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Effects of an L-glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), on the growth patterns of two human… Lang, Lye Mooi 1984

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EFFECTS OF AN L-GLUTAMINE ANALOGUE, 6-DIAZ0-5-0X0-L-N0RLEUCINE (DON), ON THE GROWTH PATTERNS OF TWO HUMAN CERVICAL CARCINOMA CELL LINES by LYE MOOI LANG B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Anatomy) We accept t h i s t h e s i s as conforming t o ^ h e required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY, 1984 © L y e Mooi Lang, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 6 (3/81) ABSTRACT The objective of this study was to observe the effects of a potential chemotherapeutic drug and an inhibitor of glycosaminoglycan synthesis, 6-diazo-5-oxo-L-norleucine (DON), on the growth patterns of two human cervical carcinoma cell l ines . In part icular, the in vitro cell-shedding patterns, colony formation and aggregation of the cell lines were examined with DON treatment, to test the hypothesis that secretion of extracellular materials (ECM) by one cell l ine (C-4II) was responsible for i ts more d is -persed and in f i l t ra t i ve growth pattern. The cultured human cervical carcinoma cel ls used in this study were derived i n i t i a l l y from the same biopsy specimen. These two morphological-ly different cell l ines had dist inct ive patterns of growth both in vivo and in v i t ro . C-4I cel ls grew as round masses. In vivo, these cel ls did not in f i l t ra te into host hamster t issues, while jn_ v i t ro , few of these cel ls were shed from confluent cultures into culture medium. In contrast, C-4II cel ls did not grow as round cohesive masses, but in a dispersed man-ner. In vivo, these cel ls grew as small clumps and inf i l t rated into host t issues, while in v i t ro , such cultures shed many viable cel ls into culture medium. Previous ultrastructural analysis indicated that tumors with such dispersed growth patterns tend to have many features of secretory c e l l s , with characteristics of glandular and basal cell d i f ferent iat ion. In this study, an L-glutamine analogue, DON, was used to inhibit the secretion of ECM. DON has been shown to inhibit the formation of many glutamine-requiring metabolites in the c e l l , including glucosamine-6-phosphate, a metabolite essential for the formation of ECM. Hence, the i i effects of DON on the growth and shedding patterns of the 2 types of human cervical cancer cel ls were examined. This study showed that DON had numerous morphological effects on cultured C-4 c e l l s , other than its well-known growth-inhibitory effect . In addition, results showed that the shedding patterns of C-4 ce l l s were altered with DON treatment. By harvesting cel ls shed into culture medium, i t was determined that DON induced shedding in C-4I cultures while i t enhanced shedding in confluent C-4II cultures. This increased shedding was most l ikely caused by a decrease in c e l l - c e l l cohesion. Furthermore, the aggregation of C-4I c e l l s , treated with DON prior to dissociat ion, was greatly inhibited. Decreased c e l l - c e l l cohesion might also account for the decrease in s t rat i f icat ion of C-4I cultures, irregularly-shaped C-4I colonies and aggregates, and for the "holes" that appeared within both types of C-4 colonies treated with high doses of DON. In contrast, aggre-gation of similarly treated C-4II ce l ls was not consistently inhibited. Changes in colony forms, to more irregular shapes, were evident in both cell l ines . By examining single cel ls growing on the substratum, i t was determined that these changes might be due to DON decreasing ce l l - sub -stratum adhesion in both cell l ines . DON-treated C-4 ce l ls were signif icantly larger when projected two-dimensionally. This was due, at least in part, to increases in cell volume, as detected on the Coulter counter and size distribution analyzer. The results of this study do not support the previous hypothesis that the more dispersed and in f i l t ra t i ve growth pattern of C-4II tumors, as compared to C-4I tumors, was due to the secretion of ECM. This hypo-thesis predicted a decrease in the shedding of viable cel ls and a change i i i from a dispersed to a compact growth pattern in DON-treated cultures. Instead, the results indicate the contrary. The results suggest that cel ls may be secreting DON-sensitive cell surface-associated ECM that are responsible for the cohesive nature of C-4I c e l l s . C-4II cel ls may pro-duce less of this cohesive material, resulting in less cohesive cultures and more dispersed growth. Hence, with DON treatment, C-4I cultures were altered, resembling C-4II cultures, in terms of their shedding patterns, aggregabi1ity and morphology of aggregates and colonies. