , was bottled and stored at 4 ° C .  To prepare  the gels,  a solution of medium 199  combined with 0.34N NaOH i n a r a t i o solution.  l e f t , to  This was added to 1.6  ml of the  together i n a tube on ice to prevent then transferred  of 2:1  concentration)  to make a t o t a l collagen  premature  to a 3 5 mm P e t r i d i s h .  (10X  of 0.4  solution and  gelling.  was  ml of stirred  The solution was  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 s o l u t i o n .  placed i n an incubator one  (National,  Inc.)  at  37°C,  The culture  dishes were  5$ C O 2 and 9 5 $ a i r  for  29  -  h to allow complete g e l l i n g  of the  -  solution.  After that  time,  2 ml of  experimental medium was added to the 3 5 mm P e t r i dishes and 0 . 5 ml to  the  wells for 2 4 h p r i o r to the experiment to allow e q u i l i b r a t i o n .  C e l l Culture Procedure 5  Initial  cultures  were  seeded  at  of  2  X 10  2-2.5  cells/cm  hydrated collagen gels i n 3 5 mm P e t r i dishes on w e l l s .  The cultures  CO2 and 9 5 $ a i r and observed d a i l y . day at phase were  the  same time during  contrast  microscope,  approximately  50$  identified  as  analysis.  The f i r s t  between  days  culture.  growing  9-11,  A third  stationary.  the  it  cultures  cultures  destined  and  were incubated at  With d a i l y  was determined at  the  37°C, 5 $  The medium was changed every second  day.  confluent  and  observation under  what point the  confluent.  The  as  stationary  cultures  former  between day 3 and 4 , the  depending  growth  stage,  between- days  Morphologically,  the  rate was  17-18,  cultures  appeared  of  each  second  particular  identified 85$  were  by DNA  stage was attained the  the  cultures  100$  latter  on  the  Identical  f o r DNA determination were grown concurrently with the for glycosaminoglycan a n a l y s i s .  onto  as  late  epithelial.  At  3  these  times,  the  medium  (S.A.=30-60  medium  contained  growth).  cultures  was  MCi/mmol; only  labelled  with  NEN, DuPont  insulin  (5wg/ml)  H-glucosamine  Canada, and  Inc.).  5 $ PCS  in  growth  The  growth  (necessary  for  No hormones were added to stimulate d i f f e r e n t i a t i o n . 5  Cultures  were  also  seeded  at  a  higher  density  of  5«0  x  10  2  cells/cm .  These cultures were i d e n t i f i e d  as  stationary  at  day 4 by DNA  analysis. Radiolabelling Procedures All 1.5ml  experiments  were  of growth medium.  l a b e l l e d with  H-glucosamine at  Two experiments  were  100  l a b e l l e d with  35  yCi/ml  in  S-sulphate  - 30 at  100  viCi/ml  (NEN, c a r r i e r - f r e e )  instead  of  H-glucosamine.  This was  done as an alternate means of i d e n t i f y i n g synthesized sulphated GAG.  All  l a b e l l i n g was done f o r a 24 h time period.  Resolution into Fractions - Medium, C e l l and E x t r a c e l l u l a r Matrix After l a b e l l i n g the medium was removed from the culture ice i n a 15 ml centrifuge tube. tube  (VWR S c i e n t i f i c ,  Scientific  Services)  The g e l was placed i n a 1.5 ml Eppendorf  Inc.) and centrifuged at  13,000xg  to  the  i n a mirocentrifuge  f o r 5 min at  squeeze any medium out of the i n t e r s t i t i a l removed and added  and stored on  40°C.  spaces.  o r i g i n a l medium.  This  (Western  was done  to  This medium was then  The g e l was then  rinsed i n  approximately 1 ml of T r i s - s a l i n e s o l u t i o n , pH 7«4 (Tris lOmM, 0.85$ NaCl) for 30 min to remove a d d i t i o n a l trapped medium. ice  during the time period and vortexed  This tube was also kept on  every few minutes.  The g e l was  then spun down at 13,000xg f o r 15 min i n the microcentrifuge at 4 ° C . The supernatant was added to the o r i g i n a l medium tube. To lyse the c e l l s , the gels were extracted on i c e with 1 ml of detergent solution (Appendix 5) and vortexed gels  were then centrifuged a f i n a l  The supernatant  constituted  called the e x t r a c e l l u l a r  occasionally over a 30 min period. time at  13,000xg  The  f o r 15 min at 4 ° C .  the soluble c e l l f r a c t i o n while the p e l l e t was  matrix  fraction.  They were  separated  and placed  into 15 ml centrifuge tubes and stored on i c e .  Extraction of Glycosaminoglycans To  each  solution  of  (Sigma,  these type  lOmg/ml i n lOmM T r i s ,  three  fractions  was added  XIV, Streptomyces pH 7.4.  griceus)  a at  nonspecific  protease  a concentration  of  One ml of protease solution was added f o r  - 31 every  2 ml of f r a c t i o n sample.  added to each tube to prevent incubated  in  a  water  bath  A drop of bacterial  at  were compared to  growth.  38°-41°C  added a f t e r 24 h of incubation.  sodium azide  for  (0.01$ w/v)  A l l fractions  48  h.  Fresh  were  then  protease  Some samples were l e f t for 60-72 h .  those that were  incubated  for  the  48  was  was These  h time period  to  ensure no further enzyme action took place a f t e r that time. Digests were precipitated with 10$ t r i c h l o r o a c e t i c at  acid (Sigma)  for 1 h  centrifuge  (Western  They were then centrifuged using a tabletop  4°C.  S c i e n t i f i c Services)  at  saved and the p e l l e t s  200-250 Xg for 10 minutes. discarded.  The supernatants  The supernatants  were  were divided into  1.5  ml Eppendorf tubes (VWR S c i e n t i f i c ) with each tube receiving 0.3 ml of the solution.  The tubes were c l e a r l y 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  GAGs:  of  the  following  chondroitin sulfate cartilage)  and heparin  (Sigma,  The  labelled  (Sigma,  porcine  intestinal  umbilical  type B (Sigma, mucosa).  each Eppendorf tube.  cord),  This  shark  Twenty-five  acted  as  a cold  to help "bring down" labelled GAG. Each tube was diluted with 3  volumes of 90$ ethanol/l.4$ ml.  acid  type A (Sigma, whale c a r t i l a g e ) ,  ul of this mix was added to carrier  hyaluronic  potassium acetate to give a f i n a l volume of  ethanol/potassium GAG.  Each  tube  acetate  solution  was vortexed  acted  to  and allowed to  help sit  1.5  precipitate  for  24  h  at  -20°C. Precipitated GAGs were collected by centrifuging a l l Eppendorf tubes for 30 minutes i n the microcentrifuge were discarded and the p e l l e t s  at  4°C and 13,000xg.  resuspended i n 0.3  The  supernatants  ml of glucosamine  mM) i n water using a Pasteur p i p e t . ' The tubes were then allowed to s i t 1 h at  4°C to  ensure  that  the  pellet  completely  dissolved.  (0.1 for  Afterwards  - 32 another 3 volumes of 90$  ethanol/l.4$  tube to bring the t o t a l  volume to 1.5  was repeated,  all  -20°C. total.  that  The whole  is,  tubes  precipitation  potassium acetate was added to ml.  The procedure  were vortexed procedure  A f t e r the f i n a l centrifugation,  was  the p e l l e t s  to 12 Eppendorf tubes may contain p e l l e t s  described above  and stored carried  each  for  out  3  24  h  at  times  in  from one f r a c t i o n  (up  from one fraction)  were pooled  and the t o t a l 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 i d e n t i f i c a t i o n of GAG procedure began.  Analysis of GAG by Enzyme Digestion  ( l h y a l u r o n i c Acid I d e n t i f i c a t i o n Streptomyces  hyaluronidase (500  of 50 mM sodium acetate at Eppendorf tubes of NaCl (1.5  by adding 100  M).  i n water at  cool completely.  The assay was carried  y l of sample,  30  A 25  40°C for 4 h . 100°C  for  out i n 1.5  The samples  The enzyme was then  15 min.  ml  ul of enzyme and 15 Ml  A l l samples were run i n t r i p l i c a t e .  enzyme were incubated at by b o i l i n g  pH 5 « 0 .  u n i t s / m l , Sigma) was dissolved i n 1 ml  The samples  were  plus  inactivated allowed  to  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 centrifuged (microcentrifuge, and p e l l e t s counting).  separated  and  overnight at  13,000xg, counted  One tube containing the  ECM) was kept for the next assay.  (to  -20°C.  A l l tubes were then  30 min, 4°C) and the be  pellet  described  under  supernatants scintillation  from each sample (MED.,  CELL,  - 33 -  I n i t i a l Sample ( 1 . 5 ml)  MED. CELL ECM  HS Enzyme Assay divided into  HA Enzyme Assay  Pellet' undigested GAG so not HS  (Supernatant discarded) Pellet resuspended  Divided into  / \  Pellet =undigested GAG so not HA  Supernatant =digested GAG identified as HA  CS ABC Enzyme Assay  Pellet undigested GAG so not CS ABC  Supernatant =undigested GAG identified as HS  Supernatant =digested GAG so identified as CS 4,6 or DS  CS AC ErLZ"<nne Assav  Pellet =undigested GAG so not CS 4 or 6  Fig. 5 Flow Chart of Identification of GAG by Enzymes  Supernatant =digested GAG so identified as CS 4+6  - 34 (2) C h o n d r o i t i n S u l f a t e Two  Identification  enzymes were used t o 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 ) c h o n d r o i t i n a s e ABC ( i i ) c h o n d r o i t i n a s e AC (not Because  (Sigma, 10 u n i t s / m l ) - degrades a l l CS (Sigma,  10 u n i t s / m l ) - degrades  c h o n d r o i t i n a s e ABC  triplicate  resuspended  4 and  6  DS) will  also  digest  t h e s e a s s a y s must have HA removed f i r s t . the  CS  tubes  from  Ml o f  i n 400  the  HA  the samples  used  for  T h e r e f o r e , the p e l l e t from one  enzyme  distilled  HA,  assay  water.  f o r each  These  were  fraction  used  of was  f o r the  CS  assays. Both  enzymes  (CS  ABC,  CS  water (Sigma, 10 u n i t s / m l ) . tubes  by  adding  (MED., CELL,  60  ECM),  (Appendix 6) a t pH 8.0. All boiled cooling  Ml  were  dissolved  The a s s a y was  Ml o f 60  AC)  the of  carried  resuspended  the  enzyme  in  distilled  out i n 1.5  ml  Eppendorf  pellet  and  20  from  the  Ml o f  HA  samples  "enriched"  Tris  A l l samples were done i n d u p l i c a t e .  samples were i n c u b a t e d a t 37°C f o r 4 h a f t e r which time t h e y were i n water  at  lOO^C  c o m p l e t e l y , 25  f o r 15  Ml o f GAG  min mix  to  inactivate  (img/ml  allowed  centrifuged  to  s i t overnight at  -20°C.  the  o f each  each tube f o l l o w e d by 1 ml of 90$ E t 0 H / l . 4 $ KAC. and  separately  The  GAG)  and  scintillation  counting).  (3)Heparan S u l f a t e One hundred  pellets  separated  and  counted  was  After  added  to  A l l tubes were v o r t e x e d next  day  i n the m i c r o c e n t r i f u g e a t 13,000xg f o r 30 min  supernatants  enzyme. •  ( t o be  a l l tubes a t 4°C  and  described  were the under  Identification  Ml o f n i t r o u s a c i d  ( p r e p a r e d by a d d i n g 0.3  M sodium  nitrate  - 35 and 2.9 M acetic acid) was added to 50 ul of the i n i t i a l CELL and ECM fractions i n 1.5 ml Eppendorf tubes. duplicate.  sample of MED.,  A l l samples were done i n  After incubating the tubes at room temperature  reaction was stopped by the addition  of 50  ul of ammonium sulfamate  M) to each tube and incubated at room temperature ml of 90$ EtOH/l.4$ KAC was added to vortexed and stored (microcentrifuge,  overnight at JO  13,000xg,  min  for another 30 min.  each of the  -20°C. at  for 80 min, the  tubes which were  (2 One  then  The tubes were then centrifuged 4°C)  and  the  supernatant  and  p e l l e t s separated and counted.  S c i n t i l l a t i o n Counting Supernatants Products)  and  were 15  ml  placed of  in  20 ml  glass  scintillation fluid  s c i n t i l l a t i o n v i a l s (NEN (Aquasol,  P e l l e t s were dissolved i n 200 ul of d i s t i l l e d  water  NEW) was  added.  and placed i n 6 ml  p l a s t i c s c i n t i l l a t i o n v i a l s (NEN Products) to which 5.8 ml of s c i n t i l l a t i o n fluid  was added  (Aquasol, NEN).  S c i n t i l l a t i o n Counter for 4 min.  A l l vials  were  counted  i n a Mark  A quench curve was constructed  II  from six  3 known  samples  containing  H-solutions with  various  amounts  of  quench.  These samples were commercially purchased (Amersham/Searle, L t d . and NEN, DuPont Canada, I n c . ) . read with every  set  A control containing only of v i a l s  to  scintillation fluid  indicate background r a d i o a c t i v i t y .  was 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 S results were read using a 14C quench as a factor i n the r e s u l t s . quench curve and standards commercially purchased (Amersham/Searle, L t d . ) .  - 36 -  j  >  i  !_  2  4  6  8  Time  Fig.  6  (hours)  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 s a m p l e and t h e optimum t i m e f o r maximum d e g r a d a t i o n o f t h e s u b s t r a t e was c h o s e n f o r subsequent assays.  - 37 C a l c u l a t i o n of Assay Results The supernatant  contained  the amount of GAG which was degraded by the  p a r t i c u l a r enzyme used and the p e l l e t contained the amount of GAG which was not degraded by the enzyme.  The r a t i o of the degraded to non-degraded was  c a l c u l a t e d 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 p a r t i c u l a r enzyme.  A l l r a t i o s were compared to standard  which were run concurrently with a l l samples and contained  samples  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 c a r r i e d out over various incubation times (Fig.  6 ) and the times  chosen f o r subsequent assays were those  permitted maximum degradation. the GAG i n question,  To ensure that the enzymes were  degrading  a sample of each GAG at Img/ml was spotted  Whatman f i l t e r paper and stained with A l c i a n blue  that  on a  i n water, pH 2.5.  Enzyme was then added to each known sample and the incubation times c a r r i e d out as described. and  Afterwards  a second spot was placed  stained with A l c i a n blue.  second spot occurred.  d i d not s t a i n ,  A positive result  indicating  on a f i l t e r  was obtained  the degradation  of that  This procedure was a l s o c a r r i e d out between a l l known  and a l l enzymes to determine the s p e c i f i c i t y of each enzyme. that  streptomyces hyaluronidase  was s p e c i f i c  degraded HA, CS 4+6 and DS, chondroitinase  to HA,  when the GAG had standards  I t was noted  chondroitinase  AC degraded  paper  ABC  CS 4+6 and HA  ( s l i g h t decrease i n s t a i n i n g a f t e r enzyme) and n i t r o u s a c i d degraded only HS. A l l enzyme assays were c a r r i e d out i n duplicates or t r i p l i c a t e s .  - 38 I d e n t i f i c a t i o n of GAG Using E l e c t r o p h o r e s i s The GAG present i n each sample was a l s o i d e n t i f i e d by c e l l u l o s e 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, b l o t t e d dry and marked a t one end as "standard" or "sample". They were then placed i n an e l e c t r o p h o r e t i c u n i t (Gelman Sciences, Inc.) containing 1 l i t e r of 0.15 M z i n c acetate, pH 5.8, so that both ends were i n contact with the b u f f e r . At  the midpoint  of the s t r i p ,  5  Ml of sample and  3  Ml of standard  s o l u t i o n 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 a t 2 mA/strip and allowed to run f o r 3 h.  A f t e r the a l l o t t e d  time, the s t r i p s were removed and  placed i n a s o l u t i o n containing 500 ml of 1% A l c i a n blue, 5% a c e t i c and 10$ EtOH f o r 0.5 h.  The s t r i p s were then rinsed overnight i n 5% a c e t i c  a c i d and water and dried f l a t . known q u a n t i t i e s  acid  of each  A l l standards were determined  separate  GAG  and  staining  by running  as o u t l i n e d .  The  stained areas, corresponding to the known standards, were c a r e f 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.  F i l m Developing f o r Autoradiography Several identified  samples of each using  fraction  the e l e c t r o p h o r e t i c  (MED., CELL, ECM) and  staining  above where f u r t h e r i d e n t i f i e d by autoradiography.  separated  procedures  and  described  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  foil  and  stored  in 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 c o o l dark place f o r up to 6 wk  - 39 undisturbed. 2  wk.  The s t r i p s from the nongrowing cultures were developed  Leaving  autoradiographs.  these  (up  However, the s t r i p s  developed before 6 wk. stationary  longer  culture  to  4  wk)  did  counts  per  were  found  to contain  Ml sample  applied  was  the  could not be  Even at 6 wk, the autoradiographs were f a i n t .  strips  5  improve  from growing cultures  The  5 - f o l d higher counts i n  each band representing a GAG then growing culture 1000  not  after  strips.  necessary  A minimum of to  affect  the  H-sensitive f i l m . The f i l m was developed i n developer (Picker International) water (1:4) f o r 4 min, stopped i n Rapid Fix) f o r 10 min.  in distilled  5% acetic acid i n water and fixed (Kodak  The f i l m was then rinsed f o r 3 0 min i n running  water and allowed to dry overnight.  DNA Determination Cultures f o r DNA assay were frozen p r i o r to DNA analysis to membrane l y s i n g .  Thawed gels  were sonicated  facilitate  i n 1 ml of Na^HPO^ b u f f e r ,  pH 7 « 4 (Appendix 7) f o r 3 min. Afterwards, a 300 Ml aliquot was removed, combined with 150  Ml of a Horchst dye s o l u t i o n  (1 mg/ml of water) and  vortexed to mix thoroughly (Labarca et a l . , 1980).  A l l assays were done i n  triplicate. The  solutions  were  read  immediately  in  a  fluorospectrophotometer  (American Instrumentation Co.) with an a c t i v a t i n g wavelength of 356 nm and a  fluorescence  standards  wavelength  of  458  nm.  The results  were  ranging from 10 - 100 Mg of DNA using a c a l f  (Sigma) at 100 Mg/ml.  compared  with  thymus DNA stock  Calculations of c e l l number were based on the fact  that 7pg = 1 mammary e p i t h e l i a l c e l l (Kraenbuhl et a l . , 1981).  - 40 E l e c t r o n Microscopy Cultures  f o r e l e c t r o n microscopy were prepared by removing  and washing each g e l 3 times with Karnovsky's l e f t f o r 1 h i n this solution. 0.1 M sodium cacodylate, embedding.  solution, (Appendix 8) then  A f t e r t h i s , the gels were washed 3 times i n  pH 7.4, and stored i n t h i s s o l u t i o n at 4°C u n t i l  The embedding, sectioning and photography  Mrs. H e l l a Prochaska.  the medium  were c a r r i e d out by  RESULTS  Growth Normal  human  mammary  epithelial  cells  were  seeded  onto  5 gel-coated  35 mm  Petri  dishes  a t 2-2.5 X  m a i n t a i n e d i n c u l t u r e f o r 17-18 days. cells  formed  sub-confluent  stratification. and  patches  collagen  2  10  cells  /cm .  They  with  little  evidence  of  L a t e r i n c u l t u r e , a t days 9 - 11, t h e c e l l s were  gels  cells  had r e t r a c t e d 10-15$  of t h e i r  were  from  were  E a r l y i n c u l t u r e , days 3 - 4 , these  monolayers  of s t r a t i f i e d  approximately  collagen  evident.  the c u l t u r e  original  size  By days dish  17  cell  confluent  -  18, t h e  and c o n t r a c t e d  and t h e c e l l s  could  to  n o t be  v i s u a l i z e d by p h a s e - c o n t r a s t m i c r o s c o p y . Growth s t u d i e s active  7 ) show t h a t  phase o f growth.  stationary Cultures  growth varied  sub-confluent identical To  (Figure  by  2  -  c u l t u r e s were i n a n  Cultures  terminated  on days  indicated  by those  terminated  3  days  (50$) and c o n f l u e n t  i n t h e time  9 - 1 1  were  on days  i t took  them  in a  17 -18. to  reach  s t a t e s , as a l l c u l t u r e s d i d n o t grow a t  rates.  determine  cultures  phase,  days 3 - 4  were  i f high  seeded  density 5  a t 5 X 10  inhibited 2 cells/cm .  growth  i n culture,  No growth  several  had o c c u r r e d  t h e s e c u l t u r e s by day 4 and t h e c e l l number was comparable t o t h a t d e n s i t y c u l t u r e s terminated  a t day 9 - 11.  I t was concluded t h a t  in  o f low cultures  seeded a t h i g h d e n s i t y appeared t o be i n a s t a t i o n a r y growth phase. • T o t a l Glycosaminoglycans Cultures glucosamine  of were  human  Synthesis mammary  terminated  at  Ml  epithelial 3  time  cells points.  incubated Table  2  with  H-  shows t h e  - 42 -  2.0  -  3-4 Time  Fig.  7  9-11 (days)  Growth study 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 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 g r o w i n g ; d a y 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 d e t e r m i n e d by DNA a s s a y . E r r o r s bars i n d i c a t e SEM. (N = 3)  17-18  - 43 percentages of t o t a l fractions (days  at  during these  time points.  the  of  3 - 4 )  fraction.  