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Regulation of neutral proteinase and plasminogen activator secretion by epithelial cells in vitro Hong, Hee Ling 1985

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REGULATION OF NEUTRAL PROTEINASE AND PLASMINOGEN ACTIVATOR SECRETION BY EPITHELIAL CELLS IN VITRO by HEE-LING HONG B.D.S., The U n i v e r s i t y of Singapore, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n FACULTY OF GRADUATE STUDIES Department of Oral Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1985 © Uee Ling Hong, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of flft/K. j>iQUO&S The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D E - 6 n/R 'n ABSTRACT The aim of t h i s t hesis was to study the regulation of proteinase secretion by e p i t h e l i a l c e l l s ( E - c e l l s ) derived from the e p i t h e l i a l c e l l r e s t s of Malassez. Since these e p i t h e l i a l c e l l r ests are present only i n small numbers in- v i v o , E - c e l l s derived from porcine c e l l rests were cultured according to Brunette et a l . (1976) and conditions chosen so that detectable amounts of the proteinases, neutral proteinase and plasminogen a c t i v a t o r , could be obtained. The regulation of the secre-ti o n of these enzymes was investigated by varying the c e l l population density, adding E . C o l i lipopolysaccharide to the cultures and a l t e r i n g the shape of the E - c e l l s by both chemical and physical means. C e l l population density modulated both neutral proteinase and plasminogen activator secretion. Neutral proteinase secretion was highest at low c e l l population d e n s i t i e s and the a c t i v i t y decreased with increasing c e l l population density. Plasminogen a c t i v a t o r secre-t i o n followed a s i m i l a r pattern. E s c h e r i c h i a c o l i lipopolysaccharide ( E . c o l i LPS) stimulated both neutral proteinase and plasminogen activator secretion. LPS extracted by the phenol method and LPS extracted by the t r i c h l o r o a c e t i c a c i d method caused s i m i l a r increases i n neutral proteinase a c t i v i t y but the increase i n plasminogen a c t i v a t o r a c t i v i t y was greater when the t r i c h l o r o a c e t i c acid extracted LPS was used. These findings support the proposal that b a c t e r i a l LPS i n contact with p e r i a p i c a l tissues could stimulate the e p i t h e l i a l c e l l rests into increased production of proteinases, thereby contributing to the degradation of connective tissue associated with dental cyst formation. - i i -E - c e l l shape was a l t e r e d by physical and chemical means. Addition of cholera toxin and d i b u t y r y l cAMP caused E - c e l l s to f l a t t e n . Phorbol myristate acetate, however, caused the c e l l s to r e t r a c t s l i g h t l y . Mechanical stretching was applied to the c e l l s to cause c e l l f l a t t e n i n g , and c e l l rounding was effected by mechanical r e l a x a t i o n . Another method made use of E - c e l l s grown on a substrate with V-shaped grooves which caused the c e l l s to adopt a rounder shape more frequently than c e l l s grown on a f l a t substrate. In addition, dishes coated with increasing concentrations of poly(HEMA) solu t i o n , which a l t e r e d d i s h adhesivity to the c e l l , caused the c e l l s to become less well-spread. In a l l experiments, a more f l a t t e n e d c e l l shape cor r e l a t e d with a reduced l e v e l of neutral proteinase and plasminogen ac t i v a t o r secretion while a more rounded shape correlated with increased amounts of neutral proteinase and plasminogen ac t i v a t o r secretion. - i i i -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENT i x CHAPTER 1 - INTRODUCTION GENERAL INTRODUCTION 1 LITERATURE REVIEW 3 I) C e l l Shape and i t s Relationship to C e l l Function 3 II) E p i t h e l i a l C e l l Rests of Malassez 6 II I ) Connective Tissue Degradation 9 1) I n t r a c e l l u l a r Degradation 10 2) E x t r a c e l l u l a r Degradation - P r o t e o l y t i c Enzymes .... 11 a) Collagenase 11 b) Neutreal Proteinase 12 c) Plasminogen Activator 13 IV) Regulation of Proteinase A c t i v i t y 14 1) Control of Enzyme A c t i v i t y 15 a) Neutral Proteinase 15 i ) Tissue I n h i b i t o r of Metalloproteinases ...... 15 i i ) Latent Enzyme 16 i i i ) Serum I n h i b i t o r s 18 b) Plasminogen A c t i v a t o r 18 i ) Tissue I n h i b i t o r 18 i i ) Latent Enzyme 19 i i i ) Serum Inhibitors 20 2) Control of Enzyme Synthesis 21 a) In Vivo and In V i t r o Studies 21 - i v -Page b) Factors Implicated i n the Regulation of P.A. Synthesis 22 c) Factors Implicated i n the Regulation of N.P. Synthesis 25 PROBLEM FORMULATION 28 CHAPTER 2 - MATERIALS AND METHODS I) C e l l Culture 30 1) Procedures 30 2) Determination of C e l l Number 31 II) Chemicals 31 III ) Methods of Alter, ing C e l l Shape 31 1) Mechanical Stretching 31 2) Preparation of Grooved Substratum 35 3) Preparation of poly(HEMA) Plates 36 IV) Morphological Techniques • 36 1) Transmission Electron Microscopic Techniques 36 2) Richardson's Stain 38 3) Scoring of C e l l Spreading 38 V) Preparation of Media f o r Enzyme Assays 39 VI) Assays 39 1) C y c l i c AMP Assay 39 2) Assay for Neutral Proteinase 40 3) A c t i v a t i o n and C h a r a c t e r i s t i c s of the Neutral Proteinase 43 4) Plasminogen A c t i v a t o r Assay 45 VII) Error Study and S t a t i s t i c a l Analysis 47 - v -Page CHAPTER 3 - RESULTS I) Effect of C e l l Population Density 51 II) Effect of E.coli LPS 54 I I I ) C e l l Shape and Proteinase Secretion 58 1) Cholera Toxin 59 2) Dibutyryl cAMP 61 3) Phorbol Myristate Acetate 61 4) Mechanical Stretching 64 5) Grooved Substrata 67 6) Poly(HEMA)-Coated Dishes 69 CHAPTER 4 - DISCUSSION I) C e l l Population Density 73 II) E . c o l i LPS 76 I I I ) C e l l Growth and Proteinase Secretion 77 IV) C e l l Shape and Proteinase Secretion 78 1) C e l l shape and the Cytoskeleton 82 2) Penman's Hypothesis on the Relationship Between the Cytoskeleton and Nuclear Metabolism 86 V) Relationship Between N.P. and P.A. Secretion 87 VI) Future Work 89 1) Improvements i n Techniques 89 2) Proposed Experiments 91 SUMMARY AND CONCLUSIONS 92 REFERENCES 94 - v i -LIST OF TABLES Page Table I E f f e c t of D i f f e r e n t I n h i b i t o r s on Neutral Proteinase A c t i v i t y 46 Table II E f f e c t of E . c o l i LPS (Phenol Extract) on Neutral Proteinase A c t i v i t y 55 Table III E f f e c t of Cholera Toxin (C.T.) lng/ml 60 Table IV E f f e c t of Dibutyryl C y c l i c AMP ( B t 2 cAMP) 62 Table V E f f e c t of Phorbol Myristate Acetate (PMA) 63 Table VI E f f e c t of Mechanical Stretching on E-Cells 66 Table VII E f f e c t of Relaxing E - C e l l s Cultured on Stretched Membranes 66 Table VIII E f f e c t of Grooved Substrata 68 - v i i -LIST OF FIGURES Page F i g . 1 Technique for mechanical stretching of c e l l s grown on a petriperm d i s h 32 F i g . 2 Design of the mechanical stretching experiment 34 F i g . 3 Time course of neutral proteinase assay 41 F i g . 4 Standard curve of a z o c o l l f u l l y digested 42 F i g . 5 Dependence of a z o c o l l degradation on enzyme concentration 42 F i g . 6 Comparison of mersalyl acid and TPCK tryp s i n as a c t i v a t o r s of neutral proteinase 44 F i g . 7 Standard curve of Streptokinase a c t i v i t y 48 F i g . 8 Absorbance change as a function of the amount of added conditioned media 49 F i g . 9 The e f f e c t of c e l l population density on N.P. a c t i v i t y ... 52 F i g . 10 The e f f e c t of c e l l population density on P.A. a c t i v i t y ... 53 F i g . 11 E f f e c t of E . c o l i LPS (TCA extract) and E . c o l i LPS (phenol extract) on N.P. a c t i v i t y 56 F i g . 12 E f f e c t of E . c o l i LPS (TCA extract) and E . c o l i LPS (phenol extract) on P.A. a c t i v i t y 57 F i g . 13 Electron microscopic section of E - c e l l s grown on a petriperm dish 65 F i g . 14 E f f e c t of poly(HEMA) concentration on N.P. a c t i v i t y at day 3 71 F i g . 15 E f f e c t of poly(HEMA) concentration on P.A. a c t i v i t y at day 3 72 - v i i i -ACKNOWLEDGEMENTS I am very g r a t e f u l to Dr. D. Brunette, my supervisor, for his guidance throughout t h i s project, patience i n reading my numerous i n i t i a l d r a f t s , and suggestions while I was writing t h i s t h e s i s . I wish to also thank a l l the people i n the Department of Oral Biology who have made my introduction to research very pleasant: e s p e c i a l l y Holly Maledy and Dianne P r i c e f o r t h e i r help and friendship; and Tony Ng for his generous help when 'things around the place broke down'. I am g r a t e f u l to Lesley Weston who did the e l e c t r o n microscopic sections and Dianne who c a r r i e d out the cAMP assays and did the fi g u r e s . And to Soo Inn, who believed i n me and encouraged my venture i n t o the laboratory, I am e s p e c i a l l y g r a t e f u l . I wish to also thank the Medical Research Council of Canada f or th e i r f i n a n c i a l support. - i x -1. CHAPTER 1 - INTRODUCTION GENERAL INTRODUCTION " C e l l s are normally very s p e c i a l and well behaved They follow rules and are receptive to signals and requests from one another. The rules govern both growth and t e r r i t o r y , and they have to be followed p e r f e c t l y . Otherwise we wouldn't end up divided into fingers and toes, eyes and brain; we would end up a hopeless and shapeless jumble.." L.L. Larison Cudmore (1977) The rules governing c e l l growth have been the subject of i n v e s t i -gation f o r many decades. Most of the work have concerned chemical mediators including macromolecules and simple compounds such as c y c l i c nucleotides, but physical s t i m u l i such as mechanical stretching or gas tension can also a f f e c t c e l l d i v i s i o n and behaviour. Of p a r t i c u l a r relevance to t h i s thesis i s the work of Folkman and Moscona (1978) who have shown that c e l l shape can regulate c e l l p r o l i f e r a t i o n . For example, f i b r o b l a s t s grown on t i s s u e culture dishes f l a t t e n out and p r o l i f e r a t e whereas the same population shows reduced growth when placed on dishes treated i n such a way that the c e l l s are more rounded. Folkman and Moscona concluded from these experiments that the shape of the c e l l regulates DNA synthesis and growth of normal c e l l s . In contrast, the growth of transformed c e l l s or tumour-like c e l l s i s not regulated by i n d i v i d u a l c e l l shape. Folkman and Moscona found that SV 3T3 transformed c e l l s , maintained at d i f f e r e n t degrees of f l a t n e s s , grew at s i m i l a r rates. Another c h a r a c t e r i s t i c of transformed c e l l s i s t h e i r tendency to secrete elevated l e v e l s of plasminogen a c t i v a t o r . For example, a good number of transformed c e l l s l i n e s and a v a r i e t y of human cancers have 2. shown increased plasminogen a c t i v a t o r secretion compared to normal c e l l s . It has been suggested that the elevated l e v e l s of plasminogen ac t i v a t o r have an important role i n tumour invasion and metastasis; plasminogen a c t i v a t o r and i t s product plasmin act to a i d tumour c e l l s i n detaching from th e i r primary s i t e and penetrating through the extra-c e l l u l a r matrix (Reich, 1977; Ng & Kellen, 1983). Thus both the l o s s of growth control and increased plasminogen a c t i v a t o r secretion are c h a r a c t e r i s t i c s of transformed and neoplastic c e l l s . From the above, i t appears that there i s a complex but not neces-s a r i l y causal r e l a t i o n s h i p between growth, c e l l shape and proteinase secretion i n tumour c e l l s . It i s thus of i n t e r e s t to determine whether c e l l shape regulates growth and or proteinase secretion i n normal c e l l s . For f i b r o b l a s t s , t h i s appears to be the case for the proteinase collagenase (Aggeler et a l . , 1984). The r e l a t i o n s h i p between e p i t h e l i a l c e l l shape and proteinase secretion has not been studied d i r e c t l y and i s the topic of t h i s t h e s i s . The c e l l s used i n my study were e p i t h e l i a l c e l l s ( E - c e l l s ) derived from the e p i t h e l i a l c e l l r e s t s of Malassez and the enzymes selected for study were neutral proteinase and plasminogen a c t i v a t o r . By analogy with other c e l l types, i t was hypothesized that proteinase secretion by e p i t h e l i a l c e l l s would be regulated by c e l l population density, b a c t e r i a l lipopolysaccharides and treatments which a f f e c t c e l l shape. The background information for these studies i s presented i n the following order. I) C e l l shape and i t s r e l a t i o n s h i p to c e l l function II) E p i t h e l i a l c e l l r e s t s of Malassez 3 . I l l ) Connective ti s s u e degradation 1) I n t r a c e l l u l a r degradation 2) E x t r a c e l l u l a r degradation - p r o t e o l y t i c enzymes IV Regulation of Proteinase a c t i v i t y 1) Control of enzyme a c t i v i t y 2) Control of enzyme synthesis LITERATURE REVIEW I) C e l l Shape and i t s Relationship to C e l l Function C e l l shape has been found to regulate a number of c e l l functions. Folkman and Moscona (1978) showed that the shape of endothelial c e l l s was t i g h t l y coupled to DNA synthesis and growth. These investigators varied t i s s u e culture d i s h adhesivity by precoating the dishes with d i f f e r e n t concentrations of poly (2-hydroxyethyl methacrylate) (poly (HEMA)) solutions. The extent of c e l l spreading was thus c o n t r o l l e d and measured i n terms of c e l l diameter or c e l l height. DNA synthesis, measured by the incorporation of 3H-thymidine, was found to be inversely proportional to the height of the c e l l . Thus, the more fl a t t e n e d the c e l l , the greater the DNA synthesis. Folkman et a l further showed that the phenomenon of density dependent i n h i b i t i o n of c e l l growth may be mediated by c e l l shape. E n d o t h e l i a l c e l l s grown to confluence on tissue culture dishes were compared with sparsely populated c e l l s grown on poly(HEMA) coated dishes. Sparsely populated c e l l s with the same height as t h e i r confluent counterparts showed s i m i l a r rates of 3H-thymidine incorporation. Thus the crowded c e l l s on p l a s t i c which were at a s i m i l a r height as the sparsely populated c e l l s on poly(HEMA) dishes showed s i m i l a r rates of DNA synthesis. 4. Ben-Ze'ev et a l . (1980) investigated the r e l a t i o n s h i p between c e l l shape and DNA, rRNA, mRNA and protein synthesis. The authors showed that mouse f i b r o b l a s t s i n suspension demonstrated a strong i n h i b i t i o n of DNA, mRNA, rRNA and protein synthesis. Upon r e p l a t i n g on p l a s t i c dishes which allowed the c e l l s to f l a t t e n , the synthesis of these macromolecules was restored. Using the poly(HEMA) system of Folkman, these authors showed that the amount of DNA synthesis restored was correlated with c e l l shape over a c e r t a i n range. Restoration of mRNA synthesis was s e n s i t i v e to c e l l shape too. Reattachment of suspended c e l l s on poly(HEMA) dishes (which caused c e l l rounding) resulted i n a suppression of the mRNA synthesis compared to c e l l s which were allowed to reattach to p l a s t i c dishes (which caused c e l l f l a t t e n i n g ) . The ribosomal RNA synthesis was s i m i l a r l y a f f e c t e d by c e l l shape. Protein synthesis, however, was not affected by changes i n c e l l shape; as long as the c e l l was i n contact with the dish, protein synthesis proceeded at the same rate. There are some i n d i c a t i o n s that c e l l shape may play a r o l e i n enzyme secretion. Lawrence et a l . (1979) reported that several t r e a t -ments which caused r a t ovarian granulosa c e l l s to round up also r a i s e d plasminogen a c t i v a t o r (P.A.) l e v e l s . Aggeler et a l . (1984) attempted to study the r e l a t i o n s h i p between c e l l shape and collagenase secretion by using various chemical agents which a l t e r c e l l shape. C e l l rounding caused by tryp s i n , phorbol myristate acetate and cytochalasin B was correlated with an increase i n collagenase secretion. Although suggestive, these r e s u l t s are not d e f i n i t i v e because the chemical agents used are known to cause multiple e f f e c t s on c e l l function. 5. S t u d i e s i n v e s t i g a t i n g t h e i n d u c t i o n o f d i f f e r e n t i a t i o n o f mouse mammary c e l l s i n d i c a t e t h a t c e l l shape i s an important f a c t o r i n t h i s p r o c e s s . D i f f e r e n t i a t i o n o f mammary e p i t h e l i a l c e l l s has been shown t o be i n f l u e n c e d by s e v e r a l hormones. E x p l a n t s of mouse mammary gla n d s m a i n t a i n e d i n d e f i n e d medium c o n t a i n i n g l a c t o g e n i c hormones r e s u l t i n m o r p h o l o g i c a l and b i o c h e m i c a l changes i n d i c a t i v e of f u n c t i o n a l d i f f e r -e n t i a t i o n ( r e v i e w o f Saacke and Heard, 1974; F o r s y t h , 1971). But mammary e p i t h e l i a l c e l l s grown on p l a s t i c or g l a s s , i n the presence o f l a c t o g e n i c hormones show l i t t l e d i f f e r e n t i a t i o n . However, Emerman e t a l . (1978) showed t h a t mammary e p i t h e l i a l c e l l s grown on f l o a t i n g c o l l a g e n g e l s , i n t h e pr e s e n c e o f l a c t o g e n i c hormones, show m o r p h o l o g i c a l and b i o c h e m i c a l d i f f e r e n t i a t i o n . F l o a t i n g c o l l a g e n g e l s have t h r e e advantages o v e r p l a s t i c o r g l a s s . 1) The f l o a t i n g g e l per m i t s a c c e s s i b i l i t y of n u t r i e n t s t o b a s o l a t e r a l c e l l s s u r f a c e s . 2) T h e r e i s e p i t h e l i a l c e l l i n t e r a c t i o n w i t h c o l l a g e n . 