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES vi i i LIST OF FIGURES ix ACKNOWLEDGEMENTS xi INTRODUCTION 1 MATERIALS AND METHODS 8 I. Cell culture 8 II. Fixation and staining 9 III. Drug preparation and administration 9 IV. Stabi l i ty of DON at 37°C 10 V. Cell growth on plast ic surfaces 10 1. Dose-response 11 2. Colony form 11 3. Morphology of single adherent cel ls 12 4. Cell area 12 5. Cell volumue 13 VI. Shedding of cel ls into the culture medium 13 1. C-4I and non-confluent C-4II cultures 13 2. C-4II (confluent) cultures 14 VII. Ce l l - ce l l aggregation 14 1. Cells pretreated with DON prior to dissociation . . . . 14 2. Cells not pretreated with DON prior to dissociation . . 15 3. Reversal of the DON effect 16 RESULTS I. Variations in growth pattern of C-4 cel l lines 17 v Page. II. DON stabi l i ty 17 III. Dose response of C-4 cel ls 21 1. Growth inhibit ion 21 2. Morphology 22 3. Cel1-substratum adhesion 31 IV. Effects of 1 ug/ml of DON on the growth patterns of C-4I and C-4II l ines . Colony form 36 A. Cell shedding 40 B. Ce l l - ce l l aggregation 41 1. Aggregation of cel ls pretreated with DON prior to trypsin/EGTA dissociation 41 2. Aggregation of cel l treated with DON after trypsin/EGTA dissociation (Cells not pretreated with DON prior to dissociation) 48 3. Effects of glucosamine on aggregation of cel ls treated with DON after trypsin/EGTA dissociation . . . 56 V. Morphology of single adherent cel ls treated with 1 ug/ml DON 59 VI. Cell area 63 VII. Cell volume 66 VIII. Summary of the properties of control C-4 cel ls used in this study 69 IX. Summary of the effects of DON treatment on C-4I and C-4II ce l ls 70 DISCUSSION I. Control C-4I and C-4II cel ls used in this study 77 II. DON stabi l i ty 80 III. DON-induced changes in growth pattern of C-4 c e l l s , as represented by changes in colony form 80 IV. Effect of DON on cel l shedding 82 V. Cell aggregation 83 vi Page VI. Morphology of single adherent cel ls 88 VII. Effect of DON on projected surface areas of cel ls within colonies 89 VIII. Effect of DON on cell volume 90 IX. Relationship between colony form and s ize , cell s ize, and dose of DON 90 X. Conclusion 91 SUMMARY 93 BIBLIOGRAPHY 96 APPENDIX I. Change in variance in colony form ( x 10~3) 102 vi i LIST OF TABLES Table Page 1 Stabi l i ty of DON at 37°C (calculated from F ig . 5) . . . 20 2 Inhibition of cel l growth 24 3 Effect of DON on growth 25 4 C-4I: Change in form of colonies treated with 1 ug/ml of DON 38 5 C-4II: Change in form of colonies treated with 1 ug/ml of DON 39 6 Absolute and relative number and v iabi l i ty of shed cel ls per culture 42 7 C-4I: Aggregation of cel ls pretreated with DON . . . . 44 8 C-4II: Aggregation of cel ls pretreated with DON . . . . 46 9 C-4II: Aggregation of cel ls treated with DON after trypsin/EGTA dissociation 51 10 C-4I: Aggregation of cel ls treated with DON and/or GSA after trypsin/EGTA dissociation 54 11 C-4I: Effect of GSA on aggregation of cel ls treated with DON after trypsin/EGTA dissociation (% clumps of more than 20 u diameter) 55 12 C-4I: Change in cel l area 64 13 C-4II: Change in cell area 65 14 C-4I: Effect of DON on cell volume 67 15 C-4II: Effect of DON on cell volume 68 16 Summary of properties of control C-4I and C-4II cel ls 72 17 Summary of the effects of DON treatment on C-4I ce l l s 75 18 Summary of the effects of DON treatment on C-4II ce l ls 76 vi i i LIST OF FIGURES Figure Page 1 Structures of DON (an L-glutamine analogue) and L-glutamine 7 2 Metabolic pathway for synthesis of aminosugars . . . . 