GAG synthesized present  majority  i n the medium, c e l l and ECM  While the  synthesized  GAG was  The ECM contained approximately 1/5  this stage of growth.  were no longer growing (  cells  were  found  proliferating in  the medium  of the t o t a l GAG synthesized  When the cultures  reached a confluent state and  Table 2 -  stationary  early  culture and late stationary - days 17 - 18 i n culture  -  days  ),  9  -  11  in  the ECM contained  approximately 1/2 of a l l the synthesized GAG. To  determine  if  the  differences  seen  in  localization  and  type  of  glycosaminoglycan synthesized by cultures of human mammary e p i t h e l i a l c e l l s were due to t h e i r growth status and not a phenomenon of time i n culture, 5 two  cultures  were  seeded  terminated on day 4. a pattern  at  high  density  X  10  cells/cm )  and  The cultures, which were not growing on day 4, showed  of GAG d i s t r i b u t i o n s i m i l a r to  (days 9 - 1 1  (5  2  and l a t e r )  seeded i n i t i a l l y  that at  of the nongrowing cultures  low density (Table 2).  suggests that GAG l o c a l i z a t i o n was dependent  on growth phase rather  This than  time i n culture. When t o t a l (Figure  8  ),  GAG synthesized the  in  each  growing cultures  fraction  showed the  majority of the GAG into the medium, a 4 found i n the ECM. showed  greater  growing culture.' stationary  expressed  cells  were  fold increase  amounts  releasing  cell the  over the amount  of  synthesized this  GAG /  stage.  cell  indicating  The GAG /  that  In contrast, was 8 -  synthesized GAG /  fold  greater  stationary  than that  cultures  the  c e l l i n the ECM i n early of growing  Also by the stationary phase the ECM had more synthesized GAG / The late  more  c e l l found i n the  than 1-fold greater than that found i n the medium of  cultures  did the medium.  per  By the early stationary growth phase both medium and ECM  overall GAG was synthesized at medium was less  is  (days  17 -  18)  cultures. c e l l than  showed  the  -  44  -  D i s t r i b u t i o n of H-glucosamine l a b e l l e d glycosaminoglycans In normal human mammary e p i t h e l i a l c e l l s in c u l t u r e . 3  Culture Conditions  Medium  Cell  ECM  % incorporation o LOW DENSITY:^  3 + 0.54  16 + 1.59  9 + 1.22  51 + 0.96  44 + 2.26'  6 + 0.91  46 + 2.25  28 + 0.70  4 + 0.70  68 + 1.00  Growing  81 + 1.50  Early stationary  39  Late s t a t i o n a r y  1  0.76  High d e n s i t y : Stationary 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  glycosaminoglycans normal The  human  medium,  analysed  at  s t a t i o n a r y and  l a t e  . E r r o r  mammary c e l l  growing (days  of  ECM  i n t o  of c e l l s .  f r a c t i o n s  (days  3-4),  were  e a r l y  9-11)  i n d i c a t e  d e t e r m i n a t i o n s  c u l t u r e s e p i t h e l i a l  and  s t a t i o n a r y  bars  i n c o p o r a t i o n  i n  (days S.E.M.  one  17-18) of  phases.  three  experiment.  - 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 l e s s than the amount of GAG synthesized 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 . and 11 are representative of one experiment.  Figures 8, 9  P o s s i b l e explanations f o r the  v a r i a t i o n s i n the l a t e s t a t i o n a r y c u l t u r e s can be found i n the Discussion. The c u l t u r e s that were seeded a t high density and terminated  day 4  c l o s e l y resemble the e a r l y s t a t i o n a r y c u l t u r e s i n both o v e r a l l l o c a l i z a t i o n of synthesezed  GAG ( Table 2 ) and synthesized GAG / c e l l ( F i g . 11a and b)  i n d i c a t i n g that these patterns were r e l a t e d to growth status and not time i n culture.  Hyaluronic Acid Synthesis Hyaluronic a c i d (HA) was the most predominant GAG present i n the medium regardless of the growth state of the c u l t u r e .  In growing c u l t u r e s the  medium had more than a 7 - f o l d greater percentage of HA then GAG.  In the s t a t i o n a r y c u l t u r e s the medium contained  a 4 - fold  sulphated 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  f r 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 did  not change g r e a t l y between growing and confluent stages,  amount synthesized / c e l l d i d vary ( Figure 9 )•  fraction  the a c t u a l  During growth the amount  of HA was 5 - f o l d higher i n the medium than i n the ECM on a per c e l l basis.  Once  the s t a t i o n a r y growth  phase had been reached, while a l l  f r a c t i o n s 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 in  the ECM during  growth while  the medium  showed l e s s than  a 1-fold  increase.  This brought the HA / c e l l i n the medium and the ECM c l o s e r to a  1:1 r a t i o  (Figure 9, 11a and b ) . I n the growing phase, c e l l s  synthesized  twice as much HA / c e l l when compared to the synthesis of sulphated By  s t a t i o n a r y growth, the ECM had approximately  sulphated  GAGs.  This pattern was quite d i s t i n c t  the medium which showed predominately nonsulphated  to sulphated  a 1:1 r a t i o  GAGs.  of HA to  from GAG synthesis i n t o  synthesized HA, and had a r a t i o of  GAGs c l o s e r to 3:1 throughout a l l stages  of  growth. In the c u l t u r e s seeded at high density, where no growth occurred, the amount of HA synthesized  / cell  was very  similar  to the low density  s t a t i o n a r y c u l t u r e s (Figure 11a and b ) . In the medium, the high density c u l t u r e s 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 The  total  Synthesis  percentage  of GAG  identified  as CS was  less  than  that  i d e n t i f i e d as HA i n a l l f r a c t i o n s regardless of growth state ( Table 3 )• This d i f f e r e n c e was between 1.5 and 8 f o l d . of GAG i d e n t i f i e d  When comparing the percentage  as CS among the f r a c t i o n s analysed,  the ECM had the  greatest percentage of CS regardless of the growth s t a t e , between 1.5 - 3 fold  higher  than  that  found  synthesized HA l o c a l i z a t i o n .  i n the medium.  This  i s i n contrast t o  The greatest percentage of HA was found i n  the medium regardless of growth s t a t e .  Table 3 also shows the percentages  of i d e n t i f i e d GAG synthesized by high density c u l t u r e s .  The percentage of  CS i n the 2 f r a c t i o n s of high density c u l t u r e s were s i m i l a r to that of the stationary cultures.  - 48 When t h e amount o f s y n t h e s i z e d CS was expressed  / cell  g r o w i n g c u l t u r e s showed t h a t a l l f r a c t i o n s c o n t a i n e d s i m i l a r amounts.  ECM  f r a c t i o n had t h e g r e a t e s t  increase,  growth ceased, t h e ECM a l s o c o n t a i n e d did  relatively  small but  By t h e s t a t i o n a r y growth phase, w h i l e t h e medium and c e l l  f r a c t i o n s showed some i n c r e a s e i n t h e CS s y n t h e s i z e d the  ( F i g u r e 10) t h e  t h e medium.  / cell  (1.5 - 2 f o l d )  10 - 12 f o l d .  By t h e time  a 3 - f o l d g r e a t e r amount o f CS t h a n  The p e r c e n t a g e o f s y n t h e s i z e d  CS i n t h e ECM f r a c t i o n s o f  g r o w i n g and e a r l y s t a t i o n a r y phases showed o n l y a 1% i n c r e a s e  (Table  T h i s was due t o t h e l a r g e i n c r e a s e i n GAGs s y n t h e s i z e d i n t h e ECM 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 .  3).  fraction  The a c t u a l amount o f CS s y n t h e s i z e d  /  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 v e r s u s 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 (Table  a t day 4 ,the percentage o f CS  3) and t h e amount o f CS s y n t h e s i z e d  similar,  i n b o t h medium and ECM  cultures.  This  synthesized  GAG  indicates  / cell  fractions,  again  that  ( F i g u r e 11a and b) were  t o t h e low d e n s i t y s t a t i o n a r y  the  type  and  localization  of  a r e f u n c t i o n s o f growth s t a t u s and n o t a t i m e - i n - c u l t u r e  phenomenon. The r a t i o o f n o n s u l f a t e d t o s u l f a t e d GAGs has been d e s c r i b e d under t h e HA Synthesis s e c t i o n o f the R e s u l t s . Dermatan S u l f a t e S y n t h e s i s In  a l l cultures, regardless  o f growth  h i g h e s t p e r c e n t a g e s o f s y n t h e s i z e d DS. higher  than  that  found  i n t h e medium  accounted f o r 19 - 20 p e r c e n t . o f  showed p e r c e n t a g e s  contained  the  T h i s amount ranged from 4 .- 7 f o l d o f any  for 8 - 1 0  t h e medium, a d i f f e r e n c e o f a p p r o x i m a t e l y  cultures  t h e ECM  cultures  (Table  3).  DS  t h e s u l f a t e d GAGs i n t h e ECM r e g a r d l e s s o f  growth phase w h i l e o n l y a c c o u n t i n g in  status,  o f DS s i m i l a r  percent 2 fold.  o f t h e s u l f a t e d GAG The h i g h  density  to the s t a t i o n a r y c u l t u r e s f o r  - 49 -  TARI  E  5,  ^H-glucosamine incorporation into i n d i v i d u a l glycosaminoglycans 1n normal human mammary ep i me "Hal c e l l s in c u l t u r e .  Culture Conditions  Fraction^  Hyaluronic Acid  Chondroitin Sulfate  Dermatan Sulfate  Heparan Sulfate  % incorporation 3  Low density: Growing  Early stationary  Late stationary  10+0.84  1+0.00  1+0.43  60+1 .97  26+0.68  7+1.59  3+1.37  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  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  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  Medium  88+0 . 8 3  ECM  2  High density: Stationary  1. Cultures were resolved into 3 f r a c t i o n s : Medium, C e l l and ECM. The d i s t r i b u t i o n of alycosaminoglycan did not d i f f e r in the c e l l f r a c t i o n (HA, 76-78%; CS, 15-19%; DS, 1-3%; HS, 4-5%) regardless of growth conditions so the data from t h i s f r a c t i o n i s not shown. 2. Mean + S.E.M. ^  defers  to  seeding.  - 50 -  Fig.  9  3  H-glucosamine incoporation i n t o HA/ c e l l . T h e m e d i u m , c e l l a n d ECM f r a c t i o n s w e r e analysed i n growing (day 4), e a r l y s t a t i o n a r y ( d a y 10) a n d l a t e stationary ( d a y 1 7 ) c u l t u r e s o f n o r m a l h u m a n mammary epithelial cells. Error bars indicate 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 experiment.  - 51 *  0  4  8  12  16  20  Time (Days)  Fig.  10  3  H-glucosamine incoporation i n t o CS/ c e l l . T h e m e d i u m , c e l l a n d ECM f r a c t i o n s w e r e analysed i n growing (day 4 ) , e a r l y s t a t i o n a r y ( d a y 10) a n d l a t e stationary ( d a y 1 7 ) c u l t u r e s o f n o r m a l h u m a n mammary epithelial cells. Error bars indicate 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 experiment.  - 52 -  Fig.  11 a) 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, CS, and DS / c e l l i n t h e medium f r a c t i o n o f t r i 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 g r o w i n g , 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) 3  Fig.  11 b) 3 H - g l u c o s a r a i n e 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 o f t r i p l i c a t e c u l t u r e s o f n o r m a l 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 ) 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.(N = 3)  - 54 -  Time (days)  Fig.  12  3  H-glucosamine incoporation i n t o DS/ c e l l . T h e m e d i u m , c e l l a n d ECM f r a c t i o n s w e r e analysed i n growing (day 4 ) , e a r l y s t a t i o n a r y ( d a y 10) a n d l a t e stationary ( d a y 1 7 ) c u l t u r e s o f n o r m a l h u m a n mammary epithelial cells. Error bars indicate 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 experiment.  - 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 f r a c t i o n s showed equally low amounts  i n the growing c u l t u r e s (Figure 11a and b, 12). When growth ceased, DS had increased by 10 f o l d i n the ECM f r a c t i o n while the medium f r a c t i o n remained low, i n c r e a s i n g by only 1 - 2 f o l d .  Heparan S u l f a t e 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 f r a c t i o n a t any time point (Table 3 ) . However, the a c t u a l amount of HS synthesized i s higher i n the ECM f r a c t i o n than the medium f r a c t i o n of stationary cultures.  This i s not the case i n the growing  c u l t u r e s where  a l l f r a c t i o n s had only a small amount of synthesized HS (Table 3)» The high density c u l t u r e s showed s i m i l a r r e s u l t s  (Table 3 ) ; that i s ,  uniformly low percentages i n a l l f r a c t i o n s .  Electrophoresis Results GAGs  i n a l l fractions  electrophoresis  (Table  4).  were The  identified amount  of  by  cellulose  H-glucosamine  acetate labelled  m a t e r i a l applied to the c e l l u l o s e acetate s t r i p was up to 20 f o l d  higher  f o r the s t a t i o n a r y c u l t u r e s compared to growing c u l t u r e s due to the greater amount of t o t a l GAG synthesized by confluent c u l t u r e s . f o r some discrepancy between the e l e c t r o p h o r e t i c r e s u l t s assay  results,  e s p e c i a l l y i n the growth stage.  m a t e r i a l applied t o the s t r i p  the more accurate  having l e s s than 1000 DPM was not useable.  This may account and the enzyme  Since the more l a b e l l e d the r e s u l t s ,  any s t r i p  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 o f GAG (% o f T o t a l ) Culture Conditions  Sample  %HA  Low Density: Growing  MED CELL ECM  100 +0.00 100 +0.00 79 +2.64  ND ND 4 +0.96  ND ND 17 +1.80  Low Density: Early Stationary  MED CELL ECM  89 +1.36 86 +1.79 58 +1.84  3 +1.07 3 +0.77 9 +1.08  8 +1.17 11 +1.24 33 +1.55  Low Density: Late Stationary  MED CELL ECM  89 +1.04 90 +1.36 71 +1.98  2 +0.51 1 +0.43 8 +1.11  9 +1.22 9 +1.40 21 +1.67  High Density: Stationary  MED CELL ECM  85 +1.54 81 +1.21 ' 56 +2.01  1 +0.23 ND 16 +0.54  14 +0.83 19 +0.85 28 +1.07  1  ND = l e v e l i s n o t d e t e c t a b l e Mean o f 3 + SEM 1 - r e f e r s to s e e d i n g  7.HS/DS  7.CS  - 57 labelled material,  such as i n the case of sulfated GAGs i n the medium of  growing  resulted  cultures,  in  majority of the electrophoresis the enzyme assay results  non-detectable  quantities.  However,  the  results compared within 10 - 15 percent  (Table 4).  of  The d i s t r i b u t i o n 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.  regardless  The  medium  of growth status.  showed  as they co-migrate  largest  percentage  The ECM showed the largest  regardless of the growth status. together  the  The percentage  on the  The medium and c e l l  the  percentage  of HS and DS i n the ECM was due to the increased percent  was  seen with the  in  HS and DS while  all  enzyme assay  cases.  results.  the  Presumably  The high  ECM f r a c t i o n the  density  compared with the stationary cultures i n a l l GAG percentages the enzyme assay r e s u l t s .  of CS  fractions  contained  percent  HA  of HS and DS were combined  only  largest  of  percentage  contained  as  small percentages  strip.  of  increased of DS  cultures  as was seen i n  ,  Cultures Labelled with ^ S - Sulfate Normal human mammary e p i t h e l i a l c e l l s 5 2 (high  density)  and 2 X 10  cells/cm  seeded  (low  at  density)  5  X 10  were  5  cells/cm  2  l a b e l l e d 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 10  f~  period while cultures 2 cells/cm to 4 X  provided information on the Again,  only  the  seeded at low density had increased from 2 X 5 2 10 cells/cm by day 4. These cultures synthesis  of sulfated GAGs -  medium and ECM fractions  are  CS, DS and HS.  significant  as  the  cell  results varied only s l i g h t l y between the two densities and were n e g l i g i b l e  - 58 -  TABLE 5:  P e r c e n t a g e o f S u l f a t e d GAG i n t h e Medium and ECM o f High and Low Density Cultures l a b e l l e d with 55s-Sulfate.  Experiment  Fraction  High Density  Med. ECM-  71 + 0 . 5 4 72 + 0.59  Low Density  Med. ECM  77 + 0.57 65 + 0.96  1  Mean o f 3 + SEM.  %  CS  %  1  DS  % HS  8 + 0.23 18 + .0.67  2 + 0.21 2 +_ 0.37  7 + 0.57 17 + 0.59  —  T  59 -  .0005f  High  density  Low  density  •*•*•* 0  0  o°o  0  .0004  .0003 ° o° °o° °o° °o° °o° °o° °o° °o° °o° °o° °o° °o° ~o —2—  .0002  •0001  oo 'oo  0  .MED  ECM  MED  CS  Fig.  13  ECM  DS  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 D S / 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 h u m a n mammary e p i t h e l i a l c e l l s a thigh (5 x 1 0 cells/cm ) andlow (2 x 10 cells/cm ).  3 5  s  2  5  2  - 60 in  amounts  (Table  5)-  S  35  -sulfate labelled  GAGs  demonstrated  similar  3 patterns  to  the  H-glucosamine  labelled  sulfated  GAGs  i n growing  and  35 s t a t i o n a r y c u l t u r e s . I n the  S-sulfate 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 t h a n DS i n t h e medium f r a c t i o n and 4 - f o l d t h a n DS i n t h e ECM f r a c t i o n .  greater  T h i s was comparable t o an 8 - f o l d  and 4. -  f o l d g r e a t e r amount o f CS over DS i n t h e medium and ECM r e s p e c t i v e l y i n t h e 3 H-glucosamine ' l o w  density  stationary  cultures.  In  the  low  density  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 h i g h e r t h a n DS i n t h e medium f r a c t i o n and 4 - f o l d h i g h e r t h a n DS i n t h e ECM f r a c t i o n . This  3 a g a i n compared f a v o u r a b l y w i t h t h e H-glucosamine l a b e l l e d low d e n s i t y g r o w i n g 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 o f 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 GAG  i n the  / cell  (Figure  i n both  13).  detectable  H-glucosamine  HS  5 5  labelled  c u l t u r e s , t h e predominant s u l f a t e d  S-sulfate labelled  was  very  low  i n t h e low d e n s i t y  c u l t u r e s was  i n the high culture.  Also  CS f o l l o w e d  density  culture  comparable  was  by DS  and n o t that  the  amounts o f CS and DS / c e l l were always g r e a t e r i n t h e ECM f r a c t i o n t h a n i n the  medium  contained  fraction  regardless  more s u l f a t e d GAG  o f growth  i n the high  status.  The  medium  fraction  d e n s i t y c u l t u r e t h a n i n t h e low  d e n s i t y , however t h i s was n o t a g r e a t amount when compared t o t h a t i n the ECM (20 f o l d g r e a t e r i n t h e ECM).  Autoradiography GAGs s e p a r a t e d autoradiography  by c e l l u l o s e a c e t a t e  (Figure 14).  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  The s t a t i o n a r y c u l t u r e s showed t h e g r e a t e s t  3 concentration of  H - glucosamine l a b e l l e d  GAGs i n t h e ECM f r a c t i o n .  DS  I  - 61 was n o t d e t e c t a b l e u s i n g t h i s t e c h n i q u e . the  medium f r a c t i o n  and t h e g r e a t e s t  The growing c u l t u r e s showed  concentration  of labelled  only  material  t h e r e was i d e n t i f i e d as HA.  Electron Electron  Microscopy microscopy  characteristics apical  surfaces.  throughout  the  revealed  human  ( F i g . 15) i n c l u d i n g t i g h t  mammary  cells  with  epithelial  j u n c t i o n s and m i c r o v i l l i a t t h e  These were t y p i c a l f i n d i n g s i n t h e predominant c e l l cultures,  both  growing  and  e p i t h e l i a l t o f i b r o b l a s t c e l l s was a p p r o x i m a t e l y  stationary. 4 t o 1.  The  ratio  type of  - 62  MEDIUM  CELL  ECM  -  I  ^  ) i r CS  HA  F i g .  14  Autoradiography of H-glucosamine i n c o p o r a t e d GAG i n t h e m e d i u m , c e l l and and ECM f r a c t i o n s on an 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 human mammary e p i t h e l i a l c e l l s . 3  - 63  Fig.  -  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 n o r m a l human mammary c e l l s i n c u l t u r e d e m o n s t r a t i n g an e p i t h e l i a l nature.  N = nucleus TJ = t i g h t j u n c t i o n MV = m i c r o 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  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 were shown to vary the growth status of the c u l t u r e .  The  hy with  r e s u l t s w i l l be discussed under the  f o l l o w i n g 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) o v e r a l l amount of synthesized The  discussion  will  be  generated by. the research  completed  GAG  with  a  brief  presented i n t h i s  outline  t h e s i s and  the  of  questions  d i r e c t i o n 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  thesis are growing or nongrowing ( s t a t i o n a r y ) .  