3) The s u b s t r a t e i s f l e x i b l e , a l l o w i n g the c e l l s t o assume a c u b o i d a l t o columnar shape w i t h rounded a p i c a l s u r f a c e s . I n c o n t r a s t , c e l l s grown on p l a s t i c o r g l a s s a r e f l a t t e n e d and have broad a p i c a l s u r f a c e s . Emerman e t a l . t e s t e d t h e s i g n i f i c a n c e o f each o f t h e s e f e a t u r e s i n r e l a t i o n to mammary c e l l d i f f e r e n t i a t i o n . S u b s t r a t e f l e x i b i l i t y a l l o w i n g f o r c e l l shape changes was found t o be i m p o r t a n t f o r f u n c -t i o n a l d i f f e r e n t i a t i o n i n mammary e p i t h e l i a l c e l l s i n - v i t r o . That t h e a b i l i t y o f t h e s e c e l l s t o assume a c u b o i d a l o r columnar shape p l a y s an important r o l e i n the i n d u c t i o n o f d i f f e r e n t i a t i o n has been c o n f i r m e d by t h e ex p e r i m e n t s o f Shannon and P i t e l k a ( 1 9 8 1 ) . 6. I I ) E p i t h e l i a l C e l l Rests of Malassez The e p i t h e l i a l rests of Malassez (ERM) are a group of e p i t h e l i a l c e l l s derived from Hertwig's e p i t h e l i a l root sheath which plays an important role i n root formation. The development of the root i s described by Bhussry (1976). The a p i c a l elongation of Hertwig's e p i t h e l i a l root sheath, which consists of an inner and outer e p i t h e l i a l layer, delineates the shape, siz e and number of roots. C e l l p r o l i f e r a -t i o n at the t i p of the short, h o r i z o n t a l , leading component of the root sheath r e s u l t s i n root sheath elongation. At the same time, the adjacent connective tissue c e l l s (both on i t s inner and outer surface) p r o l i f e r a t e and the inner connective t i s s u e c e l l s adjacent to the root sheath d i f f e r e n t i a t e into odontoblasts and form dentine. As soon as t h i s happens, the e p i t h e l i a l c e l l s of the root sheath becomes disrupted, leaving a network of e p i t h e l i a l c e l l s . The connective t i s s u e c e l l s adjacent to the outer e p i t h e l i a l layer of the root sheath move through the openings to l i n e up along the dentine surface as cementoblasts, secreting cementum. The remnants of the root sheath are present as c e l l rests throughout the l i f e t i m e of the tooth, t h e i r number decreasing with age, but reaching a plateau around age 30 years (Reeve and Wentz, 1962; Simpson, 1965). There i s general agreement that the ERM e x i s t s as i n a network of c e l l s (Provenza, 1972; Sicher and Bhaskaram, 1972) but whether t h i s network i s continuous with attachment epithelium or not i s open to debate. Simpson (1965) and Valderhaug et a l . (1966) show that part of the ERM appears to be continuous while Scott and Symons (1974) describe them as " i s o l a t e d columns" or "an incomplete network". The r e l a t i o n -ship of the ERM to the attachment epithelium (or j u n c t i o n a l epithelium) 7. at the marginal region has recently been investigated by Spouge (1984), who studied eight porcine molars and t h e i r supporting t i s s u e s . Spouge reports that the coronal margin of the network of e p i t h e l i a l r e s t s was connected to the junctional epithelium i n intermitent i n t e r v a l s . This r e l a t i o n s h i p between the j u n c t i o n a l epithelium and the network of ERM has been previously described by Spouge (1980) as a 'basketball hoop' with the j u n c t i o n a l epithelium resembling the hoop and the network of c e l l rests suspended from the hoop, forming the basket. The d e t a i l e d configuration of ERM i n humans remains to be explored. The p h y s i o l o g i c a l function of the ERM i s not known. Suggested r o l e s include an endocrine function (Higaki, 1931; Orban, 1953), forma-t i o n of enamel pearls (Gottlieb, 1921), a protective role against root resorption (Waerhaug, 1958), and the maintenance of the periodontal ligament space (Loe and Waerhaug, 1961). The ERM has been shown to be able to p r o l i f e r a t e i n inflammation and contribute to the formation of dental cysts (Valderhaug, 1972). Har r i s and T o l l e r (1975) have suggested that the e p i t h e l i a l layer i n the cyst serves as a b a r r i e r i s o l a t i n g pulpal i r r i t a n t s from the r e s t of the connective t i s s u e . As dental cysts are the most common benign, destructive lesions of the skeleton (Harris and T o l l e r , 1975), the study of the regulation of p r o l i f e r a t i o n of the ERM would appear to be important. In the l i g h t of the a b i l i t y of the ERM to p r o l i f e r a t e i n response to the Inflammatory stimulus, and the anatomical proximity of the c e l l s to the attachment epithelium, Spouge (1980) has suggested that the a p i c a l migration of the attachment epithelium i n chronic p e r i o d o n t i t i s could be f a c i l i t a t e d by the p r o l i f e r a t i o n of the ERM already present. 8. Thus the response of the ERM at the marginal region to inflammation could aid i n pocket formation i n periodontal disease. Several aspects of ERM behaviour i n - v i t r o have been studied. Grupe et a l . (1967) were the f i r s t to observe ERM p r o l i f e r a t i o n i n t i s s u e as opposed to c e l l c u l t u r e . They found that ERM i n tissue culture i n contrast to ERM in-vivo showed DNA synthesis and a decrease i n nuclear/cytoplasmic r a t i o . There was an absence of glycogen, l i t t l e s u c cinic dehydrogenase a c t i v i t y and an accummulation of fat droplets i n d i c a t i n g that the metabolism had s h i f t e d from a c i t r i c a c i d cycle to a hexose monophosphate shunt. Since then, i t has been found that these e p i t h e l i a l c e l l s can be i s o l a t e d and grown i n c e l l c u l t u r e . Brunette et a l . (1976) reported a technique f o r c u l t u r i n g e p i t h e l i a l - l i k e c e l l s from porcine periodontal ligament. Moreover the e p i t h e l i a l c e l l s could be separated from f i b r o -b l a s t s by a t r y p s i n - c i t r a t e s a l i n e s o l u t i o n (Owens, 1974). F i b r o b l a s t -l i k e c e l l s rounded up a f t e r 5 to 10 minutes while the e p i t h e l i a l - l i k e c e l l s took twice as long to detach from the dish. Brunette et a l . (1977) further observed that monkey periodontal ligament i n culture produced outgrowths of multilayered e p i t h e l i a l and f i b r o b l a s t c e l l s . The e p i t h e l i a l c e l l s i n multilayers demonstrated attachment by desmosomes on the v e n t r a l and dorsal surfaces. No such structure was observed between epithelium and f i b r o b l a s t l a y e r s . E p i t h e l i a l c e l l s were also found sandwiched between two f i b r o b l a s t l a y e r s , mimicking t h e i r in-vivo r e l a t i o n s h i p where e p i t h e l i a l c e l l r e s t s of Malassez are surrounded by f i b r o b l a s t c e l l s of the periodontal ligament. These porcine 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 further 9. characterized by Brunette et a l . (1979). The c e l l s showed a d i p l o i d karyotype with 38 chromosomes, t h e i r size remaining constant over 9 subcultures. The e p i t h e l i a l c e l l s also secreted several proteins and prostaglandins E and F. Birek et a l . (1983) showed that these e p i t h e l i a l c e l l s i n culture produce f a c t o r / f a c t o r s which cause bone resorption I n - v i t r o . The addi-t i o n of indomethacin to the culture r e s u l t e d i n an i n h i b i t i o n of bone resorption i n d i c a t i n g that the prostaglandin produced by the e p i t h e l i a l c e l l s contributed to bone resorption. These r e s u l t s suggest that the ERM may play a d i r e c t role i n the bone resorption seen i n dental cysts. These c e l l s have also been found to be able to phagocytose collagen (Birek et a l . , 1980) and to secrete neutral proteinase and l a t e n t collagenase i n - v i t r o (Pettigrew, Ph.D. Thesis; Limeback and Brunette, 1981). Thus the secretion of proteinases together with endocytosis by e p i t h e l i a l c e l l s may be one of the mechanisms involved i n connective tissue destruction observed i n the pathogenesis of the dental cyst. I l l ) Connective T i ssue Degradation Collagen, the major component of connective tissue can be degraded through two pathways: 1) i n t r a c e l l u l a r degradation following phagocytosis by the c e l l ; 2) degradation of collagen by p r o t e o l y t i c enzymes secreted by the c e l l into the e x t r a c e l l u l a r environment. 10. 1) I n t r a c e l l u l a r Degradation Whether c e l l s can phagocytose e x t r a c e l l u l a r collagen has been investigated by numerous workers. Electron microscopic evidence have led to the suggestion that f i b r o b l a s t s (Ten Cate et a l . , 1976) and macrophages (Parakkal, 1969) can ingest and degrade collagen. However, the p o s s i b i l i t y of the i n t r a c e l l u l a r collagen observed i n e l e c t r o n microscopic sections r e s u l t i n g from endogenously synthesized collagen could not be discounted. The i n - v i t r o experiments of Svoboda et a l . (1979) have shown c l e a r l y that f i b r o b l a s t s can phagocytose collagen f i b r i l s . S e r i a l e l ectron microscopic sections of f i b r o b l a s t s which had been c u l t i v a t e d i n the presence of rat t a i l collagen, showed large banded collagen f i b r i l s within the c e l l s . In contrast, f i b r o b l a s t s cultured without exogenous collagen did not contain large i n t r a c e l l u l a r accummulations of collagen f i b r i l s . Phagocytosis of collagen by f i b r o b l a s t s has also been shown by Yajima and Rose (1977). These investigators c u l t i v a t e d human g i n g i v a l f i b r o b l a s t s on collagen coated c o v e r s l i p s . E l e c t r o n microscopic sections of the c e l l s showed the presence of collagen f i b r i l s surrounded by dense bodies. Using a s i m i l a r experimental system as Svoboda et a l , Birek et a l . (1980) showed that E - c e l l s derived from ERM could also phagocytose rat t a i l collagen. It had been previously reported that the i n t r a c e l l u l a r degradation of collagen was c a r r i e d out by h y d r o l y t i c enzymes i n the lysosome (Deporter and Ten Cate, 1973). Birek et a l . therefore tested for acid phosphatase, a lysosomal enzyme, and found a p o s i t i v e r e a c t i o n i n v e s i c l e s associated with i n t r a c e l l u l a r collagen. Isolated lysosomal 11. f r a c t i o n s from E - c e l l s were found to consist of cathepsin Bj, cathepsin D, and other lysosomal enzymes. Furthermore, the addition of t h i s Isolated lysosomal f r a c t i o n to rat t a i l collagen resulted i n the degra-dation of collagen into smaller fragments. Thus Birek et a l . concluded that ERM can phagocytose collagen and that the ERM possess the mechanisms for the d i g e s t i o n of phagocytosed collagen. The available evidence suggests that e p i t h e l i a l c e l l s can c o n t r i -bute to connective ti s s u e degradation by phagocytosis of collagen f i b r i l s . In addition, there i s evidence that a s i g n i f i c a n t amount of newly synthesized collagen i s degraded before i t reaches the extra-c e l l u l a r space (Bienkowski et a l . , 1978; Baum et a l . , 1980). 2) E x t r a c e l l u l a r Degradation - P r o t e o l y t i c Enzymes E x t r a c e l l u l a r degradation of collagen involves p r o t e o l y t i c enzymes, the most important being collagenase and neutral proteinase. Plasminogen ac t i v a t o r i s also thought to have a role i n the degradation of connective t i s s u e . a) Collagenase Collagenase i s defined as an enzyme capable of degrading native collagen at p h y s i o l o g i c a l pH and temperatures (Harris and Cartwright, 1977). In-vivo, collagen e x i s t s as aggregates of f i b r i l s , each f i b r i l containing collagen molecules with cross-l i n k s between them. Collagen f i b r i l s with intermolecular cross-l i n k s are more r e s i s t a n t to collagenases than are non-cross-linked f i b r i l s , but i t i s not known whether other enzymes are needed to cleave the c r o s s - l i n k s before the collagenase can act on the collagen molecule. 12. The collagenase cleaves the collagen molecule i n t o 2 f r a g -ments: the 3/4 fragment, TC^, and the 1/4 fragment, TC B. The collagenase has been shown to be unable to degrade the 2 fragments further (McCroskery et a l . , 1975; Gross et a l . , 1974). Once cleaved, the fragments then denature and become s o l u b i l i s e d as g e l a t i n polypeptides (Sakai and Gross, 1967; Mc Croskery et a l . , 1973). The collagenases are metallo-enzymes, Z n + + and Ca"*"*" being e s s e n t i a l for the i r a c t i v i t y . Thus agents l i k e EDTA, i n h i b i t collagenase a c t i v i t y , presumably by chelating C a + + and or Zn*"*". b) Neutral Proteinase While collagenases are defined as acting s p e c i f i c a l l y on native collagen at p h y s i o l o g i c a l pH and temperatures, the neutral proteinases act to further degrade the products of primary co l l a g e n o l y s i s (Harris and Cartwright, 1977). Neutral proteinase has been reported to be able to degrade g e l a t i n , proteoglycan, a z o c o l l and azocasein (Evans et a l . , 1981). It i s not. clear whether t h i s represents a s i n g l e enzyme with wide substrate s p e c i f i c i t y , or d i f f e r e n t enzymes with d i f f e r e n t substrate s p e c i f i c i t i e s but possessing s i m i l a r c h a r a c t e r i s t i c s . There i s one report ( S e l l e r s et a l . , 1978) that the neutral proteinase that degrades g e l a t i n can be separated chromatographic-a l l y from that which attacks c a r t i l a g e proteoglycan. The g e l a t i n -degrading enzyme did not degrade collagen, c a r t i l a g e proteoglycan or azocasein but a z o c o l l was degraded to a small degree. The other neutral proteinase degraded c a r t i l a g e proteoglycan, 13. azocasein and a z o c o l l but neither collagen nor g e l a t i n . Other investigators have been unable to separate the g e l a t i n degrading and the proteoglycan degrading neutral proteinases (Cartwright et a l . (1983) and Murphy et a l . (1981)). Neutral proteinase shares many c h a r a c t e r i s t i c s with collagenase. Both are metallo-enzymes, requiring Ca for a c t i v i t y and both are act i v e at neutral pH. They are i n h i b i t e d by the same agents, including EDTA, cysteine and some serum proteins. The l a t e n t forms can be activated by t r y p s i n or APMA. Because the two enzymes are often found together, i t has been suggested that collagenase and neutral proteinase are secreted together as a co l l a g e n o l y t i c package (Pettigrew et a l . , 1978). c) Plasminogen Ac t i v a t o r (P.A.) By d e f i n i t i o n , a plasminogen a c t i v a t o r i s an enzyme that converts plasminogen into the active form, plasmin. Plasminogen ac t i v a t o r s have usually been c l a s s i f i e d i n t o four categories: c i r c u l a t i n g P.A., tissue P.A., urinary P.A. (urokinase) and tiss u e c u l t u r e P.A. How these d i f f e r e n t P.A. are rela t e d to each other i s not known but urokinase has been shown to be immunologically d i s t i n c t from tis s u e P.A. Like other serine proteinases, P.A. i s in h i b i t e d by di i s o p r o p y l phosphofluoridate. It i s also i n h i b i t e d by some serum proteins. P.A. i s thought to be involved i n a host of p h y s i o l o g i c a l and pathological processes including: f i b r i n o l y s i s , p r o t e o l y t i c processing of c e l l u l a r and serum proteins ( i n complement a c t i v a t i o n , k i n i n formation and p r o i n s u l i n formation), migration 14. of macrophages during inflammation, t i s s u e remodelling, and neoplasia (Gelehter et a l . , 1983). Of p a r t i c u l a r relevance to t h i s t hesis i s the suggestion that P.A. i s involved i n e x t r a c e l l u l a r p r o t e o l y s i s of connective t i s s u e . Reich (1978) has observed that processes such as inflam-mation, ovulation, trophoblast implantation, and neoplasia share i n common, events l i k e t i s s u e remodelling and c e l l migration, where connective tissue degradation takes place. Furthermore Reich has shown that there i s a c o r r e l a t i o n between these events and increased secretion of P.A. Reich therefore suggests that the secretion of P.A. provides a mechanism which enables tumour c e l l s and inflammatory c e l l s to migrate, and allows the degradation of the ovarian f o l l i c l e w a l l during ovulation. He further postulates that the secretion of P.A. could be part of a mechanism for l o c a l -i s e d e x t r a c e l l u l a r p r o t e o l y s i s of connective t i s s u e . S i m i l a r l y , Ossowski et a l . (1979) have also shown that there i s a large increase i n P.A. following the i n v o l u t i o n of mammary glands, during which there i s degeneration of the secretory epithelium. A second r o l e f o r P.A. i n connective tissue destruction has been put forward by Werb et a l . (1977). Their f i n d i n g that rheumatoid synovial c e l l s could produce plasmin i n the presence of plasminogen, and the a b i l i t y of these c e l l s i n the presence of plasminogen to degrade collagen suggests that the plasmin a c t i v a -ted the latent collagenase which i s also secreted by these c e l l s . IV) Regulat ion of Proteinase A c t i v i t y Proteinases are subject to regulation on two l e v e l s : 1) con t r o l of the enzyme a c t i v i t y ; 2) con t r o l of synthesis. 15. 1. Control of Enzyme A c t i v i t y a) Neutral Proteinase i ) Tissue I n h i b i t o r of Metalloproteinase Tissue i n h i b i t o r s play an important role i n regulating the a c t i v i t y of the neutral proteinase. S e l l e r s et a l . (1979) f i r s t showed that the collagenase I n h i b i t o r secreted by rabbit bone explants also i n h i b i t e d the g e l a t i n and proteoglycan-degrading neutral proteinase. The rabbit bone collagenase i n h i b i t o r p u r i f i e d by Cawston et a l . (1981) migrated as a s i n g l e band at Molecular Weight (MW) 28000 on the sodium dodecyl sulphate (SDS) polyacrylamide g e l . This p u r i f i e d i n h i b i t o r named Tissue Inhibi t o r of Metalloproteinases (TIMP) blocked the a c t i v i t i e s of collagenase, gelatinase and proteoglycan-degrading neutral proteinase. Its a c t i v i t y was l o s t with t r y p s i n treatment (250 ug/ml) for 30 minutes, heat treatment at 90°C for 1 hour and a f t e r reduction and a l k y l a t i o n . I n h i b i t o r s with s i m i l a r properties as TIMP have been i s o l a t e d from human amniotic f l u i d (Murphy et a l , 1981a); from human synovium explants of normal, rheumatoid, and o s t e o a r t h r i t i c patients (Murphy et a l . , 1981b); and from human g i n g i v a l f i b r o b l a s t s (Heath et a l . , 1982). The presence of considerable amounts of the free t i s s u e i n h i b i t o r appears to be c o r r e l a t e d with e i t h e r the absence or low l e v e l s of the enzyme. Meikle et a l . (1980) showed that explants of rabbit c a l v a r i a secrete elevated l e v e l s of the i n h i b i t o r i n the f i r s t two days of Culture when no neutral proteinase could be detected. With the appearance of the l a t e n t neutral proteinase, the l e v e l of the i n h i b i t o r dropped markedly. Cambray et a l . 