7 3 Metabolic pathway of the amide nitrogen of L-glutamine 7 4 Growth patterns of C-4 cel l l ines 18 5 Stabi l i ty of DON with time, at 37°C 19 6 Effects of DON on growth 23 7 Effects of varying concentrations of DON on C-4I cultures (day 4) 26 8 Effect of multiple doses of 1 ug/ml DON on C-4I cultures 27 9 Effects of varying concentrations of DON on C-4II cultures (day 8) 28 10, 11 Effects of multiple doses of 1 ug/ml DON on C-4II cultures 29-30 12 Morphologies of single cel l (indicative of cell-substratum interactions) 32 13 Effect of different concentrations of DON on the percentage of flattened cel ls 33 14 Effect of different concentrations of DON on the percentage of irregularly-shaped cel ls 34 15 Effect of different concentrations of DON on the percentage of spherical cel ls 35 16 C-4I: Aggregation of cel ls pretreated with DON (1 ug/ml) 45 17 C-4II: Aggregation of cel ls pretreated with DON (1 ug/ml) 47 ix Figure Page 18 C-4II: Aggregation of cel ls treated with DON after trypsin/EGTA dissociation 52 19 C-4II: Aggregation of cel ls treated with DON or DON plus GSA after trypsin/EGTA dissociation . . . . 53 20 C-4I: Aggregation of cel ls pretreated with DON after trypsin/EGTA dissociation 58 21-23 Effect of DON on the morphology of single cel ls . . . . 60-62 24-25 C-4I and C-4II clumps, hours after t rypsiniat ion, respectively 73-74 x ACKNOWLEDGEMENTS I wish to express my sincere appreciation to: Dr. N. Auersperg, Department of Anatomy, for her continual guidance and interest throughout this investigation. Drs. B. Crawford and L. Jasch, Department of Anatomy, for their interest and their advice. Dr. D. Brunette, Faculty of Dentistry, for the use of his Coulter counter, size distribution analyzer and recorder. The staff of the Biological Sciences Data Center, Dr. J . Petkau and Ms. H. Crapeau, Department of Mathematics, and Dr. J . Weinberg, Department of Anatomy, for their assistance in s tat is t ica l analysis. xi INTRODUCTION One of the major d i f f i c u l t i e s encountered i n cancer research i s that cancer i s not one dis e a s e , but ra t h e r , c o n s i s t s of numerous di s e a s e s . This unique heterogenous nature of tumors has unf o r t u n a t e l y complicated the treatment and prognosis of cancer p a t i e n t s . Since tumors may have d i f f e r e n t subpopulations of c e l l s with d i f f e r e n t growth and invasion pat-terns and with d i f f e r e n t m etastatic p o t e n t i a l s ( F i d l e r and Hart, 1981; Marx, 1982), any one treatment may not be e f f e c t i v e i n combating the tumorous growth and metastases (Marx, 1982). Tumor heterogeneity has been observed i n poorly d i f f e r e n t i a t e d human c e r v i c a l i n v a s i v e squamous carcinomas, which d i s p l a y a v a r i e t y of i n v i v o and i n v i t r o patterns of growth and i n v a s i o n , and d i f f e r e n t i a t i o n (Auer-sperg and Worth, 1966; Auersperg et a l _ . , 1973). I t has been shown t h a t even though such tumors are h i s t o l o g i c a l l y c l a s s i f i e d as being poorly d i f f e r e n t i a t e d , they s t i l l r e t a i n some u l t r a s t r u c t u r a l t r a i t s of normal d i f f e r e n t i a t i o n , which may in f l u e n c e t h e i r growth p a t t e r n s . For example, tumors with spinous t r a i t s , such as glycogen accumulations, t o n o f i b r i l s and desmosomes, have a compact growth p a t t e r n , c e n t r a l areas of necr o s i s and l e u c o c y t i c responses. Tumors with glandular or basal t r a i t s , such as secretory v e s i c l e s , Golgi complexes, or basement membranes, have a d i f f u s e growth p a t t e r n , or an i n f i l t r a t i v e method of spreading ( i n small groups or strands of c e l l s ) , no ce n t r a l necrosis and extensive i n v a s i o n of the host connective t i s s u e s (Auersperg et a l _ . , 1973). Not only are tumors heterogenous i n t h e i r t r a i t s of d i f f e r e n t i a t i o n and i n t h e i r growth patterns i n v i v o , but they are a l s o heterogenous i n - 1 -- 2 -their growth patterns in v i t ro . A correlation study by Auersperg and Erber (1976) has shown that cervical carcinomas with spinous t ra i ts of differentiation grow cohesively in vivo as well as in v i t ro . Most carc in-omas with basal or glandular t ra i ts have a dispersed growth pattern in  vivo. However, they grow more variably in culture, producing completely and part ia l ly dispersed as well as cohesive cultures. Differentiated