i n this  B r i e f l y , growing c u l t u r e s 5  are  3 - 4 2 cells/cm ).  density density  day  old  cultures  seeded  at  low  density  X  10  Stationary cultures are 9 -11 day old cultures seeded at low 5 2 (2.0-2.5 X 10 cells/cm ) and 4 day old c u l t u r e s seeded at high 5 2 (5«0  X  10  cells/cm ).  The  GAG  profile  of  i s the same regardless of time i n c u l t u r e suggesting and  (2.0-2.5  localization  of  synthesized  GAG  depends on  culture and not time spent i n c u l t u r e .  stationary  cultures  that the type, amount  the  growth  status  of  a  This f i n d i n g supports the work of  Cohn et a l . (1976). The 10 )  17 - 18 day old cultures i n i t i a l l y seeded at low density (2.0-2.5 X (late  stationary  cultures)  have  varied  GAG  patterns  and  will  be  - 65 discussed separately from the other nongrowing c u l t u r e s .  2) D i s t r i b u t i o n of Synthesized GAGs The  c u l t u r e s were separated  into  (soluble c e l l f r a c t i o n only) and previously However,  described i t should  equivalent.  by be  Parry  3 fractions,  the ECM  that  medium, the  cell  ( i n c l u d i n g the c e l l membrane) as  et a l . (1985)  noted  the  cell  and  surface  Nevo and  et  al.  ECM  GAG  (1984). are  not  Recent studies have shown that c e r t a i n types of GAGs are  cell  surface s p e c i f i c and are not found i n the ECM and v i c e versa (Rapraeger and Bemfield,  1985).  al.,  Angello et a l . , 1982;  use  1979; one,  two,  Many studies do not  or a l l of the  define an ECM  component (Cohn et  Chandrasekaran et a l . , 1979)  following - c e l l ,  cell  but instead  surface and  medium.  Most of the synthesized GAGs i n t h i s study were e i t h e r i n the medium or the ECM  f r a c t i o n with very l i t t l e i n the c e l l  (Table 2) i n d i c a t i n g that  these  molecules were not accumulating  there during any stage of growth.  Parry et  al.  accumulation  i n mouse  (1984) a l s o  found  little  of  synthesized  mammary e p i t h e l i a l c e l l s cultured on collagen g e l s . al.  (1976) found a  large amount (up  present ' i n t h e i r 3T3  cells  found equal amounts of GAG  to 90  Conversely,  percent)  cultured on p l a s t i c and i n the c e l l  and  GAG  Cohn et  of synthesized  GAG  Parry et a l . (1985)  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 t i s s u e _in v i v o , i t has been c l e a r l y demonstrated that migrate  toward  or  create  their  conducive to continued growth.  own  suitable extracellular  reached (Hay,  1982).  these No  environment  In-embryonic t i s s u e s release of GAGs allow  migratory pathways to form (Toole et a l . , 1971). observed moving along  cells  pathways u n t i l  Growing c e l l s have been  their  final  destination i s  f u r t h e r release of the migration-stimulating  GAG  - 66 occurs  and the c e l l s  begin to d i f f e r e n t i a t e  and accumulate  an ECM.  In  culture, the c e l l s may also be releasing synthesized GAG into the medium to create  a  suitable  accumulated  most  environment  of the synthesized  cessation  of growth,  contained  a substantial  There  still  f o r growth.  remained  a portion  growth  the  GAG i n the medium (Table  the l o c a l i z a t i o n increase  During  of GAG changed  so  cultures 2).  that  With  the  ECM  i n the percentage  of synthesized GAG.  of the synthesized  GAG i n the medium,  however this was h a l f of what i t was during growth. Nevo et a l . (1984) suggest  i n t h e i r study on bovine corneal endothelial  c e l l s i n culture that the ECM does not play a role i n ' e a r l y growth.  They  grew c e l l s on p l a s t i c and on ECM and noted that the growth rate f o r 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 f o r cultures on the ECM i s 18 percent higher than cultures on p l a s t i c .  They conclude that the major function of the ECM occurs  confluent, c u l t u r e .  An ECM may permit higher c e l l densities,  in a  however, as a  confluent state i s not attained u n t i l day 4 and greater growth was noted for the cultures confluent s t a t e . cells  on the ECM than on p l a s t i c f o r the 24 hours p r i o r to the Therefore,  an ECM may not be necessary  i n culture need not incorporate  f o r growth and  synthesized GAG into an ECM during  t h i s stage. From t h e i r experiments on a normal human breast c e l l l i n e (HBL-100) and two  malignant  human  breast  cell  lines  (MDA-MB-231  and  MCF-7),  Chandrasekaran et a l . (1979) show that the majority of synthesized GAG i s found  in  the  medium.  Their  cultures  confluent and terminated 48 h l a t e r . paper, results  were  labelled  when  80  Although i t i s not stated i n their'  the cultures appear to be growing at the time of l a b e l l i n g . compare  favourably  with  the  percent  results  of  growing  These  human mammary  - 67 e p i t h e l i a l c e l l s described  i n this thesis..  Cohn e t a l . (1976) found t h a t t h e medium c o n t a i n s  l e s s t h a n 20 p e r c e n t  4 of  the  synthesized  GAGs  when  their  3T3  mouse  cells  3« 5  are  X  10  2 cells/cm cells  (not confluent)and  a r e 7.0  percentage  X  a higher  10^ c e l l s / c m ^  (confluent).  o f GAGs i n t h e medium  presence  o f GAG  i n t h e medium.  r e s u l t s d i f f e r from those r e p o r t e d cell  density  d i s t r i b u t i o n of synthesized  they  when t h e  the increase conclude  in  that the  d e n s i t y may be r e l a t e d t o t h e A l t h o u g h t h e i r methodology and  i n this  and d e n s i t y - d e p e n d e n t  percent)  Although  i s not l a r g e ,  i n h i b i t i o n o f t h e growth o f c e l l s a t h i g h e r increased  (35  percentage  t h e s i s , i t seems p o s s i b l e  growth  inhibition  alter  that  t h e type and  GAGs i n t h e i r work as i t appeared t o 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  that  the d e n s i t y changes as growth o c c u r r s .  Because o f t h i s , i t i s d i f f i c u l t t o  assess  the  which  synthesis.  of  Cohn  the  factors  plays  e t a l . (1976)  relates  major  many  role  i n altering  of the a l t e r e d  GAG  synthesis  p a t t e r n s seen i n t h e c u l t u r e d 3T3 c e l l s t o d i f f e r e n c e s i n c e l l d e n s i t y .  It  would i n d e e d seem r e a s o n a b l e t o assume t h a t d e n s i t y does p l a y a r o l e i n GAG synthesis. compatible  High with  cell  density  nongrowth.  i n cultures Nongrowing  s y n t h e s i s compared t o g r o w i n g .  studied  cultures  f o r this show  an  research  is  altered  GAG  The growing c u l t u r e s have a much l o w e r  cell  density. A l t h o u g h low and h i g h d e n s i t y c u l t u r e s a r e e i t h e r growing o r 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 t o d e n s i t y  b e s i d e s growth.  a r e growing a r e g e n e r a l l y  surface. non  C e l l s that  migrating  across  T h e r e f o r e t h e l o c a t i o n o f GAGs may d i f f e r between m i g r a t i n g  migrating  cells-.  C e l l s i n contact  with  other  cells  alter their  a  and shape  - 68 r e s u l t i n g i n a l t e r e d receptor shape or d i s t r i b u t i o n .  Both of these f a c t o r s  may a f f e c t synthesis and d i s t r i b u t i o n of d i f f e r e n t GAGs. Factors i n a d d i t i o n t o growth and density may a f f e c t the l o c a t i o n of synthesized  GAGs.  For example, Parry et a l . (1985) looked  c u l t u r e s of mouse mammary e p i t h e l i a l c e l l s and  found  that  synthesized GAG.  the type  of . substrate  on three  influences  at confluent  d i f f e r e n t substrates localization  of newly  C e l l s on p l a s t i c release the majority of t h e i r GAGs i n t o  the medium while the c e l l s on collagen gels release the majority of t h e i r GAGs i n t o the ECM. synthesized growing.  C e l l s grown on p l a s t i c may not be able to incorporate  GAGs i n t o an ECM despite  the f a c t that they are no  The human mammary e p i t h e l i a l  cells  longer  cultured on collagen  gels  incorporate GAG i n t o an ECM a t a s t a t i o n a r y growth state (Table 2), j u s t as mouse mammary c e l l s cultured on collagen gels do. In the l a t e s t a t i o n a r y 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 ( c u l t u r e s seeded a t 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 s t a t i o n a r y c u l t u r e s .  However, the  a  t o t a l GAG sythesis v a r i e s from the e a r l y s t a t i o n a r y and between the 3 l a t e s t a t i o n a r y c u l t u r e s , such that 1 c u l t u r e has a greater amount of GAG, 1 has an equal amount and 1 c u l t u r e has l e s s than the amount of GAG found i n e a r l y s t a t i o n a r y c u l t u r e s (data not shown). for  this difference.  crowded  condition;  approximately  Several f a c t o r s could account  A p o s s i b l e explanation i s that the c e l l s are i n a i n some  10 percent  cases  they  of i t s o r i g i n a l  contract size.  the collagen This  g e l to  may have made i t  d i f f i c u l t f o r normal n u t r i e n t and gas exchange to occur r e s u l t i n g i n c e l l dying and death.  Varied GAG synthesis appears to be r e l a t e d somehow to the  p h y s i c a l state of the substrate.  - 69 Types of Synthesized GAGs Hyaluronic A c i d As  discussed  i n the  Introduction, various  GAGs are  associated  with  c e r t a i n b i o l o g i c a l functions on the basis of where they are found before, during and a f t e r such functions and by the types their  synthesis  ( i e . growing c e l l s ) .  The  of c e l l responsible f o r  term "growth" i n t h i s t h e s i s  r e f e r s to an increase i n c e l l  number not  an increase i n i n d i v i u a l  size.  epithelial  cells  Normal  predominantly  human  mammary  in  culture  cell  synthesize  HA during growth and release i t to the medium (Table 3) •  It  i s p o s s i b l e that these c e l l s are synthesizing HA and r e l e a s i n g i t i n t o the medium because they are growing or migrating. migraton of t i s s u e , HA i s the most abundant GAG the  epithelial  non-migrating  cells  i n the  process  t i s s u e (Toole, 1982;  of  1979).  