16. (1981) found that explants of normal rabbit synovium produced no active nor latent enzyme but considerable amounts of the i n h i b i t o r , whereas synovium from a r t h r i t i c j o i n t s showed elevated l e v e l s of the active enzyme and a sharp decrease i n i n h i b i t o r l e v e l s . Thus Murphy and S e l l e r s (1980) suggest that a l l connective tissues secrete tissue i n h i b i t o r s to control the a c t i v i t y of the metalloproteinases. Only when there i s excess enzyme over the i n h i b i t o r would tissue breakdown take place. Control over the a c t i v i t y can be exerted through d i f f e r e n t rates of synthesis and removal of the i n h i b i t o r . i i ) Latent Neutral Proteinase Neutral proteinase i n a l a t e n t form has been reported from many tissue and c e l l culture systems. The latent enzyme can be activated by the a c t i o n of proteinases such as t r y p s i n or by the action of the t h i o l - b i n d i n g reagents such as APMA. Most of the studies on the l a t e n t enzyme have been done on collagenase. Since the neutral proteinase presents as a latent form and i s activated by the same agents, and the TIMP has been shown to i n h i b i t both collagenase and neutral proteinase ( S e l l e r s et a l . , 1979), i t i s highly probable that the information on the l a t e n t collagenase applies to the neutral proteinase too. Two mechanisms have been proposed to explain the l a t e n t form of the enzyme: 1) the latent enzyme i s a proenzyme (Zymogen) that needs proteinases for a c t i v a t i o n ; 2) the l a t e n t enzyme i s an enzyme-inhibitor complex. 17. D i f f e r e n t authors have proposed that the l a t e n t collagenase exists as a proenzyme form (Vaes, 1972; S t r i c k l i n et a l . , 1977). S t r i c k l i n et a l . p u r i f i e d procollagenase from human ski n f i b r o b l a s t s and organ cultures of human skin. Two forms of procollagenase (60,000 and 55,000 daltons) were detected on sodium dodecyl sulphate polyacrylamide gels, and a c t i v a t i o n by tr y p s i n resulted i n 50,000 and 45,000 dalton forms, r e s p e c t i v e l y . Auto-a c t i v a t i o n however, caused no detectable change i n MW of the enzyme. S t r i c k l i n et a l . suggested that t h i s could be the r e s u l t of a conformational change i n the zymogen molecule, leading to the ac t i v e s i t e being exposed without change of peptide bonds. Further evidence from V a l l e and Bauer (1979) has strengthened the hypothesis that the enzyme i s secreted i n a zymogen form. Va l l e and Bauer showed that both i n t r a c e l l u l a r and e x t r a c e l l u l a r collagenase proteins secreted by human s k i n f i b r o b l a s t s i n culture, comigrate with the two p u r i f i e d forms of procollagenase on the SDS polyacrylamide g e l . There i s also evidence f o r the second hypothesis of an enzyme-i n h i b i t o r complex. S e l l e r s et a l . (1977) have found that latent collagenase from bone explants could be acti v a t e d by t r y p s i n as well as 4-amino-phenyl mercuric accetate (APMA) and other t h i o l -blocking agents, and t h i s conversion was accompanied by a decrease i n MW of 8,000 to 15,000. A collagenase i n h i b i t o r was i d e n t i f i e d and p a r t i a l l y p u r i f i e d . Moreover, the combination of the i n h i b i t o r and APMA-activated enzyme resulted i n a latent enzyme complex which could be reactivated by APMA or t r y p s i n . S e l l e r s et a l . therefore suggested that the latent enzyme exists as an 18. enzyme-inhibitor complex and that reagents such as APMA and thiocyanate cause a d i s s o c i a t i o n of the enzyme-inhibitor complex while proteinases act by p r e f e r e n t i a l degradation of the i n h i b i t o r . It was also s i g n i f i c a n t that the disappearance of the i n h i b i t o r i n the medium of bone explants coincided with the appearance of the latent enzyme. There i s however, a discrepancy i n MW data which i s hard to explain. The combination of the i n h i b i t o r (MW 30,000) with the active collagenase (MW 30,000) yiel d e d a l a t e n t enzyme of MW 40,000. i i i ) Serum I n h i b i t o r s H a r r i s and Krane (1972) report that the neutral proteinase from rheumatoid synovial tissues i s i n h i b i t e d by serum proteins. Murphy et a l . (1982) observed that a2-macroglobulin i n h i b i t e d the a c t i v i t y of the p u r i f i e d gelatinase from human polymorphonuclear leucocytes. Thus serum i n h i b i t o r s play a r o l e i n c o n t r o l l i n g the a c t i v i t y of the neutral proteinase. Therefore medium containing serum used f o r maintaining c e l l s i n c u l t u r e makes i t d i f f i c u l t f o r the presence of enzyme a c t i v i t y to be detected. b) Plasminogen Ac t i v a t o r i ) Tissue I n h i b i t o r s Tissue i n h i b i t o r s to P.A. have been found i n a number of c e l l systems. Loskutoff and Edgington (1981) report the presence of an i n h i b i t o r to tissue P.A. and urokinase i n the c y t o s o l of rabbit endothelial c e l l s . Bovine endothelial c e l l s have also been shown to secrete an i n h i b i t o r to both human ti s s u e P.A. and urokinase (Loskutoff et a l . , 1983) which has an apparent MW of 55,000 and which i s stable to reducing agents, denaturants, and extremes of pH. 19. Protease nexin, an i n h i b i t o r secreted by human f i b r o b l a s t s , has been extensively studied. It i n h i b i t s both urokinase and thrombin, and i t resembles antithrombin I I I i n having a high a f f i n i t y heparin binding s i t e (Baker et a l . , 1980). However, i t i s e l e c t r o p h o r e t i c a l l y and immunologically d i s t i n c t from a n t i -thrombin I I I . P h i l l i p s et a l . (1984) have demonstrated an i n h i b i t o r d i f f e r e n t from protease nexin. They report an i n h i b i t o r secreted from human endothelial c e l l s that showed a very low a s s o c i a t i o n rate with thrombin unlike protease nexin. Also while protease nexin has a high a f f i n i t y heparin binding s i t e , the en d o t h e l i a l c e l l i n h i b i t o r has a low a f f i n i t y f o r heparin. Coleman et a l . (1982) report that hepatoma c e l l s secrete an i n h i b i t o r to urokinase-type P.A. Subsequent studies showed that this i n h i b i t o r was also d i f f e r e n t from protease nexin, having no e f f e c t on thrombin a c t i v i t y . Hoal et a l . (1983) report a d i f f e r e n t mechanism which regulates P.A. a c t i v i t y . These authors found that melanoma c e l l s which secrete P.A., when c o - c u l t i v a t e d with f i b r o b l a s t s , showed a decrease i n P.A. a c t i v i t y . Evidence was presented which suggests that the P.A. secreted i n t o the medium could become bound to f i b r o b l a s t s and become in a c t i v a t e d . i i ) Inactive or Latent PA Levin (1983) reported that human endothelial c e l l s from the umbilical vein secrete a P.A. that i s immunologically s i m i l a r to t i s s u e P.A. It i s i n a c t i v e i n the media, has an apparent MW of 100,000 and i s not s e n s i t i v e to diisopropylfluorophosphate. Levin 2 0 . suggests that t h i s i s a t i s s u e P.A.-inhibitor complex based on the following evidence: 1) conditions that d i s s o c i a t e other serine protease-inhibitor complexes r e s u l t i n a reduction of a n t i - t i s s u e P.A. immunoprecipitable material from 100,000 MW to 60,000 MW; 2) radioiodinated melanoma c e l l t i s s s u e P.A. mixed with the conditioned medium from endothelial c e l l s showed an increase i n MW from 70,000 to 110,000 i n d i c a t i n g that the conditioned medium contained an i n h i b i t o r capable of complexing tissue P.A. Levin also showed that the i n a c t i v e form of t i s s u e P.A. could be activated by SDS treatment. A t i s s u e type P.A.-inhibitor complex has also been demonstrated to be secreted by human endothelial c e l l s ( P h i l l i p s et a l . , 1984). Serum free conditioned media showed no a c t i v i t y on f i l m gels but SDS treatment converted the complex from a 95,000 to 135,000 MW to a 72,000 MW form which was a c t i v e . The MW of the i n h i b i t o r component was estimated at 50,000 to 70,000. i i i ) Serum I n h i b i t o r s Serum has been shown to be able to suppress c e l l associated PA a c t i v i t y i n endothelial c e l l s i n - v i v o (Levin and Loskutoff, 1980). Urokinase has been found to be slowly inactivated by several plasma protease i n h i b i t o r s : o^-antiplasmin, antithrombin I I I , and 0 4 - a n t i t r y p s i n (Moroi and Aoki, 1976; Clemmensen and Christensen, 1976). Tissue type P.A. was found to be i n h i b i t e d by ct^ -antiplasmin and to a l e s s e r extent by o^-macroglobulin (Korninger and Collen, 1981). In another study, Rijken et a l . (1983) found that the tissue type PA i n plasma formed a complex with ct2-antiplasmin and a j - a n t i t r y p s i n . Cont ro l of Proteinase Synthesis a) In- Vivo and In V i t r o Studies A study that involved measuring the amount of neutral proteinase i n extracts of tissue from patients has shown that the l e v e l of neutral proteinase was higher i n g i n g i v a l samples that were c l e a r l y inflammed compared to non-Inflammed samples (Uitto et a l . , 1981). The usefulness of t h i s study i s i n confirming that tissues i n vivo secrete neutral proteinase and that the l e v e l i s elevated i n the diseased state. However, the l i m i t a t i o n s of such studies include d i f f i c u l t i e s i n determining the c e l l u l a r source of the enzyme and determining regulatory f a c t o r s that modulate enzyme secretion. In order to study f a c t o r s that regulate proteinase secretion, i n v i t r o c e l l culture provides a suitable model. C e l l culture y i e l d s a defined population of c e l l s and the e f f e c t of ah added agent on c e l l behaviour can be studied and the l e v e l of enzyme secreted e a s i l y measured. However, the i n v i t r o state i s not necessarily i d e n t i c a l to the i n vivo state with i t s c i r c u l a t o r y system and other p h y s i o l o g i c a l metabolites. One of the goals of c e l l culture i s to be able to grow c e l l s i n a serum-free medium because serum contains a host of fa c t o r s which makes i t d i f f i c u l t to i n t e r p r e t studies where an agent added to the culture r e s u l t s i n a measurable e f f e c t . Of p a r t i c u l a r relevance to this study i s the fact that serum contains a 2 -macroglobulin, an i n h i b i t o r of neutral proteinase a c t i v i t y , and other serum i n h i b i t o r s which i n a c t i v a t e P.A. a c t i v i t y . Recently Brunette (1984a) has developed a low serum media (0.5% dialysed f o e t a l c a l f serum) containing low Ca^and K +, which maintains e p i t h e l i a l c e l l s In c u l t u r e . This has made i t possible to grow e p i t h e l i a l c e l l s and to c o l l e c t the secreted proteinases i n t h i s medium. b) Factors Implicated i n the Regulation of P.A. Synthesis C e l l culture conditions can a f f e c t P.A. secretion, one v a r i -able being serum. The presence of serum has been found to depress the P.A. a c t i v i t y i n keratinocyte c u l t u r e s , as measured by the rate of f i b r i n l y s i s (Birkedahl-Hansen and Taylor, 1983). Levin and Loskutoff (1980) also report that when 0.1% f o e t a l c a l f serum was added to bovine a o r t i c endothelial c e l l cultures, the c e l l -associated P.A. a c t i v i t y decreased by 50%. Serum from horse, goat, r a t , and humans also caused a suppression i n P.A. a c t i v i t y . Thus the c o l l e c t i o n of P.A. should preferably be done i n serum free media which circumvents the problem of serum i n h i b i t i o n of P.A. a c t i v i t y . Another important v a r i a b l e i n c e l l c u l t u r e a f f e c t i n g P.A. production i s c e l l population density which refers to the number of c e l l s per u n i t area. L i u et a l . (1982) found that i n t h e i r transformed c e l l l i n e , there was an increasing amount of i n t r a -c e l l u l a r P.A. a c t i v i t y per c e l l with a decreasing c e l l density up to a point, when the P.A. a c t i v i t y per c e l l then became independ-ent of the c e l l density. The authors showed that the decrease i n P.A. a c t i v i t y at higher c e l l d e n s i t i e s was not because of the release of soluble i n h i b i t o r s and they suggest the e f f e c t i s 23. mediated by c e l l - t o - c e l l contact. Another study by Rohrlich and R i f k i n (1977) showed that the amount of P.A. secreted by normal embryo f i b r o b l a s t s increased with increasing c e l l density, reach-ing a plateau as the c e l l s reach confluence. The data from these two studies cannot be d i r e c t l y compared because of the d i f f e r e n t c e l l l i n e s used and the difference i n P.A. l o c a t i o n , one being i n t r a c e l l u l a r and the other e x t r a c e l l u l a r . However, the data indicates that c e l l population density should be taken into account i n studies of P.A. a c t i v i t y . Also, the r e l a t i o n s h i p between c e l l population density and P.A. a c t i v i t y can provide information f o r optimizing conditions to give maximum enzyme a c t i v i t y . A t h i r d f a c t o r a f f e c t i n g P.A. production i s the growth state of the c e l l s . This e f f e c t has been studied by Chou et a l . (1976). They measured the d a i l y P.A. secretion as a group of c e l l s grew to confluence and compared the P.A. a c t i v i t y of growing c e l l s versus confluent c e l l s which showed no growth. The secreted P.A. from confluent c e l l s was s i x to ten f o l d less than the secreted P.A. from growing c e l l s . C e l l associated P.A. however, remained r e l a -t i v e l y consistent. This data i s d i f f i c u l t to i n t e r p r e t because the c e l l population density e f f e c t has not been disso c i a t e d from the growth state of the c e l l s which was of i n t e r e s t i n t h i s study. Aggeler et a l . (1982) found that e x t r a c e l l u l a r P.A. a c t i v i t y increased during the G2 and M phases of the c e l l cycle of Chinese hamster ovary f i b r o b l a s t s , i n d i c a t i n g that increased P.A. i s related to c e l l d i v i s i o n . Peritoneal macrophages obtained from mice that had been 2 4 . i n j e c t e d with endotoxin showed more P.A. secretion compared to control macrophages (Gordon et a l . , 1974). Exposure to endotoxin and subsequent phagocytosis of late x p a r t i c l e s had a strong stimu-latory e f f e c t on the macrophage secretion of P.A. A number of other agents such as mineral o i l , s a l i n e , BCG antigen, and f o e t a l c a l f serum i n combination with latex p a r t i c l e s also Induced increased secretion of P.A. but endotoxin proved to be the best priming stimulus. Agents shown to stimulate P.A. secretion i n human epidermal c e l l s include c o l c h i c i n e , cholera toxin, and epidermal growth fac t o r (Hashimoto et a l . , 1983). P.A. secretion can be suppressed by gl u c o c o r t i c o i d s such as dexamethasone. Organ cultures of rat tongue, macrophages, human polymorphonuclear leucocytes, r a t hepatoma c e l l s , bovine endothelial c e l l s , and tumour c e l l s have been shown to respond to dexamethasone (Wunschmann-Henderson and Astrup, 1974; V a s s a l i and Reich, 1976; Granelli-Piperno et a l . , 1977; Gelehrter et a l . , 1983; Levin and Loskutoff, 1982). Laug et a l . (1983) showed that bovine endothelial c e l l s secrete both urokinase-type and t i s s u e type P.A., and that dexamethazone, cortisone, and methyl prednisolone i n h i b i t c e l l u l a r and secreted P.A. Urokinase-type P.A. appeared to be s e l e c t i v e l y i n h i b i t e d by dexamethasone while tissue P.A. seemed to be unaffected. The i n h i b i t i o n of P.A. a c t i v i t y i n r a t hepatoma c e l l s i n the presence of dexamethasone has been shown to be due to the s e c r e t i o n of an i n h i b i t o r ( S e i f e r t and Gelehrter, 1978). Other 25. agents found to cause i n h i b i t i o n of P.A. secretion i n bovine endothelial c e l l s include thrombin which caused a decrease i n both c e l l u l a r and secreted P.A., and c o l c h i c i n e which blocked the secretion of P.A. but appeared to increase i n t r a c e l l u l a r P.A. (Levin and Loskutoff, 1982). c) Factors Implicated i n the Regulation of Neutral Proteinase  Secretion The synovial j o i n t system contains many d i f f e r e n t c e l l types but i t has been shown that macrophages and chondrocytes are able to secrete neutral proteinase. C e l l - t o - c e l l i n t e r a c t i o n i s one factor that regulates the neutral proteinase secretion i n t h i s system. Lymphocytes, producing lympokines (soluble lymphocyte factors) have been shown to stimulate macrophages into producing increased l e v e l s of neutral proteinase (Hauser and Vaes, 1979). In addition, macrophages have been shown to be able to stimulate other c e l l types to secrete proteinases. Deshmukh-Pradke et a l . (1978) report that chondrocytes from normal rabbit a r t i c u l a r c a r t i l a g e and endotoxin-treated r a b b i t peritoneal macrophages separately secrete small amounts of neutral proteinase but when the conditioned medium from the macrophages was added to a culture of chondrocytes, increased secretion of neutral proteinase was induced. The