cultures of human invasive squamous cervical carcinomas are derived mainly from differentiated tumors, while poorly differentiated tumors produce mainly poorly differentiated cultures (Auersperg and Worth, 1966). Tumor cel l properties that may influence the spread of cancer ce l l s are moti l i ty , and adhesion to other tumor cel ls and to host t issues. In addition, tumor cel ls interact with extracellular products of tumor or host origin (Skyvova et al_., 1973; Toole et aj_., 1979; Roblin, 1981). Speci f ica l ly , previous studies of carcinomas of the uterine cervix (Auersperg, 1969a, 1969b; Auersperg et al_., 1973; Auersperg and Erber, 1976) have shown that: 1) cultured cel ls which prefered to adhere to one another, rather than to substrata, grew as compact masses in vivo and in v i t ro . However, cultured cel ls that adhered preferentially to substrata grew more di f fusely. 2) tumors that in f i l t rated into host tissues were associated with more basement membranes and amorphous extracellular materials than non-i n f i l t r a t i v e ones. 3) tumor cel ls that grew diffusely in vivo, tended to shed more viable cel ls into culture medium. This shedding of cel ls was accompanied by an increase in intercel lu lar spaces. It was postulated that extracellular materials or secretory products of - 3 -tumor cel ls may aid in c e l l - c e l l separation and hence, in tumor cell invasiveness as well as in contributing to a diffuse growth pattern. The above hypothesis was tested in the present study. Two human cervical carcinoma cel l l ines were used to represent two different types of poorly differentiated cervical carcinomas (Auersperg, 1969a, 1969b). The significance of using these two cell l ines as a model was that certain in vivo tumor cell properties were reproduced in v i t ro . Even though both cel l l ines were different in their in vivo and in  vitro growth patterns and invasive patterns, metabolism and ultrastruc-tural features, they originated from the same tumor biopsy. The C-4I cel l l ine represents tumors that are histological ly cohe-sive. These cel ls have retained some tra i ts of spinous d i f ferent iat ion— cel ls strat i fy and flatten apical ly , have a lower nuclear/cytoplasmic ratio than basal c e l l s , lack organelle polar i ty , have abundant tonof ibr i ls and tonofilament-associated desmosomes, wide intercel lu lar spaces and glycogen granules. C-4I cel ls lack junctional complexes and, in vivo, have fewer areas with basement membranes than C-4II c e l l s . In culture, C-4I cel ls grow as cohesive round masses and tend to overcome crowding by st rat i fy ing . In vivo (in hamster cheek pouches), the growth pattern correlates with that seen in v i t ro , that i s , the tumors are cohesive, compact and have necrotic centers. The C-4II cel l l ine represents tumors with a diffuse growth pattern. The cel ls in culture are basically monolayered, less cohesive, and form irregularly-shaped colonies. C-4II cel ls have retained some tra i ts of basal cel l d i f ferent iat ion, such as a high nuclear/cytoplasmic rat io , organelle polar i ty , and secretory vesicles. Unlike C-4I c e l l s , C-4II ce l ls have few desmosomes, more complete and extensive basement membranes, - 4 -no glycogen, narrower intercel lu lar spaces and have retained the capacity to form tight junctions and junctional complexes in an in vitro l iquid environment. As C-4II cel l cultures get crowded, flattened ce l ls become more columnar, intercel lu lar spaces widen, viable cel ls are shed into the culture medium, and domes form (which indicate secretory act i v i t y ) . Based on morphological studies of single subcultured cel ls and on adhesion studies of cultured colonies, C-4II cel ls showed preferential ce l l - sub -stratum adhesion relative to c e l l - c e l l adhesion, in contrast to C-4I c e l l s . The i n i t i a l aim of this study was to determine whether secreted extra-cel lu lar materials aid in cel l separation and dispersion, and whether they affect growth patterns of tumor cel ls in culture. Since 6-diazo-5-oxo-L-norleucine (DON) has been used in the past to inhibit glycoprotein, glycol ipid and glycosaminoglycan (GAG) synthesis (Pratt et_ al_., 1973; Spooner and Conrad, 1975; Greene and Pratt, 1977; Hurmerinta et_ a]_., 