In embryological growth and being synthesized by both  migrating  and  the  surrounding  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 f o r locomotion, as seen i n r a p i d l y moving f i b r o b l a s t s where i t i s concentrated i n the extending l a m e l l a and r e t r a c t i o n f i b e r s (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 v a r i e t y of c e l l s which detach the presence of HA  et a l . , 1982), c) by hydrating  large-areas  of t i s s u e , c r e a t i n g open spaces which c e l l s can move through,  as seen i n  neural  crest  (Abatangelo  readily i n  cells  (Tosney,  d i f f e r e n t i a t i o n (Toole, 1977).  1978)  and  d)  by  preventing  early  Toole (1972) shows that a d d i t i o n of HA i n t o  chick embryo somite c e l l s beginning to d i f f e r e n t i a t e stop f u r t h e r c a r t i l a g e formation. HA Silberstein techniques  synthesis i s not only associated with embryological and  Daniels  (1982,  that mouse mammary gland  1984)  demonstrate  i n vivo synthesize  by HA.  growth.  histochemical This  HA  is  - 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 t i s s u e . that t h i s s p e c i f i c GAG  They also show  synthesis and deposition i s present i n s e r i a l l y aged  glands and i s thus r e l a t e d to growth status and not t i s s u e age. In v i t r o , the a d d i t i o n of HA promotes locomotion (SV40-3T3, chick heart  f i b r o b l a s t s ) across  i n c l u d i n g collagen and Other c e l l s  do  not  and  into  f i b r o n e c t i n (Turley, 1984;  respond  to  the  various  types  substrates,  Bernanke et a l . , 1979)•  a d d i t i o n of HA,  (Turley, 1984), leucocytes (Forrester et a l . , 1981) (Newgreen et a l . , 1982).  of various c e l l  such as  3T3  cells  and neural crest c e l l s  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 a v a i l a b l e receptors to HA  (Turley, 1984;  U n d e r h i l l et a l . , 1981).  HA binding receptors may  only  be present at c e r t a i n times during a c e l l ' s development and a d d i t i o n of HA during inappropriate times may cells.  account f o r the lack of response i n these  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 f o r l a m i n i n , a glycoprotein found i n the ECM Rosso et a l . , 1981). to have a major  Laminin,  role,  by  as discussed i n the Introduction, i s known  binding  with  securing c e l l s to a basement membrane. laminin  creates  an  environment  associated with excess HA  (Del  thereby  collagen  proteoglycans,  in  The lack of a f f i n i t y between HA  and  conducive  for  and  detachment  f a c i l i t a t i n g growth and  for  cells  / or m o t i l i t y .  For growing c e l l s i n t i s s u e i t would appear b e n e f i c i a l , i f not o b l i g a t o r y , to have an abundance* of HA  present.  This would ensure growth and  m o t i l i t y up to the point where a f i n a l d e s t i n a t i o n i s reached and any  p o s s i b i l i t y of e a r l y adhesion and  precocious  differentiation.  / or prevent It i s  - 71 likely  that normal human mammary e p i t h e l i a l c e l l s  are  able  to  create  an  environment conducive to t h e i r growth, i e . HA synthesis (Table 3). Stationary cultures,  however, also synthesize HA which i s found both i n  the medium and the ECM.  There are  once the cultures are stationary  several explanations  for t h i s .  there i s most l i k e l y an ongoing cycle  c e l l death and p r o l i f e r a t i o n that may continually be causing the of HA and i t s studies  release into the medium.  (discussed  in  the  First,  Secondly,  Introduction)  that  it  appears  HA has  of  synthesis  from recent  several  functions.  What HA may be doing for the growing c e l l may be quite d i f f e r e n t from i t s function  in a  stationary  found i n a d i f f e r e n t  or  differentiated  location,  ie.  cell,  HA present  particularly  i n the  ECM of  if  it  is  stationary  cultures (Figure 9) may have a very d i f f e r e n t function from the HA present in  the  mammary  medium of  growing  epithelial  cells  cultures. have  only  Growing  approximately  synthesized HA i n the ECM while the stationary total  HA incorporated  there.  cultures  Thirdly,  20  of  normal human  percent  of  total  cultures have 50 percent  synthesis  of HA and i t s  of  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 i n t e r e s t i n g  to  note that HA i s the only GAG of any abundance i n the medium of a l l growing and nongrowing cultures, abundant  GAG.  Parry  et  mammary e p i t h e l i a l c e l l s  3 to al.  5 times  (1985)  cultured  the  amount  demonstrate  on p l a s t i c  This i s also true of the c e l l s on attached  of  that  the  CS, for  the  next  most  confluent mouse  medium i s  rich  i n HA.  collagen gels but these cultures  show approximately equal amounts of HA i n the medium and ECM. This finding is  identical  to  the  e p i t h e l i a l cultures  findings  on attached  predominance of HA i n the  of  the  stationary  collagen g e l s .  medium, including  normal  human mammary  Other studies also show a studies  of cloned pigmented  - 72 retinal  epithelial  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) f i n d 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 f i n d  that the  percentage of HA i n the medium a c t u a l l y increases with a high density or nongrowth s i t u a t i o n . It  appears that c e l l s i n c u l t u r e p r e f e r e n t i a l l y  medium over  other  GAGs being  synthesized.  This  release HA i n t o the phenomenon appears i n  c u l t u r e s of various c e l l types both growing and nongrowing. (1976)  find  that  although  the medium  ( s t a t i o n a r y ) and low (growing) density synthesized  contains  mainly  Cohn et a l . HA  c u l t u r e s , the o v e r a l l  in  high  amount of  GAG ( p a r t i c u l a r l y . HA) i s l e s s i n the s t a t i o n a r y c u l t u r e s so,  although the percentage of HA i n the medium i s higher, the a c t u a l amount i s less.  Parry  et a l . (1985) also f i n d  that the mouse mammary e p i t h e l i a l  c e l l s synthesize l e s s HA i n the medium when they are grown on collagen gels than on p l a s t i c and are approximately present  i n the medium (CS, HS).  equal to the sum of the other GAGs  When the collagen gels are allowed to  f l o a t e a r l y i n c u l t u r e they synthesize even l e s s HA i n t o the medium and ECM when compared to the c e l l s on attached g e l s .  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 d i f f e r e n t i a t e d than c e l l s on e i t h e r p l a s t i c or attached  collagen g e l s , as analysed  by milk p r o t e i n synthesis.  I t may be that c e l l s that are f u l l y d i f f e r e n t i a t e d do not require as much HA to function or as much of any GAG, r e s u l t i n g i n l e s s synthesis and turnover  of GAGs.  t h i s research  The normal human mammary e p i t h e l i a l c e l l s studied i n  are not d i f f e r e n t i a t e d although  they reached a confluent s t a t e .  they do stop growing when  - 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  released Parry  suggested  to the medium,  that  most of the GAG synthesized  incorporated  et a l . (1985) also find  into  an ECM or rapidly degraded.  that the c e l l  f r a c t i o n contains  amount of GAG i n confluent mouse mammary e p i t h e l i a l gels. and  i s either  the least  cultures on collagen  This i s 2 - and 5 - f o l d less than the amount of GAG i n the medium  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  al.  (1976), looking at 3T3 c e l l s cultured on p l a s t i c , and Parry et a l . (1985), looking at mouse mammary c e l l s on p l a s t i c , find greater or equal amounts of t o t a l synthesized GAG i n the c e l l compared to that i n the medium regardless of their growth status. to  The reasons f o r these  differences may be related  the components of the substrate, the physical nature  of the substrate  and c e l l - c e l l i n t e r a c t i o n s . The ECM of growing human mammary e p i t h e l i a l c e l l cultures contain l i t t l e of  the t o t a l  medium.  amount of synthesized  HA, most of which i s found  in  the  As the cultures become stationary the amount of HA increases i n  the ECM by almost 4 - f o l d , r e s u l t i n g i n approximately i n the ECM and medium (Figure 11a and b).  The major function of HA seems  to be associated with growth and / or migration Underhill, 1982).  equal amounts of HA  (Toole, 1977; 1979;  1982;  Therefore, why i s the HA found i n the ECM and why i s HA  found i n cultures of nongrowing c e l l s ?  In l i g h t of recent developments on  binding s i t e s f o r various GAGs, HA may play a much more varied role i n 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 s i t e s on c e l l  - 74 surfaces such as f i b r o b l a s t s (Underhill, 1982). bind  to f i b r o n e c t i n (Yamanda et a l . , 1980).  thought to represent The  HA binding  HA has also been shown to  S p e c i f i c proteins which are  s i t e s have been i s o l a t e d (Turley,  binding properties of HA may relate to available receptor  1982).  s i t e s , the  dependency of the p a r t i c u l a r c e l l type on HA mediated adhesion, the nature of the growth surface and the stage of adhesion to the underlying (Turley, 1984).  substrate  Turley suggests that HA may be involved i n early adhesion  formation and be replaced over time by other GAGs or possibly glycoproteins. In the normal human mammary e p i t h e l i a l very l i t t l e HS i s synthesized. associated 1982).  with  strong  cells  As pointed  cell-matrix  cultured  f o r this  study,  out i n the Introduction, HS i s  adhesion  properties  (Hook  et a l . ,  I t 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  cell  surfaces  together.  The mouse mammary e p i t h e l i a l c e l l  has  recently been shown to have d i s t i n c t  l i n e NMuMG  cell-associated HS acting as an  anchoring mechanism between the c e l l and underlying ECM (Rapraeger et a l . , 1985). the  I t may be that, i n the absence of HS, cell-associated HA (found i n  ECM fraction) may perform  the same service  f o r the c e l l .  