stimulatory f a c t o r was heat stable and was l o s t by d i a l y s i s with tubing of MW cutoff 12,000. Ridge et a l . (1980) also demonstrated that a macrophage fa c t o r could induce the synthesis of neutral proteinase from chondrocytes as well as a r t i c u l a r c a r t i l a g e i n c u l t u r e . I n h i b i t i o n of the secretion of 26. t h i s macrophage fa c t o r by cycloheximide indicated that i t was a protein. I t was also found to be heat stable (10 minutes at 60°C), stable to t r y p s i n treatment but p a r t i a l l y i n a c t i v a t e d at pH 2. Not only macrophages but synovial t i s s u e of undefined c e l l types have been shown to be able to induce chondrocytes to secrete increased l e v e l s of neutral proteinase ( F e l l and Jubb, 1977). It i s thought that the synovial tissue secretes a soluble f a c t o r , a protein which p r e c i p i t a t e d between 60 to 100% ammonium sulphate saturation, which was heat s e n s i t i v e (at 70°C for 10 minutes) and was digested by chymotrypsin and pancreatic elastase, but not t r y p s i n (Dingle et a l . , 1979). Synovial c e l l s themselves also respond to the macrophage factor by increasing t h e i r secretion of proteoglycan degrading ne u t r a l proteinase (Peeters-Joris and Vaes, 1984). This f a c t o r was non-dialysable through 8000 MW cutoff membrane and was p a r t i a l l y p r e c i p i t a t e d by ammonium sulphate at 60% saturation. Inflammatory s t i m u l i such as asbestos f i b r e s , b a c t e r i a l products (endotoxin, muramyl dipeptide, formyl-methionyl-peptide), phorbol myristate acetate, and concanavalin A were able to enhance the secretion of t h i s f a c t o r by macrophages. A v a r i e t y of defined factors have also been shown to induce increased secretion of neutral proteinase from f i b r o b l a s t s and tissue explants. Werb and Reynolds (1974) showed that the ingestion and subsequent i n t r a c e l l u l a r storage of l a t e x p a r t i c l e s by rabbit synovial f i b r o b l a s t s stimulated the release of neutral proteinase i n t o the medium. The l e v e l of neutral proteinase 27. secreted was dependent on the amount of p a r t i c l e s ingested. Porcine g i n g i v a l explants i n culture have been shown to respond to increased oxygen tension of the culture medium by increasing the l e v e l of neutral proteinase secreted (Pettigrew et a l . , 1978). The same explants responsed to 5 ug/ml indomethacin by a decreased synthesis of neutral proteinase, i n d i c a t i n g that prostaglandins may be involved i n the regulation. Pettigrew et a l . (1981) also found that the addition of 30 yg/ml endotoxin into the culture media of the g i n g i v a l explants resulted i n an increased synthesis of neutral proteinase but not the tissue i n h i b i t o r , which was unaffected. Meikle et a l . (1980) reported that the a p p l i c a t i o n of continuous t e n s i l e stress to rabbit c a l v a r i a explants (organ cultures) resulted i n an increase i n secretion of both g e l a t i n -degrading and proteoglycan-degrading neutral proteinase. This was accompanied by an increase also i n the tis s u e i n h i b i t o r l e v e l . The authors had previously found (Meikle et a l . , 1979) that the same explants responded to t e n s i l e stress by a 1.5 to 3 f o l d increase i n protein accummulation and a 2 f o l d increase i n collagen. However, although there was increased secretion of p r o t e o l y t i c enzymes, t h i s was not accompanied by an increase i n the degradation of s t r u c t u r a l proteins as measured by 3H-proline release. The increased amount of i n h i b i t o r synthesized was able to complex the increased l e v e l of enzyme secreted, confirming that t h i s i s one l e v e l of control exerted on the a c t i v i t y of the proteinases. Although e p i t h e l i a l c e l l s have been shown to be able to 28. secrete neutral proteinase (Pettigrew, Ph.D. t h e s i s ) , the f a c t o r s regulating i t s secretion have not been investigated i n d e t a i l . PROBLEM FORMULATION The aim of this thesis was to study the regulation, with emphasis on the r o l e of c e l l shape, of n e u t r a l proteinase and plasminogen a c t i -vator secretion by E - c e l l s i n c u l t u r e . To this end, several techniques and experimental conditions had to be developed. 1) Enzyme Assays The assays for neutral proteinase (N.P.) and P.A. a c t i v i t y had to be s e n s i t i v e enough to detect the enzyme a c t i v i t i e s i n the media. 2) C e l l Culture It was necessary to set up the c e l l c ulture system so that detect-able amounts of the enzyme a c t i v i t y were obtained. Because of i t s importance i n other systems, the e f f e c t of c e l l population density had to be investigated. The choice of the medium was also important as the r e l a t i v e l y high concentrations of f o e t a l bovine serum used i n most cultures contains s i g n i f i c a n t amount of c<2-macroglobulin and other f a c t o r s which i n h i b i t N.P. and P.A. a c t i v i t y . Moreover, I was i n t e r -ested not only i n studying the e f f e c t of d i f f e r e n t factors on the secretion of N.P. and P.A. by e p i t h e l i a l c e l l s but also the e f f e c t s of c e l l p r o l i f e r a t i o n on the regulatory process. Therefore conditions had to be devised which allowed a comparison between the c e l l s i n a p r o l i f e r a t i v e and a n o n - p r o l i f e r a t i v e state. 29. 3) Methods For A l t e r i n g C e l l Shape Various chemical agents known to cause morphological changes were applied to E - c e l l s . Cholera toxin and d i b u t y r y l c y c l i c AMP caused c e l l f l a t t e n i n g while phorbol myristate acetate caused a s l i g h t c e l l round-ing. The main problem associated with the use of these agents i s that they a f f e c t more than j u s t the c e l l shape; cholera toxi n f o r example, causes an increase i n cAMP and c e l l growth as we l l . To circumvent t h i s problem, mechanical means were also used to change c e l l shape, and the i r e f f e c t s on proteinase secretion i n v e s t i -gated. In addition, t i s s u e c u l t u r e d i s h adhesivity was a l t e r e d by pre-coating the dishes with varying concentrations of poly(HEMA) (using the technique of Folkman and Moscona, 1978). An Increasing concentra-t i o n of poly(HEMA) solution resulted i n a decrease i n E - c e l l spreading. A d e t a i l e d d e s c r i p t i o n of these techniques i s given i n the next chapter. 30. CHAPTER 2 - MATERIALS AND METHODS I) C e l l Culture 1) Procedures E p i t h e l i a l c e l l s derived from porcine c e l l r e s t s of Malassez were obtained from porcine perio°dontal ligament as described by Brunette et a l (1976). The c e l l s were cultured i n ctMEM plus 15% f o e t a l c a l f serum (FCS) (Flow, Cockeysville, MD) with p e n i c i l l i n G (Sigma, St. Louis, MO) 100 ug/ml, gentamycin (Sigma) 50 ug/ml and amphoteracin B (Gibco, Grand Island, NY) 3 ug/ml, at 37°C i n a humidified atmosphere of a i r plus 5% CO2. To subculture the E - c e l l s , the medium was removed from the dish and replaced with 10 ml of a try p s i n s o l u t i o n (0.25% t r y p s i n , Worthington Cat. No. 44521) i n c i t r a t e s a l i n e (pH 7.8). This so l u t i o n was removed and a fresh 10 ml of try p s i n solution was added and the c e l l s incubated at 37°C. A f t e r 4-5 minutes, when the c e l l s had rounded and could be detached from the dish, the c e l l s i n t r y p s i n s o l u t i o n were added to an equal volume of ctMEM + 15% FCS i n a s t e r i l e p l a s t i c tube (Falcon 2001) and the mixture centrifuged f or 15 minutes at 750 g. The p e l l e t of c e l l s was resuspended i n the medium and mixed well by pipetting the suspension up and down several times. This suspension was then used for Inoculation of c u l t u r e s . For experiments, the E - c e l l s were plated on 60 mm Falcon dishes at 4 x 10 5 c e l l s / d i s h unless otherwise stated and incubated overnight i n ctMEM +15% FCS. The medium was removed and replaced with a modified low serum medium with 1 mM K + and 0.1 mM Ca"*-*" designated 3MEM plus 0.5% dialysed FCS (described by Brunette, 1984a) for three to four days to allow s u f f i c i e n t enzyme sec r e t i o n f o r the a c t i v i t y to be assayed. The number of c e l l s i n the culture were counted at each time point of medium c o l l e c t i o n . 2) Determination of C e l l Number One half ml of the c e l l suspension to be counted were added to v i a l s containing' 9.5 ml i s o t o n i c s a l i n e (Isoton, Coulter, Hialeah, F l o r i d a ) and mixed thoroughly. Two readings were taken of each sample on an e l e c t r o n i c c e l l counter (Coulter E l e c t r o n i c s , Inc., Hialeach, F l o r i d a ) . II) Chemicals E. c o l i lipopolysaccharide serotype 0127:B8, phenolic e x t r a c t i o n and t r i c h l o r o a c e t i c acid extraction (Sigma), cholera toxin (Sigma), d i b u t y r y l c y c l i c - 3'5' - adenosine monophosphate ( B t 2 cAMP) (Sigma) and phorbol 12-myristate 12-acetate (PMA) (Sigma) were added to the medium to give the desired f i n a l concentration. III) Methods of A l t e r i n g C e l l Shape 1) Mechanical Stretching Mechanical s t r e t c h i n g was applied using a modification of the method of Brunette (1984b) developed by Hasegawa et a l . ( i n press). The f l e x i b l e p l a s t i c bottom of a Petriperm d i s h (Tekmar, Ci n c i n n a t i , OH) was stretched by putting a template with a convex surface underneath the d i s h and a lead weight on the top (see Figure 1). Twenty dishes could be stretched at the same time by F i g . 1. Technique for mechanical stretching of c e l l s grown on a p e t r i -perm dish. A convex template i s placed underneath the petriperm dish and a lead weight placed on top. 33. placing a 40 x 26 x 0.55 cm p l e x i g l a s s sheet over the dishes and then putting two 6 kg weights on the pl e x i g l a s s . A template producing a 4% increase i n surface area was used. The template was made by heating the centre of a 100 x 15 mm p e t r i dish and placing the p e t r i d i s h onto the curvature of a globe of radius 7. cm. The extent of the curved area was lim i t e d by applying a b o t t l e cap of i n t e r n a l diameter 4.6 cm. The surface curvature produced was that of an arc of 36° with a radius of 7.6 cm. The p r i n c i p l e of the method i s that stretching the f l e x i b l e p l a s t i c membrane of the Petriperm dish, which i s attached f i r m l y to the c e l l s by hemidesmosomes, r e s u l t s i n stretching the c e l l s and making them more flattened compared to the unstretched c e l l s . Conversely, growing the c e l l s on a stretched membrane and then taking the petriperm dish o ff the template allows the p l a s t i c to return to i t s i n i t i a l state and the attached c e l l s to become more rounded. Sixty mm Petriperm dishes were plated with E - c e l l s at 8 x 10 5 c e l l s / d i s h i n otMEM +15% FCS overnight. The medium was replaced with fresh medium and the c e l l s grown for an ad d i t i o n a l three day under the conditions below (see Figure 2). For the detection of proteinase secreted, the c e l l s were washed twice with 8MEM + 0.5% dialysed FCS (DFCS) and incubated i n the same medium for 2 days. i ) Unstretched controls: The c e l l s remained unstretched over the 5 day experimental period. 34. unstretched control MECHANICAL ^ STRETCHING I I stretched 2 days in a MEM 0 MEM 3 days in  + 15% FCS + 0.5% DFCS stretched control MECHANICAL RELAXATION L ^ ^ l relaxed unstretched dish stretched dish F i g . 2. Design of the mechanical stretching experiment. E - c e l l s plated at 8 x l 0 5 c e l l s / d i s h were treated by e i t h e r mechanical stretching or mechanical relaxation, with corresponding controls. 35. i i ) Stretched: The c e l l s were stretched during the two day proteinase c o l l e c t i o n period. i i i ) Stretched controls: The c e l l s were grown on a stretched membrane and remained stretched during the two day proteinase c o l l e c t i o n period. i v ) Relaxed: The c e l l s were grown on a stretched membrane and then the dishes were taken o f f the templates during the two day proteinase c o l l e c t i o n period. 2) Preparation of Grooved Substratum The amount of proteinase secreted by c e l l s cultured on grooved substrates was compared to c e l l s grown on f l a t substrates because time lapse cinemicrographic observations of c e l l s grown on grooved substrates indicate that the c e l l s assume a round shape more frequently than c e l l s grown on a f l a t substrate (Brunette -personal communication). That c e l l s grown on grooved substrates are more round than those grown on f l a t substrates has also been observed by Rovensky et a l (1971). S i l i c o n wafers with V shaped grooves were produced as described by Brunette et a l . (1983). The grooved substrates had v-shaped grooves which were 79 microns wide at the top of the v and 60 microns deep. The grooves were separated by f l a t areas which were 13 microns i n width. Impressions of the groove pattern on the s i l i c o n wafers were made with Exaflex (G-C Dental I n d u s t r i a l Corp., Japan). The groove pattern was then reproduced onto Epotek r e s i n (Epoxy tech-nology Inc., B i l l e r i c a , MA). Three ml of the r e s i n were poured onto 60 mm dishes (Falcon) and the Exaflex impressions with a 3 6 . diameter s l i g h t l y smaller than the dish were lowered onto the surface of the r e s i n . These were allowed to stand overnight at room temperature. The dishes were then placed i n a 60°C oven f o r 3 days to allow complete curing. The control dishes were made with a f l a t surface. 3) Preparation of Poly(HEMA) Plates C e l l shape was also a l t e r e d by changing the cul t u r e d i s h surface adhesivity. Following the method described by Folkman and Moscona (1978), 3 g poly(HEMA) (Polysciences, Inc., Warrington, PA) was dissolved i n 25 ml 95% ethanol by s t i r r i n g at 37°C overnight and the mixture was centrifuged f o r 30 minutes to remove undissolved p a r t i c l e s . This stock solution was then d i l u t e d i n 95% ethanol to give the desired concentrations. Following Folkman's terminology, the poly(HEMA) concentrations stated are the amounts of d i l u t i o n of the stock s o l u t i o n i n a l c o h o l . For example, to obtain a concentration of 10 - 3, 0.1 ml of the stock s o l u t i o n was added to 99.9 ml ethanol. Each 60 mm dish was precoated with 1.8 ml of poly(HEMA) solution, dried i n a s t e r i l e environment overnight and the c e l l s plated i n ctMEM + 15% FCS. Morphological Techniques 1) Transmission E l e c t r o n Microscopic Techniques E - c e l l s grown on petriperm dishes at normal density were processed f o r el e c t r o n microscopy. Each dish of c e l l s was f i x e d i n 2.5% glutaraldehyde (J.B. EM Services, Inc., Quebec) i n 0.1M sodium cacodylate at pH 7.4 f o r 1 hour on i c e , and then rinsed three times with O.lM sodium cacodylate buffer. P o s t - f i x a t i o n was c a r r i e d out i n 1% osmium tetroxide (J.B. EM Services, Inc.) i n O.lM sodium cacodylate, pH 7.4, for 30 minutes on i c e . The c e l l s were washed three times with d i s t i l l e d water and stained i n 2% aqueous tannic acid at room temperature for 20 minutes, followed by 5 washes i n d i s t i l l e d water; and 1% aqueous osmium tetroxide for 30 minutes, followed by 3 washes i n d i s t i l l e d water. The c e l l s were then dehydrated i n graded ethanol, 30% to 100%, over a time period of 35 minutes. I n f i l t r a t i o n was c a r r i e d out i n 50/50 mixture of ethanol and epon (J.B. EM Services, Inc.) and the dish allowed to stand overnight with the l i d o f f . A f t e r 2 changes of f r e s h epon mixture f o r 2 and 4 hours r e s p e c t i v e l y , the c e l l s were l e f t i n epon mixture with the accelerator, t r i (dimethyl aminomethyl) phenol (DMP 30) f o r 1 hour; replaced with a f r e s h mixture and placed i n a 37°C oven overnight and subsequently i n 60°C oven f o r 4 days. Once polymerized, the d i s h was turned over and a fresh mixture of epon and DMP 30 added to the bottom of the petriperm d i s h so that the c e l l s and the p l a s t i c layer were sandwiched between two layers of epon. The epon block was cut i n t o 5 x 5 x 10 mm blocks by a diamond t i p saw for electron microscopic sectioning. One micron sections were cut perpendicular to the p l a s t i c surface on a S o r v a l l MT2 Ultramicrotome ( S o r v a l l Inc., Connecticut), using a glass knife (LKB Produkter AB, Stockholm), stained with Richardson's s t a i n and examined to select for a suitable area. Thin sections were cut at 40 to 60 nm with a diamond k n i f e (Diatome Ltd., Bienne, 3 8 . Switzerland). Sections were picked up on 50 mesh copper grids (Fullam Inc., Schenectady, NY), coated with formvar and carbon, and stained with approximately 2% aqueous uranyl acetate f o r 20 minutes, followed by Reynolds' lead c i t r a t e for 5 minutes. The sections were then examined under a P h i l l i p s 300 Transmission Electron Microscope at 80 kV. 2) Richardson's Stain (Richardson, J a r e t t & Finke, 1960) Each dis h of c e l l s was f i x e d i n 2.5% glutaldehyde i n 0.1M sodium cacodylate at pH 7.4 on i c e . The solution was poured o f f and the c e l l s washed i n 0.1 M sodium cadodylate buffer three times and rinsed i n d i s t i l l e d water three times. Richardson's s t a i n c o n s i s t i n g of 0.5% methylene blue and 0.5% Azure 2 i n 1% borax solution was f i l t e r e d before use. Three drops of the s t a i n were placed onto the c e l l s f o r 2 minutes and then the s t a i n was washed off with d i s t i l l e d water. 3) Scoring of C e l l Spreading Cultures stained with Richardson's s t a i n to make the cytoplasm more evident were examined under the microscope. The extent of c e l l rounding was assigned a score on an a r b i t r a r y scale 0-3, based on the amount of c e l l area i n contact with the dish. C e l l s grown on tiss u e culture p l a s t i c were assigned a score of 0. They were well spread. C e l l s which were s l i g h t l y rounded compared to the c e l l s which scored 0, were given a score of 1. Round c e l l s which showed a c e l l area approximately a t h i r d of that of the c e l l s scoring 0, were assigned a score of 3. 