1979; Linsenmayer and Kochhar, 1979; Turley, 1980; Funderberg and Markwald, 1981; Hurmerinta and Thesleff , 1982), DON was used in this present study to determine i ts effects on the growth patterns of C-4I and C-4II cultures. DON (Fig. 1), being a glutamine analog, competes with L-glutamine in many L-glutamine requiring reactions in the c e l l , for example, i t inhibits the enzyme L-gl utamine-D-fructose-6-phosphate transaminase which forms glucosamine-6-phospate (Ghosh et_ al_., 1960), an essential precursor of glycoproteins, glycolipids and GAGs (Fig. 2). Examples of glucosamine-containing macromolecules are fibronectin and hyaluronic acid, both of which affect adhesion (Yamada and Olden, 1978; Mikuni-Takagaki and Toole, 1980; Culp, 1980; Mosher and Furcht, 1981). Pratt et a l . , (1973) have shown that DON-treated palates from - 5 -developing rats at the time of fusion produced less GAGs than untreated palates. In addition, Greene and Pratt (1977) have shown that not only did DON inhibit glycoprotein, glycol ipid and GAG synthesis during rat palate formation, i t also speci f ica l ly inhibited the adhesion of cultured palatal shelves. This inhibit ion was counteracted by glucosamine addi-t ion . Therefore, DON could possibly affect adhesion in other systems by similar means. Other researchers, such as Ekblom ejt al_. (1979), Turley (1980) and Hurmerinta et_ a]_. (1979) have found that DON inhibited the synthesis of extracellular matrix as well as the in vitro differentiation of kidney tubules from mouse metanephric mesenchyme, adrenocortical cel ls and mouse embryonic tooth germs respectively. Ekblom et a\_. and Turley, but not Humerinta et al_., could part ial ly reverse DON's effects by the addition of glucosamine. Funderburg and Markwald (1981) and Turley (1980) found that DON also inhibited the motil ity of cultured heart cel ls and adrenocortical cel ls respectively. These effects were again only part ial ly counteracted by glucosamine. Spooner and Conrad (1975) found that the motil ity of heart ce l ls in vitro was not inhibited by DON treatment even though the synthesis of GAGs was decreased. Even though the addition of glucosamine to DON-treated cel ls only part ia l ly increased GAG synthesis, i t had no effect on cell moti l i ty . Not only does DON inhibit the synthesis of extracellular matrix, i t also inhibits the synthesis of other L-glutamine-requiring macromolecules, for example, nucleic acids (DNA and RNA), proteins, and nicotinamide adenine dinucleotide (NAD) (Duvall, 1960) (Fig. 3). It also inhibits asparagine synthetase act iv i ty (Hiremagalur ejt aj_., 1976; Rosenbluth et - 6 -a l . , 1976), and competes with L-asparagine and L-glutamine uptake into ce l ls (Cooney ejt al_., 1976). Since DON was f i r s t discovered and isolated from an unidentified Streptomyces culture in 1956 (Ehrlich e_t al_., 1956), i ts properties have been characterized (Duvall, 1960; P i t t i l l o and Hunt, 1967; Livingstone et al_., 1970; Rando, 1975; Cabanil las, 1979; Catane et a U , 1979). DON has been found to have some ant ibacter ia l , antifungal, teratogenic and anti -neoplastic ef fects . In many earl ier t r i a l s , i t was found that the effectiveness of DON in decreasing tumor size was only transient and occured only in a small proportion of cases studied. However, i ts anti -neoplastic effects are being re-investigated (Burchenal, 1979; Overjera, 1979; Rosenfeld and Roberts, 1981). Past and recent research has shown that DON has limited but definite anti-tumor act iv i ty in certain human cancers and in certain human tumor xenografts, implanted in nude mice (Overjera, 1979). Figure 1: Structures of DON (an L-glutamine analogue) and L-glutamine. 