During  growth, as opposed to nongrowth, c e l l s require the a b i l i t y to move because of the close association between growth and m o t i l i t y . Thus, they may lack substantial amounts of HA i n the ECM at that find  the same amount of HA present  mouse mammary floating  epithelial  collagen  have a t h i r d  gels  cells  these  Parry  et a l . (1985)  i n the medium and ECM of stationary  on attached  same c e l l s ,  of the amount of synthesized  compared to 18 percent  time.  i n the attached  collagen  which  gels,  although on  are more d i f f e r e n t i a t e d ,  HA i n the ECM (6 percent as  gels) and 30 percent  more sulfated  - 75 GAG.  Cohn et a l . (1976) f i n d i n t h e i r 3T3 c e l l  c u l t u r e s that HA i s the  major c e l l - s u r f a c e GAG i n both the growing and s t a t i o n a r y cultures although i t does decrease by 20 percent when the c u l t u r e stop growing. Bernfield  (1979),  while  studying  midpregnant  mouse  Gordon and  mammary  epithelial  c e l l s , f i n d that 60 percent of the GAG i n the ECM ( i n c l u d i n g c e l l membrane) i s HA.  Crawford and Crawford (1984) f i n d that f o r cloned pigmented chick  retinal  epithelial  cells  i n c u l t u r e HA  a c t u a l l y increases  r e l a t i v e to  s u l f a t e d GAGs during d i f f e r e n t i a t i o n i n d i c a t i n g that the function of HA may be d i f f e r e n t i n d i f f e r e n t c e l l types.  Sulfated GAGs The into  small amounts of s u l f a t e d GAG compared to HA (Figure 11a) released the medium  has  been  observed  by  others  Chandrasekaran et a l . , 1979; Crawford et a l . , 1984). al.  (Cohn  et  a l . , 1976;  In contrast, Parry et  (1985) f i n d that confluent c u l t u r e s of mouse mammary e p i t h e l i a l  cells  cultured on p l a s t i c release equal amounts of HA and CS i n t o the medium, although  Cohn e t a l . (1976) f i n d mainly nonsulfated  mouse 3T3 c e l l s .  When these c e l l s  enter  GAG i n the medium of  a s t a t i o n a r y growth phase they  release an undersulfated CS that accounted f o r approximately the GAG found i n the medium.  40 percent of  They a l s o f i n d that l e s s than 5 percent of  the GAG i n the medium i s HS regardless  of growth s t a t u s .  This i s very  s i m i l a r to the f i n d i n g s of t h i s study (Table 3 ) . Although the functions of HA are s t i l l not widely understood, even l e s s 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 f o r c e r t a i n c e l l neural although  crest  cells  and leucocytes  (Hook  types such as  et a l . , 1982; Turley,  1984),  they do not appear as strong as the more h i g h l y s u l f a t e d DS and  - 76 HS.  CS has also been shown to have a stronger a f f i n i t y than HA f o r the ECM  glycoprotein laminin but not as strong as the a f f i n i t y of DS and HS ( D e l Rosso, et a l . , 1981).  I t would appear that CS may a c t as an intermediate  GAG between the functions of HA and the more h i g h l y s u l f a t e d GAGs. When monocytes change morphologically and express more macrophage-like c h a r a c t e r i s t i c s (a process equivalent to d i f 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)  f i n d that the ECM of mouse mammary e p i t h e l i a l c e l l s i n the f l a n k region o f developing mammary gland incorporates synthesized CS as the major GAG. The c e l l s i n these region are nongrowing and considered The  nongrowing  and d i f f e r e n t i a t e d  mouse  mammary  "stabilized" epithelial  c u l t u r e s studied by Parry et a l . (1985) have predominantly  tissue.  cells i n  S-GAG i n the  ECM, ranging from a 4 - f o l d greater amount than HA f o r c e l l s on p l a s t i c to a 12 - f o l d greater amount than HA f o r c e l l s on f l o a t i n g collagen g e l s . 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 g e l are s i m i l a r to those on the f l o a t i n g collagen g e l 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 s t a t i o n a r y growth state (but not d i f f e r e n t i a t e d ) and CS and HS when they are d i f f e r e n t i a t e d . f i n d i n g of t h i s work.  These r e s u l t s compare favourably with the  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 s u b s t a n t i a l amount of CS incorporated i n t o the ECM, almost 5 - f o l d higher than the amount of CS i n the ECM of growing c u l t u r e s (Figure 10). Also i n t e r e s t i n g to note i s the v i r t u a l lack of DS i n any f r a c t i o n but the ECM (Figure l l ) i n t h i s study.  Parry et a l . (1985) also f i n d that DS  - 77 is  only  present  i n appreciable  epithelial cells.  amounts i n the ECM of the mouse mammary  They find that the more d i f f e r e n t i a t e d the cultures are  the greater i s the amount of DS i n the ECM. 21 percent  This amount ranges from 1 to  of the t o t a l CS pool i n the c e l l s on p l a s t i c to 42 percent f o r  the c e l l s on f l o a t i n g collagen gels (most d i f f e r e n t i a t e d ) with the attached collagen  g e l 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 CS pool) and i n several cases i s too low to be detected.  of t o t a l  The same pattern  emerges i n the human mammary e p i t h e l i a l c e l l s cultured i n t h i s study. medium and c e l l  fractions often have nondetectable  always has most of the DS (Figure 10).  l e v e l s while  The  the ECM  DS i s most pronounced i n the ECM of  nongrowing cultures, 4 - f o l d greater than the amount of DS i n the ECM of growing cultures  (Figure  ll).  DS contains  predominately L-iduronic  residues which are epimers of the carbon-5-glucuronic a c i d .  The presence  of iduronic acid has been linked with the function of the orderly of  collagen  fibrils  (lozzo,  1985).  I t s presence  acid  arranging  i n the ECM may be  e s s e n t i a l f o r 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 and  i s demonstrating that d i f f e r e n t species  are either c e l l  surface  or ECM associated  of heparan sulfate exist (Rapraeger et a l . , 1985).  Parry et a l . (1985), as mentioned, find almost equal amounts of HS and CS i n the ECM of confluent mouse mammary c e l l cultures on collagen gels. A l l cultures  i n their  differentiation. synthesized  study Nevo  have et  a hormonal  a l . (1984)  milieu  also  find  designed 50  to stimulate  percent  of the  GAG i n the ECM of their d i f f e r e n t i a t e d bovine endothelial c e l l s  - 78 to be HS.  An i n t e r e s t i n g f i n d i n g 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 t h e i r 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 f r a c t i o n s 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 i n t o 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 c u l t u r e s studied f o r t h i s t h e s i s (Table 3).  One  explanation f o r t h i s may  to the process or maintance of not  differentiated  require HS. the use  and  be t h a t , i f HS i s r e l a t e d  d i f f e r e n t i a t i o n , the c e l l s described  therefore  are not  able  to  synthesize  or  do  are not  They are obviously able to adhere to the substrate e i t h e r by  of other proteoglycans  or glycoproteins or p o s s i b l y by  utilizing  any HS present i n the collagen g e l . Although  the  evidence  d i f f e r e n t i a t i o n i s not not  for  the  importance  of  HS  conducive to growth.  this  as  1  Mg/ml  surrogate  caused a GAG  Clark et a l . (1975) f i n d  binds  nontoxic to  cell  G^  arrest.  surfaces  morphology of the c e l l s as w e l l as causing 1973).  cellular  conclusive, s e v e r a l studies have shown that i t i s that the a d d i t i o n of  dextran s u l f a t e (an a r t i f i a l S-GAG) to c u l t u r e s of BHK low  in  c e l l s i n doses as  I t was  and  discovered  i s able- to  that  change  growth changes (Goto et a l . ,  Majacek and Bornstein (1984) conclude that components of an ECM  c o n t r o l the b i o s y n t h e t i c a c t i v i t y of c e l l s .  the  can  They observed that a d d i t i o n of  exogenous soluble HS or DS can increase the production of 2 noncollagenous proteins i n cultured r a t smooth muscle c e l l s . have t h i s a f f e c t .  via  include any the  do  not  They postulate that these two proteins played a r o l e i n  growth i n h i b i t i o n . may  Other proteoglycans  The  mode by which the HS molecule acts upon the  cell  or a l l of the f o l l o w i n g : l ) a l t e r a t i o n of the c e l l shape  cytoskeleton,  in  turn  altering  biosynthetic  abilities,  2)  via  - 79 surface receptor and a response evoked by a "second messenger" system or 3) endocytosis  of the proteoglycan  (Majacek et a l . , 1984). most l o g i c a l  and d i r e c t d e l i v e r y to i t s s i t e of a c t i o n  The f i r s t method stated would appear to be the  way f o r HS incorporated  i n t o an ECM, i n c l u d i n g  the c e l l  surface, to exert i t s e f f e c t 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 c u l t u r e s an ECM i s produced and approximately one h a l f of the synthesized GAG found there i s s u l f a t e d with the majority of that being CS (Figure l i b ) .  DS i s a l s o present i n the ECM of nongrowing cultures (Figure  10).  3) O v e r a l l Amount of Synthesized GAG The  o v e r a l l amount of GAG synthesized by c e l l s seems r e l a t e d to s e v e r a l  f a c t o r s i n c l u d i n g 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 v s . 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 n e i t h e r  growing  nor d i f f e r e n t i a t e d , synthesize  much  q u a n t i t i e s of GAG than those c u l t u r e s on collagen gels (a 4 - f o l d amount).  cultures,  phase,  synthesize  less  GAG  when  compared  a decrease of approximatley 20 percent.  study show an increase a growing  studies  greater  Cohn e t a l . (1976) also f i n d that t h e i r mouse 3T3 cells,, i n a  nongrowing  from  larger  underway  stimulated  The r e s u l t s i n t h i s  i n the amount of GAG synthesized  to nongrowing i n this  state  laboratory  (Figure indicate  to d i f f e r e n t i a t e v i a a d d i t i o n  to the growing  as the c e l l s go  7 ) . However, that  these  of hormones,  preliminary cells,  decrease  when their  - 80 o v e r a l l GAG synthesis, a phenomenon found by other researchers  (Parry et  a l . , 1984). I t 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 al.  (1984) f i n d  endothelial  this  cells.  to be They  true  compared  i n their  study  their  results  proteoglycan content of basement-membranes tissue  and  amounts.  discovered  that  (Muir, 1977). on  Nevo et  cultured  with  bovine  reports  on  i s o l a t e d from other e n d o t h e l i a l  cultured ' c e l l s  are  synthesizing  In t h i s s t u d y no a n a l y s i s of GAG from normal breast ;  greater  tissue i s  done as a comparison and there are no reports of t h i s information i n the literature.  I t would be i n t e r e s t i n g to see i f t h i s t i s s u e , l i k e  others,  showed increase GAG synthesis i n c u l t u r e . Much work has been done i n the area of malignancy and GAG synthesis.  As  t h i s was discussed a t length i n the Introduction only d i f f e r e n c e s 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 1982).  (Shishiba et a l . , 1984; Angello et a l . ,  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 t i s s u e and human t h y r o i d adenocarcinoma t i s s u e and compared them f o r t o t a l GAG synthesis. the  They f i n d a 6 to 15 f o l d greater amount of GAG i n  adenocarcinoma t i s s u e .  nonmalignant c e l l s with synomymous.  However, some studies compared malignant to  the assumption that nonmalignant and normal are  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 f a s t growing and can grow i n s o f t agar and one which i s slow growing and does not grow i n soft  agar.  They  find  that  the more  aggressive  tumor  subpopulation  - 81 synthesizes 8 times more GAG than the l e s s 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 (MDA-MB-231 synthesize  and MCF-7). equal  l i n e s , especially  They  find  that  the HBL-100  and MDA-MB-231  amount of GAG and the MCF-7 considerably so-called  lines  less.  Cell  "normal" ones, should be compared with normal  primary c e l l s with some r e s e r v a t i o n . The three  c e l l s used i n the experiments presented  different  women ( d e t a i l s  produced approximately One  i n t h i s thesis  are from  i n Appendix 9 ) . Two o f the experiments  equal amounts o f t o t a l GAG a t a l l stages of growth.  experiment produced greater amounts of GAG, up to 5 - f o l d  higher.  However, the percentages of GAG i n each f r 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 t h i s occur?  human mammary e p i t h e l i a l c e l l s s e v e r a l factors The  age of the donor may be s i g n i f i c a n t .  When dealing with normal  must be taken i n t o account.  The two experiments with  o v e r a l l GAG synthesis were from two women i n t h e i r mid to l a t e The  t h i r d experiment i s from a 19 year o l d woman.  from younger women are more m e t a b o l i c a l l y a c t i v e . for  growth ( c e l l  older women. mammary  proliferation  less  thirties.  I t may be that c e l l s The o v e r a l l  potential  and GAG synthesis) may be decreasing i n  S i l b e r s t e i n and Daniels (1984) looked a t s e r i a l l y aged mouse  ducts  autoradiographic  and  compared  GAG  and histochemcial  synthesis techniques.  and  localization  Interestingly,  they  using 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 o l d ducts but that older ducts appear to synthesize l e s s o v e r a l l GAG, i d e n t i f i e d by t h e i r decreased a b i l i t y to concentrate r a d i o l a b e l l e d m a t e r i a l .  - 82 Future  Research  The  r e s u l t s o f t h i s study provide  research.  First,  experiments  t h e framework f o r two major a r e a s o f  designed  to  achieve  a  differentiated  phenotype i n t h e normal human mammary e p i t h e l i a l c e l l _in v i t r o would a n a l y s i s o f GAG s y n t h e s i s under t h i s  permit  c o n d i t i o n . A change i n GAG s y n t h e s i s  i n d i f f e r e n t i a t i o n as opposed t o growth/nongrowth would a l l o w  f o r further  postulation  to c e l l u l a r  on t h e f u n c t i o n o f v a r i o u s  development. compared  Second,  to a  normal  malignant  GAGs as they  mammary  baseline  epithelial  i n culture  relate cells  regarding  GAG  c a n now  be  s y n t h e s i s and  localization. Expanding f u r t h e r on t h e o v e r a l l f u n c t i o n o f GAGs i t would appear, g i v e n the  s t r u c t u r a l heterogeneity  o f GAGs and p r o t e o g l y c a n s ,  that  they a r e n o t  l i m i t e d t o p r o v i d i n g h y d r a t i o n o r a d h e s i o n m o l e c u l e s t o t h e ECM. the  substrate  with  various  amounts  and  types  o f GAG  Enhancing  may  alter  the  f u n c t i o n i n g o f the c e l l s on t h a t s u b s t r a t e and a i d i n e l u c i d a t i n g t h e r o l e o f GAGs i n c e l l b e h a v i o u r . differentiate  i f  differentiation.  F o r example, growing c e l l s may s t o p growth and  placed This  on  a  should  substrate  increase  designed  understanding  to of  stimulate cell-matrix  interactions. 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 p r o t e i n core i n a p r o t e o g l y c a n .  t o GAG r e s e a r c h  i s the r o l e  of the  Recent r e s e a r c h i n d i c a t e s t h a t t h e p r o t e i n  c o r e may i n p a r t be r e s p o n s i b l e f o r t h e f i n a l d e s t i n a t i o n o f a  proteoglycan  (Nevo  changes t h e  e t a l . 1984).  Addition  p a t t e r n o f GAG s y n t h e s i s .  to cultures  B - x y l o s i d e a c t s as a "pseudo" c o r e p r o t e i n which  the  cell  al.  (1984) demonstrates t h a t t h e a d d i t i o n o f B - x y l o s i d e causes t h e c e l l s t o  release  uses t o l i n k  of a B-xyloside  most  of their  synthesized  synthesized  GAG s i d e c h a i n s .  PG i n t o  The s t u d y  t h e medium  by Nevo e t  whereas  t h e same  - 83 cells  without  treated  with  B-xyloside B-xyloside  c u l t u r e s without •beginning  incorporated have  72 p e r c e n t  B-xyloside.  t o be examined  t h e PG i n t o an ECM. of the f i n a l  The c u l t u r e s  cell  count  The importance o f t h e p r o t e i n c o r e  and t h e system  used  i n this  study  i t s e l f t o s t u d i e s aimed a t e x a m i n i n g p r o t e i n core f u n c t i o n .  of the i s just  would  lend  A corollary to  t h i s would be t o examine t h e GAGs t h a t a r e c e l l - a s s o c i a t e d a s opposed t o those t h a t a r e d e f i n i t e ECM c o n s t i t u e n t s . HA s e c t i o n , t h a t h y a l u r o n i c the p r e s e n t  a c i d i s mainly  cell  f u n c t i o n i s there  i n the  membrane a s s o c i a t e d .  time i t i s n o t c l e a r how t h e s e m o l e c u l e s a r e a t t a c h e d  plasma membrane and what t h e i r The  I t may be, as d i s c u s s e d  as opposed  system used i n t h i s s t u d y would have t o be m o d i f i e d  At  to the  t o t h e ECM.  to separate  t h e 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  f u r t h e r i n t e r e s t i n g and v a l u a b l e  presented  should  lend  themselves  experiments i n t h e c o n t i n u i n g  c e l l - E C M i n t e r a c t i o n s as t h e y r e l a t e t o c e l l f u n c t i o n s .  to  search f o r  - 84 SUMMARY This  thesis  distribution culture. the  research  was d e s i g n e d  to investigate  o f GAGs by normal human mammary  epithelial  The medium f r a c t i o n o f a l l c u l t u r e s  growth s t a t u s .  depending  on  percentage  contained  cells  and  i n tissue  HA r e g a r d l e s s  growth  status  50-60  -  t h e growing  synthesized percent.  GAG  while  cultures  had  only  the s t a t i o n a r y  Of t h e p e r c e n t a g e  o f GAG  a  small  cultures  had  i n t h e ECM o f  s t a t i o n a r y c u l t u r e s , 50 p e r c e n t were s u l f a t e d ; t h e s u l f a t e d GAG i n c l u d e d (.10%) and DS  of  The ECM f r a c t i o n v a r i e d i n t h e type and amount o f GAG  of t o t a l  approximately  the synthesis  (30%).  HS d i d n o t comprise more  than  5-6 p e r c e n t  CS  i n any  culture. To remove the. l e n g t h for  t h e changes  seen  o f time spent i n c u l t u r e a s a p o s s i b l e i n GAG  synthesis  between  growing  explanation  and  stationary  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 h i g h and  terminated  cultures and  c l o s e l y resembled  No growth o c c u r r e d . the s t a t i o n a r y  The GAG p r o f i l e  cultures  seeded  of these  a t low d e n s i t y  t e r m i n a t e d a t days 9 - 1 1 The  the  on day 4.  density  type and l o c a t i o n o f s y n t h e s i z e d  growth  status  o f the c u l t u r e .  GAG was found t o be dependent on  Sulfated  GAGs  appeared  r e l a t e d t o s t a t i o n a r y growth and were l o c a t e d  i n the ECM.  be  t h e predominant  present i n a l l stages  medium.  o f growth  and were  t o be  more  HA appeared t o GAG i n t h e  - 85 Appendix 1 Transport Medium F12:DME Hepes b u f f e r C a l f serum Insulin  -  (l:l) lOmM 5$ 5Mg/ml  DMe - Delbecco's Modified Eagles Medium  Appendix 2 D i s s o c i a t i o n Medium F12 Hepes b u f f e r BSA Insulin Collagenase Hyaluronidase  -  (1:1) lOmM 2$ 5Mg/l 300U/ml lOOU/ml  -  (1:1) lOmM 5$ 5ug/ml  Appendix 3 Growth Medium F12:DME Hepes b u f f e r F e t a l C a l f Serum Insulin  Appendix 4 Freezing Medium DME - 50$ DMSO - 6% CS ' - 44$  (dimethysulfoxide)  - 86 Appendix 5 Detergent S o l u t i o n T r i s pH 7.2 T r i t o n X-lOO Deoxycholate  - lOmM - 1% - 1%  Appendix 6 Enriched T r i s 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 b u f f e r Na H0P - 50mM NaCl - 2mM EDTA - 2mM a d j s t to pH 7.4 2  4  (tetrasodium s a l t )  b) Hoechst Dye (Calbiochem) stock = 20 yg/ml i n H 0 2  c) DNA ( c a l f thymus) stock = 100 yg/ml i n  H2PO4  Appendix 8 Karnovsky's s o l u t i o n 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  buffer  - 87 Appendix 9 Patient  Age  Reason f o r Surgery  1) M.A.S.  34  accessory breast t i s s u e  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. 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