39. V) Preparation of Media for Enzyme Assays The media i n which the c e l l s were incubated was centrifuged for 10 minutes at 500 rpm to p e l l e t any c e l l s that may have been present. M i l l i p o r e CX-10 (MW cutoff 10,000) f i l t e r s ( M i l l i p o r e Corp., Bedford, MASS) were pretreated by 1) passing through d i s t i l l e d water to remove preservatives and 2) passing through 100 ml of conditioned media per f i l t e r i n order to saturate any s i t e s on the membrane that could bind to the enzymes. To test whether the bound enzymes could be subse-quently released, f r e s h medium was concentrated using a pretreated f i l t e r and the f i l t e r e d medium assayed for neutral proteinase a c t i v i t y . No a c t i v i t y was found. Conditioned media were therefore concentrated ten to twenty times using the pretreated f i l t e r s . The samples were kept at 0°C during processing, then dialysed i n Spectrapore 2 membranes with MW cutoff 12,000 to 14,000 (Spectrum Med., Ind., LA) against 50 mM T r i s buffer containing 200 mM sodium c h l o r i d e and 5 mM calcium c h l o r i d e at pH 7.5 at 4°C. VI) Assays 1) C y c l i c AMP Assay Two dishes from each group were used f o r measurement of i n t r a -c e l l u l a r cAMP l e v e l s (Oey et a l . , 1974). The medium was removed and replaced with 2 ml of cold 5% t r i c h l o r o a c e t i c a c i d and the dishes incubated at 4°C for 1 hour. The mixtures were then centrifuged at 4°C f o r 5 min at 10,000 rpm (International Equipment Co., Needham Heights, Mass, USA), and the supernatant washed f i v e times with 2 volumes of water saturated ether a f t e r which i t was l y o p h i l i z e d . The p e l l e t was c o l l e c t e d for a protein 4 0 . assay. Cyclic AMP levels i n the supernatant were measured using the radioimmuno assay k i t [ 1 2 5 l ] (Becton Dickinson Immuno-diagnostics, Orangeburg, NY). 2) Assay for Neutral Proteinase (N.P) Neutral proteinase was assayed using azocoll (Calbiochem, San Diego, CA) as the substrate. Each assay included 450 ul of the activated sample, 4 mg azocoll and 1 ml of 50 mM T r i s buffer containing 200 mM sodium chloride and 50 mM calcium chloride, pH 7.5. The assay mixtures were incubated i n Reacti-Therm v i a l s (Pierce Chem. Co., Rockford, IL) with s t i r r i n g at 37°C. After 24 to 48 hours, the mixtures were transferred to 1.9 ml microcentri-fuge tubes (Evergreen S c i e n t i f i c , LA, CA) and centrifuged for 5 minutes i n an Eppendoff microcentrifuge (Brinkmann Instruments, Westbury, NY). The supernatant was read at 520 nm i n an SP 800 spectrophotometer (Unicam, Cambridge, England) and compared to a standard curve of the digestion of azocoll by trypsin (Figure 4). A l l assays were done i n duplicates and controls included 1) buffer only 2) fresh medium. The degradation of azocoll was li n e a r with respect to enzyme concentration (Figure 5) as we l l as incubation time up to 56 hours (Figure 3). One unit of enzyme a c t i v i t y was defined as equal to 1 mg azocoll s o l u b i l i s e d per hour at 37°C under these experimental conditions. A c t i v i t i e s were expressed as Units per 10 5 c e l l s . 41. (J) o If) O 10 2 0 3 0 4 0 5 0 6 0 Incubation time in hours F i g . 3. Time course of neutral proteinase assay. Three hundred pi of conditioned medium were activated and incubated with a z o c o l l for time periods shown. The background a c t i v i t y found i n the c o n t r o l has been subtracted to give the values shown. Each point represents the average of two determinations. 1.6 . o CM 10 Q o 1.2 0.8 0.4 0 CM 1 2 3 4 mg. Azocoll 2 8 • > '> o 4 CO 0 I I I I 100 200 300 400 conditioned media ul F i g . 4. Standard curve for the digestion of a z o c o l l . Different amounts of a z o c o l l were incubated with excess trypsin at 37°C u n t i l a l l the a z o c o l l had been digested. Each point represents the average of two determinations. F i g . 5. Dependence of a z o c o l l degradation on enzyme concentration. Increasing amounts of conditioned media were activated with mersalyl a c i d . The assays, done i n duplicate, were incubated f o r 46 hours. 43. 3) A c t i v a t i o n and C h a r a c t e r i s t i c s of the Neutral Proteinase  (N.P.) The N.P was secreted i n a l a t e n t form which could be activated by mersalyl acid or TPCK t r y p s i n or 4-aminophenyl mercuric acetate (APMA). The reagents were made up as follows. Mersalyl acid (Sigma) was added to water, the addition of a few drops of 1M NaOH helped to di s s o l v e any remaining p a r t i c l e s and the volume made up with water. APMA (Sigma) was dissolved i n 1M NaOH, t i t r a t e d with 1M HC1 u n t i l a l i t t l e p r e c i p i t a t e i s seen, then a few more drops of NaOH was added to dissolve i t and the volume made up with water. A c t i v a t i o n with mersalyl a c i d or APMA was c a r r i e d out by preincubating 300 p i of the concentrated media with 75 pi of 40 mM of the a c t i v a t o r f o r 10 minutes, followed by an ad d i t i o n of 75 p i of 1 mg/ml Soybean t r y p s i n i n h i b i t o r (STI) for a further 10 minutes. The STI i n a c t i v a t e s any traces of t r y p s i n that might have been l e f t behind from the subculture procedure. A c t i v a t i o n with t r y p s i n was also c a r r i e d out i n a s i m i l a r way using 75 p i of 0.1 mg/ml TPCK tryp s i n (Worthington Biochem. Corp., Freehold, NY) followed by a tenfold excess of STI (1 mg/ml). Figure 6 shows that maximum a c t i v i t y was achieved with the additi o n of 75 p i of 20 mM mersalyl a c i d (1 mM f i n a l concentra-t i o n ) . In contrast, 10 pg of TPCK tryp s i n (100 p i of 0.1 mg/ml TPCK trypsin) activated only 30% of the maximum a c t i v i t y . APMA at a f i n a l concentration of 2 mM activated 17% of the N.P. a c t i v i t y obtained by a c t i v a t i n g with an equivalent concentration of mersalyl a c i d . 44. 1.2 o CM LO Q O 0.8 0.4 i y o 1 0' 1 * ' X f t ' t' 0 1 ^ " i 25 50 75 • 1 100 volume of activator (MI) F i g . 6. Comparison of mersalyl acid and TPCK try p s i n as acti v a t o r s of neutral proteinase. Two hundred u l of the conditioned medium were preincubated with increasing amounts of mersalyl (20 mM)(o) or TPCK t r y p s i n (0.1 mg/ml)(x). The assays were incubated f o r 46 hours. The background a c t i v i t y found i n the co n t r o l has been subtracted to give the values shown. Each point represents the average of two determinations. 45. The activated neutral proteinase was i n h i b i t e d by EDTA (a metal chelating agent), cysteine and f o e t a l c a l f serum (Table I ) . These c h a r a c t e r i s t i c s are s i m i l a r to the neutral proteinase obtained from rheumatoid synovium, rabbit bone and rabbit synovial f i b r o b l a s t s (Harris and Krane, 1972; S e l l e r s et a l . , 1978; Werb & Reynolds, 1974). 4) Plasminogen Ac t i v a t o r (P.A.) Assay The two stage assay described by Jackson, Esmon and Tang (1981) was used. Plasminogen (Kabi, Stockholm, Sweden) was dissolved i n 50 mM T r i s buffer, pH 7.5 to a concentration of 5 U/ml and stored frozen i n 200 p i al i q u o t s . Before use, i t was d i l u t e d to 1 U/ml with 50 mM T r i s buffer + 0.5% bovine serum albumin (BSA), pH 7.5. Streptokinase (Behring I n s t i t u t e , Marburg, W. Germany) was made up to 100 IU/ml i n 50 mM T r i s buffer, pH 7.5 and stored frozen. Before use, i t was d i l u t e d to 0.5 IU/ml with 50 mM T r i s buffer + 0.5% BSA, pH 7.5. The synthetic substrate D-Val-Leu-Lys-p-nitroanilide (Kabi S2251) was dissolved i n water to a concentration of 5 mg/ml. Before use, i t was mixed with a 1.77 M NaCl i n 32 mM T r i s buffer s o l u t i o n (pH 7.5) i n a 2:3 r a t i o . The assay was c a r r i e d out i n 1.9 ml micro-centrifuge tubes (Evergreen) at 37°C i n a shaking water bath. The f i r s t stage of the reaction was i n i t i a t e d by adding 20 p i of the sample to 40 p i T r i s buffer and 40 p i of plasminogen (1 U/ml) solu t i o n . The f i r s t stage of the re a c t i o n was terminated and the second begun by adding 80 p i of the substrate S-2251 i n NaCl solution at p r e c i s e l y 46. TABLE I: EFFECT OF DIFFERENT INHIBITORS ON NEUTRAL PROTEINASE ACTIVITY Sample O D 5 2 0 % Control Activated Sample 0.59 + 0.01 100 Activated sample + EDTA (0.5 mM f i n a l concentration) 0 0 Activated sample + cysteine (0.5 mM f i n a l concentration) 0.22 + 0.01 34 Activated sample + dialysed FCS (0.5% f i n a l concentration) 0.53 + 0.02 89 Activated sample + dialysed FCS (10% f i n a l concentration) 0.21 + 0.05 35 Two hundred and seventy-five y l of the samples were preincubated with mersalyl a c i d ( f i n a l concentration 2 mM) before the d i f f e r e n t agents were added. The assays were incubated f o r 21 hours. The 0D reading represents t e s t s minus background values. The estimated v a r i a t i o n i s given as ± SEM. 47. 30 minutes a f t e r the r e a c t i o n was st a r t e d . The second stage of the reaction was terminated a f t e r a further 30 minutes by the addit i o n of 0.8 ml of 40% a c e t i c a c i d . The OD was then read at 405 nm. A l l assays were done i n duplicate and included the following controls: a) buffer only to measure any spontaneous degradation of the plasminogen; b) controls without plasminogen to measure any S-2251 degrading enzymes other than plasmin (controls a and b should read zero); c) A s e r i e s of increasing volumes of Streptokinase s o l u t i o n to give the standard curve which was used as the basis for c a l c u l a t i o n of the amount of P.A. a c t i v i t y i n the unknown samples (Figure 7). The assay was l i n e a r with respect to increasing amounts of the sample up to an absorbance of 0.9 (Figure 8). A c t i v i t i e s were expressed i n terms of IU Streptokinase /10 5 c e l l s . VII) E r r o r Study and S t a t i s t i c a l A n alysis In order to quantitate the amount of v a r i a t i o n between groups, 5 groups of 3 dishes each were set up with c e l l s plated at normal density. At day 3 of culture, the c e l l s were counted and the medium assayed for N.P. and P.A. a c t i v i t y . The N.P. a c t i v i t y was 15.6 x 1 0 - 3 U/10 5 c e l l s ± 0.23 (SEM) and the P.A. a c t i v i t y was 55.9 x 10~ 3 IU 48. 1.6 1.2 o ^-Q o 0.8 0.4 0 10 15 -3 10 ° IU streptokinase F i g . 7. Standard curve of Streptokinase a c t i v i t y . Each point repre-sents the average of two determinations. 49. 2.0 • 0 2 4 6 8 10 20 volume of conditioned media (pi) F i g . 8. Absorbance change as a function of the amount of added con-ditioned media. Increasing amounts of conditioned media from E - c e l l s cultured on f l a t substrates (•) and from E - c e l l s cultured on grooved substrates ( A ) were assayed for P.A. a c t i v i t y . 50. Streptokinase/10 5 c e l l s ± 1.4 (SEM). The c o e f f i c i e n t of v a r i a t i o n f o r N.P. and P.A. a c t i v i t y was 3% and 6% re s p e c t i v e l y . In experiments where r e p l i c a t e cultures were used, the data was analysed using one way analysis of variance. In other experiments, the sign t e s t (Zar, 1974) was used to te s t whether the differences observed were due to chance alone. The d i r e c t i o n of the differences between the treated and c o n t r o l values was noted; a p o s i t i v e sign was assigned to the difference observed which was i n the expected d i r e c t i o n . For example, cholera toxin was expected to cause a reduced l e v e l of enzyme secretion. Therefore when a reduced value was observed, a + sign was assigned. A negative sign was assigned f o r a value that showed no difference from the control or showed a difference opposite to what was expected. A sign was given f o r each p a i r of values obtained from the treated and control groups. The p r o b a b i l i t y owing to chance that the number of times + signs were obtained out of the t o t a l (N), was obtained from a table. The n u l l hypothesis was rejected i f the p r o b a b i l i t y p < 0.05. 51. CHAPTER 3 - RESULTS E - c e l l s i n culture secreted latent neutral proteinase which could be activated by eit h e r t r y p s i n , APMA, or mersalyl a c i d . I n h i b i t i o n of N.P. a c t i v i t y by EDTA, cysteine and FCS characterized i t as a metallo-neutral proteinase s i m i l a r to that secreted by other c e l l types (Harris and Krane, 1972; S e l l e r s et a l . , 1978; Werb and Reynolds, 1974). No act i v e form of the neutral proteinase was detectable. Tests for the free i n h i b i t o r of metalloproteinase i n day 3 medium proved negative. The presence of P.A. was detected by means of a chromogenic assay. The e f f e c t of c e l l population density, E . C o l i LPS and c e l l shape on proteinase secretion by E - c e l l s w i l l be presented. I. E f f e c t of C e l l Population Density The e f f e c t of c e l l population density on proteinase secretion was investigated by c u l t u r i n g E - c e l l s at 2,4,8, and 15 x 10 5 c e l l s / d i s h i n gMEM + 0.5% dlalysed FCS. These conditions kept the c e l l s i n a quiescent state but also permitted growth when the c e l l s were stimulated by cholera toxin or B t 2 cAMP. At days 3,7, and 10 of the experiment, proteinase a c t i v i t y was assayed and the c e l l s counted. Under these conditions, the number of c e l l s per culture remained constant over the ten day period. Figure 9 shows that neutral proteinase a c t i v i t y was highest at low c e l l population density, the a c t i v i t y decreasing with increasing c e l l population density. The r e l a t i o n s h i p between c e l l population density and N.P. a c t i v i t y was s i m i l a r at days 3,7, and 10. P.A. secretion exhibited a s i m i l a r pattern with the exception of day 3 (Figure 10). 52. o • • • • • • • • 2 4 6 8 10 12 14 cell density X10 5 cells/dish F i g . 9. The e f f e c t of c e l l population density on N.P. a c t i v i t y . The c e l l s were plated at 2,4,8, and 15xl0 5 c e l l s / d i s h . The medium was co l l e c t e d at days 3(o), 7(*) and 10(A), the c e l l s were counted and the N.P. a c t i v i t y determined. The data shown are from one representative experiment that was repeated and the trends were the same i n both experiments. 30 A 0 O LO o 25 20 co CO I o > o CO < 0_' 15 10 0 A ,° o o o 2 4 8 10 12 14 cell density X10 cells/dish 10. The e f f e c t of c e l l population density on P.A. a c t i v i t y . The c e l l s were plated as described i n F i g . 9 and the P.A. a c t i v i t y on days 3(o), 7(«) and 10(A) determined. The data shown are from one representative experiment that was repeated and the trends were the same i n both experiments. 54. In contrast to N.P. a c t i v i t y , P.A. a c t i v i t y i n the medium at day 3 was lower than that found on day 7 or day 10. I I . E f f e c t of E . c o l i LPS As b a c t e r i a l lipopolysaccharide has been found to cause a v a r i e t y of b i o l o g i c a l e f f e c t s such as induction of the inflammatory response (Jensen et a l . , 1966) and the stimulation of macrophages and g i n g i v a l explants to release increased amounts of metalloproteinases (Wahl et a l . , 1975; Pettigrew et a l . , 1981), i t was of i n t e r e s t to inve s t i g a t e whether LPS had a si m i l a r stimulatory e f f e c t on E - c e l l s . E . c o l i LPS (phenol extract) was tested at 3,30 and 60 pg/ml. Table I I shows that N.P. secretion by E - c e l l s was stimulated by E . c o l i LPS, 3 ug/ml being just as e f f e c t i v e as 60 pg/ml. Two preparations of LPS, a t r i c h l o r o a c e t i c a c i d (TCA) extract and a phenol extract, were compared to see i f the method of LPS preparation af f e c t e d proteinase s e c r e t i o n d i f f e r e n t l y . Figure 11 shows that both preparations of E . c o l i LPS stimulated a 2 fo l d increase i n N.P. a c t i v i t y compared to the c o n t r o l . One way ANOVA indicates that there was a s t a t i s t i c a l l y s i g n i f i c a n t difference between the treated and co n t r o l groups (p < 0.05). The TCA extract also caused an increased amount of P.A. secretion by E - c e l l s ; a cumulative increase of 3 1/2 times over the co n t r o l was seen by day 10 of culture (Figure 12). Analysis of the data shows that the difference i s s i g n i f i c a n t (p < 0.05). The phenol extract, however, caused only a small increase i n P.A. secretion (Figure 12). Components of dental plaque have been shown to i n h i b i t c e l l p r o l i -f e r a t i o n (Baloolol et a l . , 1970; Singer and Buckner, 1980). Dental 55. TABLE I I : EFFECT OF E. COLI LPS (PHENOL EXTRACT) ON NEUTRAL PROTEINASE ACTIVITY N.P. A c t i v i t y 10~3 u / 1 0 5 c e l l s day 3 day 7 day 10 cumulative a c t i v i t y Control 2 . 2 0 . 5 5 . 0 7.7 LPS 3 ug/ml 14.4 7 . 5 1 2 . 3 3 4 . 2 LPS 30 ug/ml 11.0 1 8 . 0 7 . 0 3 6 . 0 LPS 60 ug/ml 6 . 4 1 5 . 8 8 . 0 3 0 . 2 E - c e l l s were plated i n aMEM + 15% FCS overnight and then incubated i n 3MEM + 0 . 5 % DFCS. The medium was c o l l e c t e d on days 3 , 7 and 10 and assayed for N.P. a c t i v i t y . o in 3 6 10 days F i g . 11. E f f e c t of E . c o l i LPS (TCA extract) and E . c o l i LPS (phenol extract) on N.P. a c t i v i t y . Duplicate cultures were incubated i n t3MEM+0.5% DFC; and the medium co l l e c t e d and assayed for N.P. a c t i v i t y , given as cumulative values. Control (o), LPS (TCA extract) 3 ug/ml (A) and LPS (phenol extract) 3 ug/ml (•) Erro r bars i n d i c a t e SEM. 12. E f f e c t of E . c o l i LPS (TCA extract) and E . c o l i LPS (phenol extract) on P.A. a c t i v i t y . The experiment was described i n F i g . 