0 COOH N=N=CHC-CH2-CH2-CH JlH2 DON 0 COOH H 2N-C-CH 2-CH 2-CH L2 L-GLUTAMINE Figure 2: Metabolic pathway for synthesis of aminosugars. (McGuire, 1972, p. 362) Fructose-6-phosphate + L-glutamine L-glutami ne-fructose-6-phospate transamidase Glucosamine-6-phosphate + L-glutamic acid Glucosami ne-6-phosphate -> UDP-GlcNAC -> Glycol i pi ds, Glycoproteins, Glycosaminoglycans Figure 3: Metabolic pathway of the amide nitrogen of L-glutamine L-glutamine — -> other amino-acids (e.g. asparagine, h ist id ine, tryptophan) -> glucosamine-6-phosphate -> nucleic acids -> nicotinamide dinucleotide - 8 -MATERIALS AND METHODS I. Cell culture C-4I (passage 84) and C-4II (passage 97) cel ls were grown from frozen stocks, stored in l iquid nitrogen. Stock cultures were maintained in 25 cm^ tissue culture flasks (Corning), with 5 ml of Waymouth's medium (MB 752/1), supplemented with 10% fetal bovine serum, 100 U/ml of p e n i c i l l i n , and 100 ug/ml of streptomycin. The cel ls were incubated in a dry incubator at 37°C. Medium of C-4I cultures was changed almost every day, while medium of C-4II cultures was changed every 2-3 days, since the rate of lact ic acid accumulation was higher in C-4I than in C-4II cultures (Auersperg, 1972). Confluent C-4I and C-4II cultures were subcultured every 1-2 weeks, using 0.125% trypsin in C a + + , Mg + + - f ree Hanks' balanced salt solution (HBSS). Cells were centrifuged at 500-1000 rpm for 3-5 minutes and the cell pellet was taken up in 5 ml of culture medium. 20-25% of this cel l volume in 5 ml of fresh culture medium was replated into a new f lask. As C-4I cel ls were less sensitive to this form of dissociation than C-4II c e l l s , C-4I ce l ls were subcultured mainly as small to medium-sized clumps, while C-4II cel ls were subcultured mainly as single cel ls and small clumps. If dispersion into single cel ls was required, cultures were d is -sociated with 0.125% trypsin and 0.02% ethyleneglycol tetraacetic acid (EGTA) in C a + + , Mg + + - f ree HBSS, since i t was previously shown in C-4 cultures that c e l l - c e l l contacts were EGTA (Ca + + - chelator) sensit ive, while eel 1-substratum contacts were trypsin sensitive (Auersperg, 1969b; Dembitzer et a l . , 1980a). - 9 -II. Fixation and staining Cultures were fixed with 95% ethanol, after rinsing twice with C a + + , Mg + + - f ree HBSS or with Waymouth's medium. After gradual rehydration, cultures were stained with 2% toluidine blue (aqueous). III. Drug preparation and administration DON was supplied as a dry powder (Calbiochem., C a l i f . ) , and prepared in 0.9% sal ine. Stock solutions of 0.001 and 0.0001 g/ml were f i l t e r -s ter i l i zed and stored frozen (with dessicant and in the dark). Final dilutions were made from aliquots of stock solutions, and kept at 4°C for up to 2 weeks. In experiments, addition of 20 ul of DON stock solutions (0.001 and 0.0001 g/ml) to 2 mis of culture medium produced final concentrations of 10 ug DON/ml and 1 ug DON/ml of medium respective-ly . For convenience, such terms as "ug DON/ml medium" will be abbreviated "ug/ml DON". In a l l experiments, equal volumes of 0.9% saline were given to controls. Glucosamine hydrochloride (GSA) (Sigma) was also dissolved in 0.9% saline and f i l t e r - s t e r i l i z e d . It was prepared immediately prior to t reat -ment. Concentrations of GSA used were 10, 50, 100 and 200 ug/ml. In al l experiments, the time of subculture (cell dissociation and plating) was defined as day 0. In dose response experiments (page 11), the range of DON concentrations used was from 0.5 to 10 ug/ml. Subse-quently, a DON concentration of 1 ug/ml, which did not appear to be too toxic to C-4 c e l l s , was used in al l further experiments. Single (added either on day 0, 1, or 2), double (added on days 1 and 2) and t r ip le (added on days 1, 2 and 3) doses of 1 ug/ml DON were used. It was i n i t i a l l y observed that adding a single dose one day after plating (or - 10 -day 1) produced l i t t l e v is ible changes in cel l growth and in culture morphology on day 2. However, i f a second dose was added on day 2, clear changes in morphology could be observed in the treated cultures on day 3. Adding a third dose on day 3 produced even more evident results. Single doses of 1 ug/ml were added at different times (days 0, 1, or 2) to examine effects of different exposure times to a single dose of the drug. IV. Stabi l i ty of DON at 37°C 1 ug/ml DON solutions in phosphate buffer (pH 7) (Fisher Sc ient i f ic Co.) were incubated at 37°C in a humidified incubator (5% C02/air) for up to 11 days. The purpose of this experiment was to determine the stabi l i ty of DON at 37°C and to compute the concentrations of DON in cultures given single, double or t r ip le doses of 1 ug/ml DON. In the f i r s t experiment, duplicate samples of DON solutions were frozen on days 1 to 4, 6, and 8 to 11. At