11. The medium was also assayed for P.A. a c t i v i t y , given as cumulative values. Control (o), LPS (TCA extract) 3 ug/ml (A) and LPS (phenol extract) 3 ug/ml (•). Error bars indi c a t e SEM. 58. plaque has also been demonstrated to be able to a l t e r the morphology of g i n g i v a l f i b r o b l a s t s i n - v i t r o and to cause the degradation of c e l l surface proteins such as f i b r o n e c t i n (Larjava and Jalkanen, 1984). However, E . c o l i LPS at 3,30 and 60 ug/ml caused no changes i n c e l l numbers. Furthermore, no change i n c e l l morphology was observed. Some studies have demonstrated that b a c t e r i a l LPS can cause increases i n cAMP le v e l s i n c e r t a i n c e l l types (Watson, 1976; Naylor et a l . , 1978), but no s i g n i f i c a n t e f f e c t was found i n E - c e l l s . Measurements of i n t r a c e l l u l a r cAMP l e v e l s at day one of culture showed that E - c e l l s exposed to 3 ug/ml E . c o l i LPS had 0.23 ± 0.05 (S.D.) pmol cAMP/105 compared to control c e l l s which contained 0.18 ± 0.03 (S.D.) pmol cAMP/105 c e l l s . I I I . C e l l Shape and Proteinase Secretion To test the hypothesis that E - c e l l shape modulates proteinase secretion, d i f f e r e n t means of changing c e l l shape were used. Agents that increase i n t r a c e l l u l a r cAMP l e v e l s such as cholera toxin and di b u t y r y l cAMP, which have been reported to cause E - c e l l s to f l a t t e n out (Brunette, 1984a) were tested f o r t h e i r e f f e c t on proteinase secretion. A d i f f e r e n t e f f e c t was produced by phorbol myristate acetate at 10 ng/ml, which caused E - c e l l s to r e t r a c t s l i g h t l y at the cytoplasmic edges. This morphological change caused by PMA i s i n agreement with the report by Wigler and Weinstein (1976) who observed a sim i l a r e f f e c t of PMA on normal chicken embryo f i b r o b l a s t s . 59. 1) Cholera Toxin E - c e l l s were exposed to cholera t o x i n at 1 ng/ml over a ten day culture period and the proteinase a c t i v i t y assayed at days 3, 7, and 10. The number of c e l l s i n each dish was also determined at each time point of the enzyme assay. Table III shows that E - c e l l s were plated at two d i f f e r e n t c e l l d ensities and at each density, c e l l numbers increased i n the presence of cholera toxin. Control cultures showed a small decrease i n c e l l number at day 10 compared to day 3. One way ANOVA indi c a t e s that the increased growth seen with cholera t o x i n was s i g n i f i c a n t (p < 0.05). Therefore, at the time that enzyme assays were done, the cultures treated with cholera toxin had a d i f f e r e n t c e l l popula-t i o n density compared to c o n t r o l c u l t u r e s . Since i t has been shown that c e l l population density modulates proteinase s e c r e t i o n , a c o r r e c t i o n f o r the c e l l population density e f f e c t was made on the basis of the data i n Fi g s . 9 and 10. E - c e l l s i n the presence of cholera toxin, produced on average, 69% of the amount of N.P. and 29% of the P.A. produced by co n t r o l cultures (Table I I I ) . S t a t i s t i c a l a n alysis using the sign t e s t showed that the reduced amounts of N.P. and P.A. a c t i v i t y were s i g n i f i c a n t (p < 0.05). Since i t i s thought that cholera toxin mediates i t s e f f e c t on c e l l s through cAMP, i n t r a c e l l u l a r cAMP l e v e l s were measured i n E - c e l l s . At day 1 of cultu r e , E - c e l l s i n the presence of cholera 60. TABLE I I I : EFFECT OF CHOLERA TOXIN (C.T) 1 ng/ml CONTROL C.T. % CONTROL C e l l Counts 10 5 c e l l s / d i s h Expt. 1 Day 3 Day 7 Day 10 4.2 ± 0.04 3.8 ± 0.07 4.0 4.2 ± 0.07 5.7 ± 0.07 8.2 ± 0.1 100 150 205 Expt. 2 Day 3 Day 7 Day 10 2.16 ± 0.06 1.76 ± 0.22 1.24 ± 0.02 2.1 ± 0.26 2.49 ± 0.07 2.43 ± 0.15 97 141 196 N.P. A c t i v i t y 10~ 3 U/10 5 c e l l s Expt. 1 Day 3 Day 7 Day 10 6.4 5.3 3.6 (3.8) 3.8 (3.4) 3.8 (2.0) 2.7 59 72 75 Expt. 2 Day 3 Day 7 Day 10 29.5 8.9 8.1 (23.2) 23.2 (4.5) 5.7 (2.5) 5.2 79 64 64 P.A. A c t i v i t y 1 0 - 3 IU Strepto-kinase/10 5 c e l l s Expt. 1 Day 3 Day 7 Day 10 0.7 9.2 2.8 (0.1) 0.1 (0.5) 0.7 (0.5) 0.7 14 8 25 Expt. 2 Day 3 Day 7 Day 10 7.5 2.8 3.7 (0.8) 0.8 (1.3) 1.5 (1.6) 2.3 11 54 63 E - c e l l s were plated i n aMEM + 15% FCS overnight and then incubated i n 3MEM + 0.5% dialysed FCS f o r proteinase c o l l e c t i o n . C.T. was added at 1 ng/ml. Two dishes were used for c e l l counting at each time point of medium c o l l e c t i o n . The N.P. and P.A. a c t i v i t y from the C.T. treated cultures have been corrected for the c e l l population density e f f e c t . The values i n brackets represent uncorrected numbers. Estimation of v a r i a t i o n i s given as ± SEM. 61. toxin (1 ng/ml) had 0.52 pmol cAMP/105 c e l l s compared to co n t r o l c e l l s which had 0.18 ± 0.03 pmol cAMP/105 c e l l s (± S.D). This d i f f e r e n c e i n cAMP l e v e l was s t a t i s t i c a l l y s i g n i f i c a n t (p<0.01). 2) D i b u t y r y l cAMP ( B t 2 cAMP) The e f f e c t of B t 2 cAMP on E - c e l l behaviour was s i m i l a r to that of cholera toxin. C e l l numbers increased four f o l d by day 14 (Table IV). N.P. secretion by E - c e l l s exposed to B t 2 cAMP averaged 8% of the N.P. secreted by control cultures (Table IV). Thus c e l l f l a t t e n i n g caused by both cholera t o x i n and B t 2 cAMP was accompanied by a reduction i n proteinase secretion and an increase i n c e l l p r o l i f e r a t i o n . -3) Phorbol Myristate Acetate (PMA) PMA caused a s l i g h t increase i n c e l l number i n comparison to control cultures. At day 4 of the experiment, cultures with PMA at 1 ng/ml showed a s i g n i f i c a n t increase, 29% i n c e l l numbers over the control (p<0.05). PMA at 10 ng/ml however, caused no s i g n i f i -cant increases i n c e l l number. Table V shows that 10 ng/ml PMA stimulated a 1.4 to 4 f o l d increase i n N.P. and P.A. secretion. PMA at 1 ng/ml caused a smaller increase i n proteinase secretion. The increase i n proteinase secretion accompanying the s l i g h t c e l l rounding and the reduced proteinase secretion observed together with c e l l f l a t t e n i n g (cholera toxin and B t 2 cAMP experi-ment) suggests that c e l l shape influences proteinase secretion. The use of these chemical agents to modify c e l l shape however, can be c r i t i c i z e d because they a f f e c t other c e l l functions as w e l l . 6 2 . TABLE IV: EFFECT OF DIBUTYRYL CYCLIC AMP ( B t 2 cAMP) CONTROL B t 2 cAMP % CONTROL C e l l Counts Day 4 2.0 ± 0.04 2.6 ± 0.18 130 10 5 c e l l s / Day 7 2.2 ± 0.20 4.4 ± 0.20 200 dish Day 11 1.7 ± 0.22 5.0 ± 0.14 294 Day 14 1.2 ± 0.01 5.0 ± 0.11 417 N.P. A c t i v i t y Day 4 5.0 (0.4) 0.51 10 10" 3 U/10 5 Day 7 5.7 (0.74) 1.40 25 c e l l s Day 11 2.1 (0.06) 0.12 6 Day 14 6.6 (0.4) 0.83 13 E - c e l l s were plated i n aMEM + 15% FCS overnight and then incubated i n 3MEM + 0.5% dialysed FCS. B t 2 cAMP was added at 0.5 mM. The N.P. a c t i v i t y from the B t 2 cAMP-treated cultures have been corrected for the c e l l population density e f f e c t . The uncorrected values are shown i n brackets. Estimation of v a r i a t i o n i s given as ± SEM. TABLE V: EFFECT OF PHORBOL MYRISTATE ACETATE (PMA) CONTROL PMA 1 ng/ml PMA 10 ng/ml Day 1 C e l l Counts 10 5 c e l l s / Day 2 dish Day 4 5.0 ± 0.04 5.45 ± 0.25 4.65 ± 0.04 5.85 ± 0.04 5.8 ± 0.07 6.0 ± 0.18 4.1 ± 0.21 5.6 ± 0.21 5.25 ± 0.18 Day 1 N.P. A c t i v i t y 10~ 3 U/10 5 Day 2 c e l l s Day 4 5.3 6.2 7.6 17.4 9.9 11.5 21.3 11.5 10.6 P.A. A c t i v i t y 10" 3 IUStrep- Day 4 tokinase/ 10 5 c e l l s 1.8 4.4 6.7 E - c e l l s were plated i n aMEM + 15% FCS overnight and then incubated i n fiMEM +0.5% DFCS f o r proteinase secretion. PMA was added at 1 ng/ml and 10 ng/ml. Two dishes were used for c e l l counting at each time point of medium c o l l e c t i o n . P.A. a c t i v i t y at days 1 and 2 were undetectable i n both control and test groups. Estimation of v a r i a t i o n i s given as ± SEM. 64. For example, PMA i s known to cause increased membrane transport (Boutwell, 1974) and stimulation of DNA, RNA, and protein synthe-s i s (Boutwell, 1974). Therefore d i r e c t mechanical means of a l t e r -ing c e l l shape were tested and are described i n the next section. 4) Mechanical Stretching E - c e l l s from the 4th to 8th subcultures were plated onto the f l e x i b l e p l a s t i c bottoms of petriperm dishes. Electron micro-scopic sections of the i n t e r f a c e between the c e l l and p l a s t i c show the presence of hemidesmosomes (See Figure 13) i n d i c a t i n g t i g h t attachment of the c e l l s to the p l a s t i c surface. Thus as found by Brunette (1984b) stretching the dish also stretched the attached E - c e l l s , r e s u l t i n g i n the E - c e l l s becoming more f l a t t e n e d . By removing the dishes from the template, a release i n tension could be obtained allowing stretched c e l l s to relax. The amount of stretching was l i m i t e d to 4% because stretching beyond t h i s produced a l o s s of e l a s t i c i t y of the f l e x i b l e p l a s t i c bottom. However, even stretching E - c e l l s by 4% produced a 2 to 6 f o l d decrease i n P.A. s e c r e t i o n compared to unstretched c e l l s (Table VI). The e f f e c t of relaxing stretched c e l l s compared to controls which remained stretched was an increase (40 to 210%) i n P.A. secretion (Table VII). S t a t i s t i c a l analysis was done using the s i g n t e s t . The d i f f e r e n c e i n P.A. values between the treated and the control on a l l f i v e occasions that the experiments were c a r r i e d out was i n the expected d i r e c t i o n . The p r o b a b i l i t y of t h i s occurrence happening due to chance alone i s < 0.05. The e f f e c t of stretching on N.P. secretion was investigated 65. F i g . 13. Electron microscopic section of E - c e l l s grown on a petriperm dish. C e l l attachment to the p l a s t i c surface (P) was through hemidesmosomes (HD). Magnification x 83,200. 66. TABLE VI: EFFECT OF MECHANICAL STRETCHING ON E-CELLS Treatment P.A. A c t i v i t y 10~ 3 IU Streptokinase/10 5 c e l l s Experiment 1 Experiment 2 Unstretched Control 12.0 3.2 Stretched 2.0 1.8 TABLE VII: EFFECT OF RELAXING E-CELLS CULTURED ON STRETCHED MEMBRANES Treatment P.A. A c t i v i t y 10~ 3 IU Streptokinase/10 5 c e l l s Experiment 1 Experiment 2 Experiment 3 Stretched Control Relaxed 1.5 3.2 4.6 6.6 4.1 7.6 Mechanical Stretching was applied as described i n Materials and Methods The medium was c o l l e c t e d on day 2 and assayed f o r P.A. a c t i v i t y . 6 7 . using a parametric s t a t i s t i c a l t e s t i n a s i n g l e experiment i n which 10 groups of 4 dishes each were plated with c e l l s at 8x10 5 c e l l s / d i s h . Five groups were stretched and the other 5 groups remained unstretched as controls. To get s u f f i c i e n t c e l l s for th i s experiment, a 9th subculture was used, which was older than that normally used i n these experiments. The c e l l s from the 9th subculture did not demonstrate s i g n i f i c a n t amounts of P.A. a c t i -v i t y but detectable amounts of N.P. were present. The stretched c e l l s produced 5.9±0.16(SEM)xl0~ 3 U of N.P./105 . c e l l s compared to 7.7±0.16(SEM)xl0~ 3 U/10 5 c e l l s produced by contr o l cultures. The d i f f e r e n c e i s s t a t i s t i c a l l y s i g n i f i c a n t when tested by ANOVA (p<0.005). There was no s i g n i f i c a n t d i f f e r e n c e i n c e l l numbers between the treated and control groups at the time of medium c o l l e c t i o n . Thus, making the c e l l s more fl a t t e n e d by stretching resulted i n a decrease i n proteinase secretion, while making the c e l l s more rounded by relaxin g stretched c e l l s resulted i n an increased proteinase secretion. 5) Grooved Substrata This experiment makes use of an observation made previously i n our laboratory, that c e l l s grown on grooved substrates assume a round shape more frequently than do c e l l s grown on a f l a t substrate (Brunette - personal communication). Rovensky et a l . (1971) suggested that the c e l l s probably attach to the bottom of the grooves less r e a d i l y than to f l a t surfaces. Table VIII shows that the e f f e c t of the grooved substrate on proteinase s e c r e t i o n 68. TABLE VIII: EFFECT OF GROOVED SUBSTRATA FLAT GROOVED GROOVED/ FLAT Expt. 1 Day 3 0.15 0.39 2.6 N.P. A c t i v i t y Day 7 0.13 0.33 2.5 10" 3 U/10 5 Expt. 2 c e l l s Day 3 6.5 11.0 1.7 Day 7 2.5 6.2 2.5 Expt. 3 Day 3 7.45 9.1 1.2 Expt. 1 P.A. A c t i v i t y 10~ 3 IU Strepto-kinase/10 5 c e l l s Day 3 Day 7 7.25 3.4 13.5 8.1 1.9 2.4 Expt. Day 3 Day 7 2 15.3 14.3 15.0 17.5 1 1.2 Expt. 3 Day 3 4.2 7.0 1.7 E - c e l l s were plated at 8 x 10 5 c e l l s / d i s h on f l a t epoxy substrates and epoxy substrates with v-shaped grooves (top width 79.3 pm, depth 60 pm, 13.3 pm apart). Proteinase a c t i v i t y was measured at days 3 and 7. P.A. a c t i v i t y from experiment 1 i s also shown i n Figure 8. 69. was, on average, a 2 f o l d increase i n N.P. a c t i v i t y and a 1.6 f o l d increase i n P.A. a c t i v i t y compared to the c o n t r o l . Analysis of the data using the s i g n t e s t i ndicates that the increase i n N.P. a c t i v i t y was s i g n i f i c a n t (p < 0.05). No s i g n i f i c a n t d i f f e r -ences were observed i n the c e l l counts between the t e s t and control groups. 6) Poly(HEMA)-coated Dishes Increasing concentrations of poly(HEMA) stock s o l u t i o n were used to a l t e r tissue culture dish adhesivity. In agreement with the r e s u l t s of Folkman and Moscona (1978), E - c e l l s became l e s s spread out (that i s , more rounded) on dishes coated with increas-ing concentrations of poly(HEMA) stock s o l u t i o n . Cultures stained with Richardson's s t a i n were v i s u a l i s e d through the microscope and the extent of c e l l spreading was assigned a score on an a r b i t r a r y scale 0-3, based on the amount of c e l l area i n contact with the dish. C e l l s grown on p l a s t i c were well spread (score 0 ) . The c e l l s became less spread out, on dishes coated with 5 x 10 - l + d i l u -t i o n of stock poly(HEMA) so l u t i o n . These were assigned a score of 1. A much reduced c e l l area compared to score 1 was observed when c e l l s were grown on dishes coated with 10 - 3 d i l u t i o n of the poly(HEMA) stock. These were given a score 3. Thus for E - c e l l s , c e l l shape changes occurred over a narrow range of poly(HEMA) d i l u t i o n . E - c e l l s were cultured i n duplicate and the c e l l counts show that under the conditions used, there was no increase i n c e l l number. N.P. a c t i v i t y increased with increasing poly(HEMA) 70. concentration (Figure 14). C e l l s cultured on poly(HEMA)-coated dishes at 1 0 - 5 d i l u t i o n and greater showed a s i g n i f i c a n t increase i n N.P. a c t i v i t y compared to the c o n t r o l (p < 0.05 using the one way analysis of variance t e s t ) . The sharpest increase i n N.P. a c t i v i t y was observed between 5 x 1 0 - 4 and 1 0 - 3 d i l u t i o n of poly(HEMA) stock thus c o r r e l a t i n g with the sharp change i n c e l l morphology noted. Although no obvious morphological change could be detected at 1 0 - 5 d i l u t i o n of poly(HEMA), an increase i n N.P. a c t i v i t y was observed. A more s e n s i t i v e method needs to be developed to measure small changes i n c e l l shape changes. Figure 15 shows that P.A. a c t i v i t y also increased with increasing poly(HEMA) concentration, the increase being more gradual compared to N.P. a c t i v i t y . The advantages of using poly(HEMA)-coated dishes to modify c e l l shape are: 1) the use of chemical agents with multiple e f f e c t s i s avoided; 2) the method allows d i f f e r e n t degrees of c e l l spreading to be achieved. The above r e s u l t s show that both N.P. and P.A. a c t i v i t y increased as the c e l l s became less w e l l -spread. A c o r r e l a t i o n between c e l l shape and proteinase s e c r e t i o n was thus shown. 71. 12 J2 75 o i n 10 8 co I o >. [> o CO 2 - T I • } 1 T ± T T - 6 >-5 . -4 1 0 " 10 ^ 10 ^ 10 Poly( H E M A ) concentration - 3 F i g . 14. E f f e c t of poly(HEMA) concentration on N.P. a c t i v i t y at day 3. E - c e l l s were plated i n duplicate at 4 x l 0 5 c e l l s / d i s h i n dishes precoated with varying concentrations of poly(HEMA) sol u t i o n . At day 3 of culture, medium was c o l l e c t e d and assayed for N.P. a c t i v i t y , ( f ) represents data from experi-ment 1 and ( A ) represents experiment 2. Error bars i n d i c a t e SEM. 72. Q) o 10 o CO ZD CO I o > o ca < CL 2.5 2.0 2 1.5 J 1.0 0.5 T 15 i I l -6 - 5 . -4 10 ~ 10 ~ 10 ^ 10 Poly( H E M A ) concentration - 3 F i g . 15. E f f e c t of poly(HEMA) concentration on P.A. a c t i v i t y at day 3. Medium c o l l e c t e d on day 3 from the experiment described i n F i g . 14 was assayed for P.A. a c t i v i t y (•) represents data from experiment 1 and (A) represents data from experiment 2. Error bars indicate SEM. 73. CHAPTER 4 - DISCUSSION E-cells i n culture secreted latent neutral proteinase as well as plasminogen activator; the secretion of both proteinases was found to be regulated by c e l l population density, E.Coli LPS and c e l l shape changes. The significance of the results obtained w i l l be discussed i n the following order: I) c e l l population density; I I ) E.Coli LPS; I I I ) c e l l growth; IV) c e l l shape; V) relationship between N.P. and P.A. secretion. F i n a l l y experiments which w i l l build on these findings w i l l be proposed. I. C e l l Population Density In the experiments, c e l l growth was dissociated from c e l l popula-tion density by culturing E - c e l l s i n a modified medium, 3MEM + 0.5% dialysed FCS, with low Ca and K . Under these conditions, c e l l numbers were maintained over the experimental period of ten days. Thus the relationship between c e l l population density and proteinase secre-tion could be analysed. Both N.P. and P.A. secretion were dependent on c e l l population density. N.P. secretion was highest at low c e l l densities, decreasing as c e l l population densities increased. P.A. secretion showed a similar pattern except at day three, the day of the f i r s t medium c o l l e c t i o n . The regulation of proteinase secretion by c e l l population density i s not confined to E - c e l l s . Other c e l l l ines have been reported to show density-dependent regulation of P.A. The work of L i u et a l . (1982) and Rohrlich and R i f k i n (1977) which have been b r i e f l y described i n the Introduction indicate that d i f f e r e n t c e l l l i n e s can respond to c e l l population density d i f f e r e n t l y . 74. Unlike f i b r o b l a s t s which were found to secrete increasing amounts of P.A. as c e l l population density increased, reaching a maximum at confluence (Rohrlich and R i f k i n , 1977); E - c e l l s produced more proteinase a c t i v i t y at low c e l l population d e n s i t i e s . Thus, f o r studies i n v e s t i g a t i n g proteinase s e c r e t i o n by E - c e l l s , the c e l l s should be plated at appropriate low dens i t i e s to allow a good harvest of proteinase a c t i v i t y i n the medium. The reasons f o r t h i s density-dependent regulation of proteinase secretion are not c l e a r . A possible explanation could be that at higher c e l l population d e n s i t i e s , the proportion of c e l l s secreting the proteinases decreases. Some i n d i r e c t evidence for th i s hypothesis comes from the work of Birek et a l . (1980) who found that the se c r e t i o n of prostaglandins of the E serie s by E - c e l l s was density-dependent with the highest amounts secreted at low c e l l population density and smaller amounts secreted at higher d e n s i t i e s . Measurement of the percentage of hemolytic plaque forming c e l l s (which i n d i c a t e s c e l l s producing prostaglandin E) i n r e l a t i o n to c e l l population density showed that the f r a c t i o n of c e l l s not secreting prostaglandin E was higher i n dishes with higher c e l l population d e n s i t i e s . Another possible explanation f o r the density-dependent r e g u l a t i o n of proteinases could be c e l l - c e l l contact. C e l l - c e l l contact influences c e r t a i n aspects of c e l l behaviour such as contact i n h i b i t i o n of locomotion, contact i n h i b i t i o n of extension, and contact i n h i b i t i o n of phagocytosis (Brown and Middleton, 1981). C e l l - c e l l contact has also been reported to r e s u l t i n contact-induced spreading i n a v a r i e t y of e p i t h e l i a l c e l l types i n cu l t u r e (Brown and Middleton, 1981). Cell-induced spreading Is a phenomenon proposed and described by 7" 75. Middleton (1977). He showed that i s o l a t e d chick embryo pigmented r e t i n a e p i t h e l i a l c e l l s lacking contacts with other c e l l s , are normally poorly spread on the cul t u r e dish, d i s p l a y vigorous blebbing a c t i v i t y , and lack a well-defined leading lamella; but when such a c e l l comes in t o contact with one or more c e l l s , i t spreads out on the substratum, ceases blebbing, and develops a leading lamella from a part of the c e l l margin not i n contact with the other c e l l s . Thus the c e l l s i n contact with one or more c e l l s become more flattened than the i s o l a t e d c e l l s . Contact induced spreading has not been studied i n d e t a i l i n the E - c e l l s used i n this study but preliminary observations indicate that the phenomenon does occur i n these c e l l s (Brunette, i n press). The phenomenon of contact spreading plus other considerations lead to the conclusion that the e f f e c t of c e l l population density on c e l l shape i s complex. At the lowest c e l l d e n s i t i e s , most of the c e l l s would be i s o l a t e d from the other c e l l s . It would be expected that increasing the c e l l population density would r e s u l t i n more c e l l f l a t t e n i n g because an increase i n c e l l number per unit area would make contact between i s o l a t e d e p i t h e l i a l c e l l s more l i k e l y and thus promote spreading. At s t i l l higher d e n s i t i e s , a f t e r confluence has been attained, however, increasing the c e l l population density r e s u l t s i n more c e l l s being crowded in t o the same area. This crowding normally causes the c e l l s to become less spread. F i n a l l y , at higher d e n s i t i e s , there i s a tendency f o r k e r a t i n i z a t i o n to occur with the resultant production of squames or squame-like c e l l s which would not be expected to contribute to proteinase secretion. Because of these complexities, i t i s d i f f i c u l t to gain any i n s i g h t into the mechanisms regulating proteinase secretion from the data given 76. i n Figures 9 and 10 which nevertheless c l e a r l y show that c e l l popula-t i o n density modulates proteinase secretion. This data does have a p r a c t i c a l a p p l i c a t i o n i n the design of experiments i n v e s t i g a t i n g proteinase secretion because i t shows that comparisons should be made when the c e l l population d e n s i t i e s do not d i f f e r greatly or when the population densities are In the plateau region of the curves shown i n F i g s . 9 and 10. I I . E . c o l i LPS The r e s u l t s show that E . c o l i LPS stimulated both N.P. and P.A. secretion by E - c e l l s . LPS at 3 ug/ml was as e f f e c t i v e at higher doses up to 60 yg/ml. These r e s u l t s are i n agreement with those of Pettigrew et a l . (1981) who showed that LPS at 30 ug/ml stimulated increased production of the metalloproteinases by g i n g i v a l tissue explants. As described i n the Introduction, the e p i t h e l i a l c e l l rests of Malassez are known to respond to the inflammatory s t i m u l i by p r o l i f e r a t i n g and p a r t i c i p a t i n g i n the formation of dental cysts. E - c e l l s derived from the e p i t h e l i a l c e l l r e s t s of Malassez have been shown to secrete N.P. (Pettigrew, Ph.D. Thesis) and collagenase i n v i t r o (Limeback and Brunette, 1981). I t i s thus possible that endotoxins penetrating the p e r i a p i c a l tissues could stimulate the c e l l rests into increased production of enzymes, thereby contributing to the degradation of connective tissue associated with cyst formation. The LPS molecule consists of three d i s t i n c t portions, the poly-saccharide region, the core region and the l i p i d A region, linked together covalently. There i s great v a r i a t i o n i n the side chains of the LPS molecule among d i f f e r e n t s t r a i n s of bacteria. Even d i f f e r e n t methods of e x t r a c t i o n r e s u l t i n differences i n the structure of the 77. molecule. For example, the Westphal phenol-water extraction process r e s u l t s i n a loss of protein which i s retained during the TCA extrac-t i o n process (Majde and Person, 1981). The l i p i d A region of the mole-cule i s considered to be important for the expression of b i o l o g i c a l a c t i v i t y . However, the mechanisms by which the LPS molecule causes these b i o l o g i c a l e f f e c t s are not known. Endotoxin has been reported to cause alte r e d mitochondrial function, increased a c t i v i t i e s of lysosomal enzymes, increased uptake of sugar, and increased formation of l a c t i c acid (De Renzis and Chen, 1983). In addition, LPS has been shown to cause increased cAMP l e v e l s i n c e r t a i n c e l l types (Watson, 1976; Naylor et a l . , 1978), but not i n others (Graber and H e l l e r q u i s t , 1982). My res u l t s show that E . c o l i LPS at 3 ug/ml caused no s i g n i f i c a n t changes i n i n t r a c e l l u l a r cAMP l e v e l s . Thus the increased production of N.P. and P.A. induced by LPS i s l i k e l y to be independent of the cAMP pathway. I I I . C e l l Growth and Proteinase Secretion Cholera toxin and d i b u t y r y l cAMP a f f e c t c e l l s by increasing i n t r a -c e l l u l a r cAMP le v e l s but act by d i f f e r e n t mechanisms. Cholera toxin acts by a c t i v a t i n g adenylate cyclase, an enzyme that catalyses the conversion of adenosine triphosphate to cAMP. The cholera toxin mole-cule i s made up of two d i f f e r e n t subunits: A and B. The function of the B subunits i s to bind to the c e l l membrane, allowing subunit A to enter the c e l l by mechanisms which are s t i l l unclear. The r o l e of the subunit A i s to activate adenylate cyclase. Dibutyryl cAMP increases i n t r a c e l l u l a r cAMP l e v e l s by i n h i b i t i n g cAMP phosphodiesterase, an enzyme that degrades i n t r a c e l l u l a r cAMP l e v e l s (Hsie, 1982). 7 8 . As found by Brunette (1984a), cholera toxin and B t 2 cAMP treatment s i g n i f i c a n t l y increased c e l l numbers. This increase i n c e l l number necessitated increasing the values obtained f o r proteinase secretion to correct for the c e l l population density e f f e c t . Even af t e r t h i s correction, the f l a t t e n e d cholera t o x i n or B t 2 cAMP t r e a t e d - c e l l s were s t i l l found to secrete less proteinase than untreated control c e l l s . Moreover, although Aggeler et a l (1982) and Chou et a l . (1976) have suggested that c e l l p r o l i f e r a t i o n i s accompanied by increased P.A. secretion, t h i s e f f e c t was not noted i n these experiments, where the e f f e c t of c e l l shape appeared to be dominant. However cholera toxin and B t 2 cAMP have multiple c e l l u l a r e f f e c t s and further i n v e s t i g a t i o n i s needed to determine whether c e l l shape i s the only mechanism regulating proteinase s e c r e t i o n here. IV. C e l l Shape and Proteinase Secretion The r e s u l t s from the experiments using s i x methods of changing c e l l shape consistently show that i r r e s p e c t i v e of the method of chang-ing c e l l shape, the more flattened c e l l s produce reduced amounts of proteinase compared to the more rounded c e l l s . This suggests that c e l l shape may be a means of regulating N.P. and P.A. secretion i n E - c e l l s . A more d e t a i l e d discussion of these experiments follows. As reported by Brunette (1984a), treatment of E - c e l l s with cholera toxin or B t 2 cAMP caused them to become more fl a t t e n e d . This morpho-l o g i c a l change was accompanied by a reduction i n proteinase secretion; cholera toxin treated c e l l s produced 69% and 29% of the N.P. and P.A. produced by control cultures. Dibutyryl cAMP-treated c e l l s produced only 14% of the N.P. secreted by c o n t r o l cultures. 79. The morphological change i n E - c e l l s i n the presence of Bt 2 cAMP i s i n agreement with other studies. D i f f e r e n t c e l l s have been shown to become more fl a t t e n e d i n the presence of B t 2 cAMP (Pastan and Willingham, 1978; Johnson et a l . , 1971; Hsie and Puck, 1971). A study by Wilson and Reich (1979) showed that the a d d i t i o n of cholera toxin (1 ng/ml) or B t 2 cAMP ( 1 0 - 3 M) to cultures of normal chick embryo f i b r o b l a s t s caused only a small decrease i n P.A. secre-t i o n . But both v i r u s and PMA induced P.A. secretion were strongly i n h i b i t e d by cholera toxin at 0.1 and 1.0 ng/ml and by cAMP at 1 0 - 3 and lO - 1* M. Va s s a l ! et a l . (1976) found that t h i o g l y c o l l a t e - s t i m u l a t e d macro-phages responded to cholera toxin ( 1 0 - 1 2 M) by producing 10% of the P.A. secreted by con t r o l c u l t u r e s . Dibutyryl cAMP-treated macrophages showed a 50% reduction i n P.A. secretion. At the same time, macro-phages unlike E - c e l l s or f i b r o b l a s t s , became more r e t r a c t i l e and l e s s well spread compared to the control cultures. In contrast to cholera t o x i n and B t 2 cAMP, PMA at 10 ng/ml caused the E - c e l l s to r e t r a c t s l i g h t l y . The r e s u l t s show that both N.P. and P.A. secretion were increased compared to the c o n t r o l . This e f f e c t of PMA i s i n agreement with the report of Wigler and Weinstein (1976) who showed that a v a r i e t y of c e l l s including chick embryo f i b r o b l a s t s , HeLa c e l l s , and hamster embryo c e l l s respond to PMA at 1.5xl0 - 8 M by increasing P.A. secretion. The e l e v a t i o n of P.A. secretion by PMA has also been reported by T r o l l et a l . (1975) and Wilson and Reich (1979). My data on the e f f e c t of c e l l shape on N.P. secretion i s c o n s i s t -ent with the work of Aggeler et a l . (1984). In t h e i r study, the morphology of rabbit synovial f i b r o b l a s t s was a l t e r e d by d i f f e r e n t 80. agents and the degree of morphologic change was compared to the amount of collagenase induced. Agents l i k e PMA, cytochalasin B, and D, t r y p s i n , t r i f l u o p e r a z i n e , and poly(HEMA) coated dishes caused d i f f e r e n t degrees of c e l l rounding which was p o s i t i v e l y correlated with the increase i n collagenase a c t i v i t y . Agents that d i d not a l t e r c e l l shape did not induce collagenase secretion. However, no c o r r e l a t i o n between morphologic change and P.A. s e c r e t i o n was seen. A reason f o r the discrepancy between t h i s data and mine could be the d i f f e r e n t c e l l types used. Mechanical stretching made E - c e l l s more flat t e n e d and conversely relaxing c e l l s grown on stretched membranes made the E - c e l l s more rounded. I t was shown that stretched c e l l s produced l e s s proteinase and relaxed c e l l s produced more proteinase than the controls. Even though the degree of s t r e t c h i n g was small, 4%, a s i g n i f i c a n t change i n proteinase secretion was observed. This indicates that the control on proteinase secretion by c e l l shape i s t i g h t . The advantage of a l t e r i n g c e l l shape by mechanical means i s that the multiple c e l l u l a r e f f e c t s produced by the use of some chemical agents are avoided. Thus the data from t h i s experiment provides more d i r e c t evidence for the hypothesis that c e l l shape regulates proteinase secretion, with a more fl a t t e n e d c e l l shape favouring less proteinase secretion and a more rounded c e l l shape favouring increased proteinase secretion. The l i m i t a t i o n s of the present technique, however, i s the small degree of mechanical s t r e t c h -ing which can be applied. There i s one other study (Meikle et a l . , 1980) where the e f f e c t of t e n s i l e mechanical stress on the secretion of metalloproteinases by organ cultures of rabbit c r a n i a l sutures was investigated. Meikle et 81. a l . found that mechanical s t r e s s stimulated 98.2% increase i n g e l a t i n -degrading N.P. and 35.9% increase i n azocasein-degrading N.P. The explant consists of multiple c e l l types (bone, connective t i s s u e , blood c e l l s ) and moreover, i t i s not c l e a r what morphological e f f e c t the a p p l i c a t i o n of t e n s i l e mechanical s t r e s s had on the i n d i v i d u a l c e l l s i n d i f f e r e n t areas of the explant. I t i s therefore d i f f i c u l t to compare d i r e c t l y the r e s u l t s of t h i s study and mine. Another physical method of a l t e r i n g c e l l shape was the use of grooved substratum. Rovensky et a l . (1971) suggest that c e l l s attached to grooved surfaces are more round than those on f l a t surfaces. A s i m i l a r f i n d i n g has been made on E - c e l l s grown i n t h i s laboratory. E - c e l l s grown on grooved substratum demonstrated a two f o l d increase i n N.P. and a 1.6 f o l d increase i n P.A. a c t i v i t y . This r e s u l t i s c o n s i s t -ent with the hypothesis that more rounded c e l l s secrete increased amounts of proteinases. Poly(HEMA)-coated dishes reduce d i s h surface adhesivity thereby a f f e c t i n g c e l l attachment and shape. The mechanism involved i n the poly(HEMA) e f f e c t on d i s h adhesivity, however, i s not known. Folkman and Moscona (1978) suggested that poly(HEMA), having a neutral charge, may act by reducing the negative e l e c t r o s t a t i c charge of the p l a s t i c dish. Another p o s s i b i l i t y was that increasing the poly(HEMA) concen-t r a t i o n would decrease the number of a v a i l a b l e s i t e s on the p l a s t i c to which the c e l l s could attach. It i s c l e a r that increasing the concentrations of poly(HEMA) solu-t i o n decreases surface adhesivity. The degree of spread of E - c e l l s , as in d i c a t e d by the area of the c e l l i n contact with the dish, decreases with increasing poly(HEMA) concentration. 82. E - c e l l shape change occurred over a narrow range of poly(HEMA) d i l u t i o n : 10~ 5 to 10~ 3 of the stock. Measurement of the proteinase secretion showed that as the c e l l s became l e s s spread out ( i . e . more round) the N.P. and P.A. secretion increased; the increase i n P.A. being more gradual than that of N.P. A c o r r e l a t i o n between c e l l shape and proteinase secretion was thus shown; increased c e l l rounding co r r e l a t e d with increased N.P. and P.A. secretion. The advantages of the poly(HEMA)-coated dishes to modify c e l l shape are: (1) the addition of agents with known multiple b i o l o g i c a l e f f e c t s i s avoided; (2) the method allows d i f f e r e n t degrees of c e l l spreading to be achieved. Thus, evidence from these experiments, where 6 d i f f e r e n t means of a l t e r i n g c e l l shape were used, have been presented which are consistent with the hypothesis that c e l l shape regulates N.P. and P.A. secretion; the more flattened c e l l s produce less proteinase and more rounded c e l l s produce increased amounts of proteinases. However, the mechanisms involved i n t h i s regulation are not known although i t i s l i k e l y that the cytoskeleton i s involved i n some way. In the next section, the possible c y t o s k e l e t a l changes brought about by the d i f f e r e n t methods of a l t e r i n g c e l l shape w i l l be discussed and a hypothesis for the role of the cytoskeleton i n regulating proteinase secretion w i l l be presented. 1) C e l l Shape and the Cytoskeleton E l e c t r o n microscopic studies have demonstrated that B t 2 cAMP treatment of 3T3-4 c e l l s r e s u l t s i n a change i n the d i s t r i b u t i o n of microfilaments and microtubules. Willingham and Pastan (1975) showed that on c e l l f l a t t e n i n g , microfilaments under the plasma membrane became more prominent, the number of microtubules increased, and became 83. aligned with the d i r e c t i o n of the c e l l processes. 