the end of 11 days, thawed solutions were measured for DON stab i l i t y by UV absorption at 274 nm (Dion et aj_., 1956) (Gilford spectrophotometer). In the second experiment, duplicate samples of DON were not frozen, but were measured on each of the above-mentioned days. V. Cell growth on plast ic surfaces After centrifugation, subcultured cel ls were suspended in 5 ml of medium. In C-4I experiments, this volume of 5 ml was diluted with 30 to 50 ml of medium. In C-4II experiments, the 5 ml of cel ls in medium were - 11 -diluted with a smaller volume of medium (20 to 30 ml), since confluent, monolayered C-4II cultures reached a lower population density than confluent, highly s t rat i f ied C-4I cultures. Two ml of cel ls and medium were dispensed into each tissue culture dish (35 mm, NUNC). Dishes were incubated in a humidified incubator at 37°C in 5% C02/air. DON treatments were given at various times as described in section III. 1. Dose-response Cultures were grown with 0.5, 1, 5, and 10 ug/ml DON for 4 to 12 days. Fixed cultures were examined visually for any differences in amounts of growth between DON-treated and control cultures. A scale of " - " to "+++" was used, where " - " signif ied no growth inh ib i t ion , "+" slight but definite inhib i t ion, "++" marked inh ib i t ion , "+++" extreme inhibit ion ( l i t t l e growth), and "+_" borderline inh ib i t ion . Cultures were also examined microscopically for changes in cel l areas, colony forms, numbers and morphology of single c e l l s , amount of cel l debris, cel l density, s t ra t i f i ca t ion , nuclear/cytoplasmic rat ios, and intercel lu lar contacts with increasing DON concentrations. 2. Colony form Colonies, in fixed and stained cultures, were projected from an inverted Wild microscope onto the magnetic tablet of a Zeiss digi tal image analyzer, MOP 3, by use of a camera lucida. Colony forms were quantitated to determine changes in growth pattern with DON treatment. Calculations of "form": This parameter was measured by MOP 3, which assigned a value of "1" for a perfectly round c i r c l e . Hence, - 12 -any irregular outline had a value of less than 1. Decreases in assigned value were proportional to increases in i r regular i ty of colony forms. Forms of small colonies (more than 5 but less than 35 cel ls ) and of large colonies (more than 50 ce l l s ) were determined separately. The data were analyzed by computer (Biological Sciences Data Center) using 1 way analysis of variance (ANOVA) and by using Newman-Keuls test , at a probability leve l , p, of less than 0.05. 3. Morphology of single adherent cel ls In order to determine DON effects on eel 1-substratum adhesion (independently of c e l l - c e l l adhesion), the morphology of single cel ls was examined. Single cel ls adherent to plast ic in fixed cultures were c lass i f ied as being (i) f lattened, ( i i ) irregularly-shaped, or ( i i i ) spherical. 4. Cell area The average projected area of individual cel ls was determined in small (5-35 cel ls ) and large (more than 50 cel ls ) colonies, since i t appeared that DON-treated cel ls in large colonies were larger than in control colonies and in small DON-treated colonies. In small colonies, the average cell area was derived at by d iv id -ing the total colony area (measured by MOP) by the number of cel ls in that colony. The latter was determined by counting the number of nuclei since i t was d i f f i cu l t distinguishing individual c e l l s . Some large colonies were extremely s t rat i f ied at the colony rims (edges). Hence, the average cell area was derived at by dividing the - 13 -area of the center part of the colony, by the number of cel ls present in the colony center. Results were analyzed s ta t i s t i ca l l y using analysis of variance (ANOVA) test and Newman-Keuls test , at a probability level of less than 0.05. 