3T3-4 c e l l s i n the presence of B t 2 cAMP were demonstrated to become c l o s e l y opposed to the i r r e g u l a r surface of the substratum and showed mult i p l e s i t e s of c e l l attachment. Dense microfilament bundles became v i s i b l e at the edges of Bt 2 cAMP-treated c e l l s as well as the attachment points. In contrast, control c e l l s showed few attachment points; at the t i p s of c e l l processes, under the nucleus, and a few s i t e s inbetween. Willingham and Pastan suggest that cAMP stimulates microtubule assembly promoting the extension of c e l l processes and at the same time cAMP i n h i b i t s microfilament-mediated contraction of the c e l l processes, therefore the f i n a l c e l l morphology i s very f l a t . Indeed, L i et a l . (1975) and Hsie et a l . (1977) have shown that the increased cAMP l e v e l s cause the a c t i v a t i o n of a cAMP-dependent protein kinase which r e s u l t s i n protein phosphorylation and the poly-merization of microtubules. PMA i s potent tumour promoter which has been found to cause a va r i e t y of e f f e c t s on cultured c e l l s , some of these changes c o r r e l a t i n g with the features of transformed c e l l s . Of i n t e r e s t to us i s the cyt o s k e l e t a l changes caused by PMA. R i f k i n and Crowe (1979) reported that PMA at concentrations as low as 7.3xl0 -*^ M produced c y t o s k e l e t a l changes. There was a loss i n the normal structure of the a c t i n containing f i b r e s and a reduction i n the length and thickness of these f i b r e s . Sakiyama and Hiwasa (1984) also report that PMA at 20 ng/ml resulted i n a l o s s of the structured a c t i n f i b r e s . These c e l l s treated with a n t i - a c t i n antibody and examined by i n d i r e c t immunofluorescence microscopy showed a d i f f u s e a c t i n pattern. Recent work by Fey and Penman (1984) indicate that PMA and other 84. tumour promoters a f f e c t the organization of the nuclear matrix-intermediate filament s c a f f o l d , a structure obtained a f t e r the removal of the soluble proteins, phospholipids, the cytoskeleton, and chromatin f r a c t i o n s . PMA resulted i n morphological changes i n Madin-Darby canine kidney (MDCK) colonies. Normally the c e l l s i n each colony display t i g h t contact with one another. In the presence of PMA at 5 ng/ml, a breakdown i n the organization of the c e l l s was observed; the c e l l s began to spread out and develop long processes. These morphological changes were r e f l e c t e d i n the d i s t r i b u t i o n of cytokeratins i n the nuclear matrix-intermediate filament s c a f f o l d . I t i s thus c l e a r that PMA changes c e l l morphology by a f f e c t i n g the c y t o s k e l e t a l elements. In the experiment using mechanical s t r e t c h i n g , E - c e l l shape was modulated by an applied external force and i n the case of stretched c e l l s being relaxed, there was a release i n tension. In h i s experi-ments on E - c e l l s , Brunette (1984b) found an increase i n the volume f r a c t i o n of microtubules and other filamentous structures i n stretched c e l l s compared to unstretched c e l l s . Thus stretching the c e l l s resulted i n changes i n the c y t o s k e l e t a l elements. The poly(HEMA)-coated dishes a f f e c t c e l l shape by reducing dis h surface adhesivity to the c e l l s . Increasing concentrations of poly(HEMA) resulted i n E - c e l l s being le s s spread out. The r e l a t i o n s h i p between substratum adhesiveness and c e l l shape i s i n agreement with the r e s u l t s of Folkman and Moscona (1978) and Willingham et a l . (1977). Willingham et a l . (1977) measured substratum adhesiveness i n terms of the length of time needed to remove 50% of the c e l l s from the sub-stratum. They found that a f l a t t e r c e l l morphology resulted i n an 85. increase i n substratum adhesiveness. On the other hand, a rounded c e l l morphology was correlated with a decrease i n substratum adhesiveness. Willingham et a l . (1977) also showed that there was a strong cor-r e l a t i o n between the adhesive strength of the c e l l to the substratum, and the presence of microfilament bundles. Normal Balb/3T3 c e l l s grown on an adhesive substratum showed a flattened morphology and numerous bundles of microfilaments. The same c e l l s grown on a low adhesive substratum were round i n shape and showed no d i s c e r n i b l e microfilament bundles. A mutant of Balb/3T3 c e l l s , which i s defective i n i t s a b i l i t y to acetylate glucosamine-6-phosphate adhered poorly to tissue culture d i s h and showed a rounded morphology. No microfilament bundles were seen. However, on addition of 10 mM N-acetyl glucosamine,normal adhesiveness was restored and the c e l l s f l a t t e n e d out. Numerous bundles of microfilaments became evident. Furthermore, MC 5-5, a transformed c e l l l i n e which showed rounded morphology, poor adhesion to the substratum also showed no bundles of microfilaments. When treated with c e l l surface protein, a f a c t o r mediating c e l l attachment, the c e l l s flattened out and showed bundles of microfilament. It i s thus c l e a r that substratum adhesiveness a f f e c t s c e l l shape and the micro-filament bundles: a more flattened c e l l shape showing more bundles of microfilaments. From the data above, i t seems reasonable to conclude that changes i n c e l l shape observed on poly(HEMA)-coated dishes involved changes i n the c y t o s k e l e t a l elements too. 2 • 86. 2) Penman's Hypothesis on the Relationship Between the Cytoskeleton  and Nuclear Metabolism At present, there i s l i t t l e d e f i n i t i v e evidence f o r the mechanisms that could be involved i n the regulation of proteinase secretion by c e l l shape. However, a possible explanation comes from the hypothesis of Penman and colleages (1983). These authors postulate that the gene a c t i v i t y of the c e l l responds to c e l l a r c h i t ecture. Benecke et a l . (1978) showed that anchorage-dependent f i b r o b l a s t s , when placed i n suspension cultures, responded by shutting down a l l major macromole-cular processes. Cytoplasmic protein synthesis could be restored by c e l l contact with a s o l i d surface. Nuclear metabolism, such as mRNA and DNA synthesis, however, was subject to regulation by the degree of c e l l spreading (Ben Ze'ev et a l . , 1980). Furthermore, Cervera et a l . (1981) showed that i n HeLa c e l l s , a l l a c t i v e l y t r a n s l a t i n g message molecules were bound to the cytoskeleton. Upon i n f e c t i o n with a v i r u s , the ribosomes translated a mixed batch of host and virus messages, both being bound to the c y t o s k e l e t a l structures. This suggested that mRNA was translated only when bound to the c y t o s k e l e t a l framework and may be a means by which s e l e c t i o n of mRNA occurs. Further evidence comes from studies involving the i n t e r a c t i o n between the e x t r a c e l l u l a r matrix and the c e l l i n tissue development, i n which there are indications that the cytoskeleton plays an important r o l e . B i s s e l l et a l . (1982) stated i n t h e i r review, "The e x t r a c e l l u l a r matrix (ECM) i s postulated to exert physical and chemical influences on the geometry and the biochemistry of the c e l l v i a transmembrane recep-tors so as to a l t e r the pattern of gene expression by changing the ass o c i a t i o n of the cytoskeleton with the mRNA and the i n t e r a c t i o n of the chromatin with the nuclear matrix". It i s conceivable that changes 8 7 . i n c e l l shape a l t e r s the a s s o c i a t i o n of the cytoskeleton to the mRNA thereby regulating the amount of proteinase translated. But confirma-t i o n of t h i s hypothesis awaits fur t h e r research. V. Relationship between N.P. and P.A. secretion The secretion of N.P. and P.A. appeared to be l i n k e d under several of the conditions employed i n these experiments. With the exception of P.A. a c t i v i t y from day 3 cultures which w i l l be discussed l a t e r , E - c e l l s respond to c e l l population density by secreting latent N.P. and P.A. i n a s i m i l a r pattern. N.P. and P.A. a c t i v i t y was highest at low c e l l population d e n s i t i e s ; the enzyme a c t i v i t y decreased with i n c r e a s -ing c e l l population density. The s e c r e t i o n of l a t e n t N.P. and P.A. was stimulated by the addition of E - c e l l LPS to the cultures. In the experiments where d i f f e r e n t methods of a l t e r i n g c e l l shape were used, a more flattened c e l l shape correlated with a reduced l e v e l of N.P. and P.A. secretion while a more rounded c e l l shape cor r e l a t e d with an increased amount of N.P. and P.A. secretion. There are, however, reports i n l i t e r a t u r e that P.A. and the metalloproteinases show differences i n regulation. Golds et a l . (1983) report that synovial c e l l s responded to mononuclear c e l l supernatant by secreting P.A. which stopped when the stimulus was removed. Collagenase secretion occurred only a f t e r a lag of one to two days and the secretion continued even a f t e r the stimulus was removed. In addition, a l l - t r a n s r e t i n o i c a c i d caused an increase i n P.A. but a reduction i n latent collagenase. Gordon and Werb (1976) found that thioglycollate-induced macrophages reponded to colchocine by increasing the secretion of metalloproteinases but decreasing that of P.A. 88. My experiments show that under c e r t a i n conditions there are differences i n the pattern of N.P. and P.A. secretion. For example, i n day 3 cultures, c e l l population density modulated P.A. a c t i v i t y d i f f e r -ently than N.P. a c t i v i t y . N.P. a c t i v i t y decreased continuously with c e l l population density whereas P. A. a c t i v i t y was low at 1 x 10 5 c e l l s / d i s h , reached a peak at 2.4 x 10 5 c e l l s / d i s h and then decreased gradually. A possible explanation could be that E - c e l l s secreted i n h i b i t o r s to P.A. i n the early days of culture which reduced the a c t i v i t y of P.A. Figure 10 shows that the l e v e l of P.A. a c t i v i t y was lowest at day 3 compared to days 7 and 10. However, i n h i b i t o r s to P.A. have not been assayed and t h i s awaits further study. Another difference observed i s the d i f f e r e n t degree of stimulation of P.A. a c t i v i t y by the TCA extracted LPS compared to the phenol extract. The TCA extract caused a cumulative increase of 3 1/2 times i n P.A. a c t i v i t y at day 10 compared to an increase of 1 1/2 times by the phenol extract. In contrast, N.P. a c t i v i t y was stimulated to the same degree by both TCA and phenol extract. Thus, although the secretion of N.P. and P.A. appear to be linked together, there are d i f f e r e n c e s In the pattern of secretion. This indicates that the two enzymes may not be secreted together,in a p r o t e o l y t i c package, and they may respond to d i f f e r e n t s t i m u l i d i f f e r -e ntly. It has been shown that c e l l shape and E . C o l i LPS regulate proteinase secretion. The mechanism involved i n the LPS e f f e c t on proteinase secretion i s not known but i t Is probably d i f f e r e n t from the c e l l shape e f f e c t since no morphological change was observed. Thus there i s probably more than one mechanism that regulates proteinase secretion In E - c e l l s . 89. Nonetheless, since P.A. has been found to be able to a c t i v a t e latent metalloproteinases i n the presence of plasminogen (Werb et a l . , 1977; Paranjpe et a l , 1980), the secretion of P.A. i n - v i v o may be a means which ensures the a v a i l a b i l i t y of N.P. i n an active form. VI. Future Work Abnormal growth, changes i n c e l l morphology, and elevated l e v e l s of P.A. are c h a r a c t e r i s t i c s of transformed c e l l s . The r e l a t i o n s h i p of P.A. secretion to the other c h a r a c t e r i s t i c s altered i n transformed c e l l s i s not known. A long term goal would be to understand the r o l e of P.A. i n neoplasia and i n p a r t i c u l a r , the possible r e l a t i o n s h i p between c e l l morphology and P.A. i n neoplasia. These broad questions require extensive research, but based on the e x i s t i n g techniques and findings i n t h i s t h e s i s , two i n t e r e s t i n g and experimentally f e a s i b l e questions can be posed. One would be to elucidate the exact nature of the r e l a t i o n s h i p between c e l l shape and proteinase secretion. The second would be to determine whether the d i f f e r e n t methods of a l t e r i n g c e l l shape share s i m i l a r mechanisms. The d i r e c t i o n of future work to reach these goals w i l l be presented i n the following order: (1) Improvement i n techniques; (2) Proposed experiments. 1) Improvement i n Techniques F i r s t , a s e n s i t i v e method of measuring c e l l shape i s needed. One p o s s i b i l i t y would be to measure the area of the c e l l by using a camera lu c i d a to trace the c e l l outlines onto a d i g i t i z i n g tablet attached to a computer. This approach i s currently being developed i n Dr. Brunette's laboratory. If successful, c e l l shapes can be altered i n a graded sequence using the poly(HEMA) method of Folkman, and the c e l l 90. area measured and correlated with proteinase secretion. A second improvement needed i s a method which can increase the desired range of mechanical s t r e t c h i n g applied to the c e l l s . The petriperm membrane i s l i m i t e d i n the amount i n which i t can be rever-s i b l y stretched. The advantage of the mechanical stretching method i s that, whereas the poly(HEMA) method makes the c e l l s increasingly l e s s spread out than the c o n t r o l ( t i s s u e culture p l a s t i c ) , the mechanical stretching method can be used not only to make the c e l l s more fl a t t e n e d than the co n t r o l but also to make the c e l l s more rounded than the con t r o l . If the degree of stretching could be increased, i t would be possible to p l o t the amount of str e t c h i n g against proteinase s e c r e t i o n and a more complete picture of the re l a t i o n s h i p between c e l l shape and proteinase secretion obtained. One p o s s i b i l i t y i s the use of poly-urethane membranes. Preliminary experiments indicate the E - c e l l s attach to these membranes and that they can be re v e r s i b l y stretched to a greater extent than the petriperm membranes. However, a method has to be developed to f a b r i c a t e dishes with the polyurethane membrane attached. T h i r d l y , a d i f f i c u l t y enountered i n t h i s project has been the r e l a t i v e l y large numbers of c e l l s required for the experiments. For example, four 60 mm dishes of c e l l s , plated at 4 x 10 5 c e l l s / d i s h and incubated for 3 days i n 3MEM + 0.5% DFCS, are required for each experi-mental group i n order to obtain s u f f i c i e n t enzyme f o r assay. The experiments would be simpler i f the number of dishes of c e l l s could be reduced. Preliminary t e s t s i n d i c a t e that c e l l s i n serum free Dulbecco's medium produced 3 times more N.P. than c e l l s grown i n 8MEM + 0.5% DFCS. In the experiments reported here, 3MEM + 0.5% DFCS was used because i t was of i n t e r e s t to compare p r o l i f e r a t i n g c e l l s (induced by cholera toxin or B t 2 cAMP) with n o n - p r o l i f e r a t i n g c e l l s . Thus where 91. c e l l p r o l i f e r a t i o n i s not required, Dulbecco's medium could be a better a l t e r n a t i v e . 2) Proposed Experiments I t has been shown that c e l l shape modulates proteinase s e c r e t i o n but the exact nature of this r e l a t i o n s h i p i s not known. Therefore one of the short term goals would be to elucidate whether t h i s r e l a t i o n s h i p i s l i n e a r , that i s , whether a change i n c e l l shape on a quantitative scale would be accompanied by a proportional change i n magnitude i n the proteinase secretion. This could be done by a l t e r i n g c e l l shape i n a graded manner, by the use of poly(HEMA) coated dishes, combined with a s e n s i t i v e method of determining c e l l area. The c e l l shape and proteinase secretion could then be determined and the r e l a t i o n s h i p analyzed. A second goal would be to carry out some experiments to determine whether the mechanisms involved i n the d i f f e r e n t methods of a l t e r i n g c e l l shape are s i m i l a r . My r e s u l t s i n d i c a t e that N.P. and P.A. secre-t i o n are modulated by changes i n c e l l shape. But i n the experiments using agents l i k e cholera toxin, d i b u t y r y l cAMP and PMA, there i s the p o s s i b i l i t y that other mechanisms might be operative. To test the hypothesis, a standard curve of c e l l shape versus proteinase s e c r e t i o n would be obtained by the poly(HEMA) method and compared with other methods of a l t e r i n g c e l l shape. For example, PMA treated c e l l s would have t h e i r c e l l areas and proteinase a c t i v i t y measured and compared to the standard curve. If the amount of increase i n proteinase s e c r e t i o n accompanying the morphological change produced by PMA i s s u b s t a n t i a l l y d i f f e r e n t from the proteinase s e c r e t i o n of c e l l s showing an equal degree of morphological change using the poly(HEMA) technique, then the 2 mechanisms are probably at l e a s t p a r t i a l l y independent of each other. 92. SUMMARY AND CONCLUSIONS The regulation of proteinase secretion by E - c e l l s derived from the c e l l r e s t s of Malassez was studied i n c e l l culture system. 1) I t was established that E - c e l l s secrete neutral proteinase i n a latent form s i m i l a r to the neutral proteinase produced by rheuma-t o i d synovium, rabb i t bone and rabbit synovial f i b r o b l a s t s . P.A. was also secreted by E - c e l l s . 2) The secretion of N.P. and P.A. was regulated by c e l l population density i n the cult u r e system. Proteinase a c t i v i t y was highest at low c e l l population d e n s i t i e s . 3) E . c o l i LPS stimulated an increase i n N.P. and P.A. secretion. 4) In contrast to other studies reporting a p o s i t i v e c o r r e l a t i o n between p r o l i f e r a t i o n and proteinase secretion, a decrease i n proteinase secretion was observed when E - c e l l s were stimulated to grow by B t 2 cAMP or cholera toxin. Proteinase secretion i s thus not necessarily correlated with growth. 5) i ) C e l l shape was a l t e r e d by both chemical and ph y s i c a l means. Cholera toxin and d i b u t y r y l cAMP caused E - c e l l s to f l a t t e n out; while PMA caused E - c e l l s to r e t r a c t s l i g h t l y . P h ysical means included: mechanical stretching and relaxing stretched c e l l s ; growth of c e l l s on grooved substratum and growth of c e l l s on poly(HEMA)-coated dishes. 9 3 . 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