5. Cell volume To determine whether an observed effect of DON, the increase in cell area, was due to an increase in cell volume, cel ls were sized on a Coulter counter. Cultures were dissociated into single c e l l s , as described. In addition, cel ls were vigorously syringed with a 21 gauge needle. After centrifugation, cel ls were resuspended in 20 ml of isoton and sized on a Coulter counter. For a diameter range of 10-20 um, ampli-fication=8, current=2, the number of cel ls sized was up to 1024/ channel; and for a range of 20-40 um, amplification=8, current=32, the number of cel ls sized was up to 64/channel. VI. Shedding of ce l ls into the culture medium 1. C-4I and non-confluent C-4II cultures Cells were plated into 6 f lasks. On day 1, cel ls growing in 2 flasks were harvested by trypsin/EGTA treatment and counted with a hemocytometer, to determine the number of adherent cel ls per flask at the time of drug addition. The number and v iab i l i t y (by exclusion of 0.4% eosin) of cel ls shed were also determined. Also on day 1, 1 ug/ml DON or 0.9% saline was added to f lasks. On days 2, 4, and 6, - 14 -medium was collected from the f lasks, and numbers and v iab i l i ty of shed cel ls were determined. Cultures were fed new medium with DON or saline on days 2 and 4. On day 6, al l cel ls adhering to the flasks were counted. Two and three separate experiments were completed for C-4I and C-4II cultures respectively. 2. C-4II (confluent) cultures Cultures were grown until they were almost confluent, at which time (days 6 and 11 after subculture in 2 experiments) 1 ug/ml DON or 0.9% saline was added. On days 2, 4, and 6 after the day of i n i t i a l DON addition, the cultures were treated as described above (Section VI.1). The reason for examining confluent C-4II cultures separately from non-confluent ones was that the shedding pattern of confluent and non-confluent C-4II cel ls di f fers (Auersperg, 1969a). VII. Ce l l - ce l l aggregation Ce l l - ce l l adhesion was examined in gyratory shaker culture. Two series of experiments were carried out: in one, the cel ls were pre-treated with DON prior to dissociation and transferred to shaker cu l -ture; in the other, they were not pretreated. 1. Cells pretreated with DON prior to dissociation C-4I cultures and C-4II cultures were grown in f lasks , as described in section I, for 6 days. DON was added on days 1 or 5 (5 day and 1 day pretreatment respectively). On day 6, the cultures were - 15 -dissociated into single ce l l s . Five ml of cel ls and medium (0.9X10^ cells/ml) were added to 50 ml Erlenmeyer f lasks, which were incubated at 37°C for up to 2 days on a gyratory shaker (New Brunswick Labora-tory Rotator model G2), rotating at 70 rpm (revolutions per minute). At the end of the experiments the cel ls were fixed with formol-saline, and c lass i f ied into single cel ls or clumps. The aggregation of cel ls not pretreated with DON was also ex-amined, to determine whether pretreatment for 1 and 5 day periods could have caused cel l damage, thus inhibit ing cel l aggregation. 2. Cells not pretreated with DON prior to dissociation The following is a flow diagram of the procedure used: rapid size slow size confluent dissociate single cel ls shaking cel l shaking cel l cultures > in flasks > clumps >clumps (200 rpm) (70 rpm) for 24 hours 24 hours Drugs were added during this period Confluent cultures were dissociated into single cel ls as described. Five ml of medium (450,000 cells/ml) were dispensed into 50 ml Erlenmeyer f lasks , which were incubated at 37°C for 24 hours on a gyratory shaker, shaking at 200 rpm. The purpose of this rapid shaking was to allow for recovery of trypsinized cel l surface com-ponents while minimizing c e l l - c e l l aggregation (Takeichi, 1977). DON was added at various times during this period of rapid shaking, that i s , 0, 6, 12, 18 and 24 hours after subculture. This period of rapid shaking was followed by another 24 hour period of shaking at 70 rpm. The purpose of the slow period was to allow aggregation to occur, after repair of trypsinized cell surface components. Live clumps were - 16 -sized at (i) the end of 24 hours at 200 rpm and ( i i ) the end of 24 hours at 70 rpm (or a total of 48 hours of incubation), on a 100 u aperture Coulter counter (model Zf ) , P54 size distribution analyzer, and an xy recorder (number 4) (Coulter Electronics, Hialeah, F lor -ida) . In addition, large C-4I clumps were sized on an Artek counter (model 980, Artek Systems, N.Y.) after being fixed (2% glutaraldehyde in Mil lonig's buffer) and stained (0.4% eosin). 3. Reversal of the DON effect GSA (10, 50, 100 and 200 ug/ml) was added simultaneously with DON, during aggregation experiments in an attempt to reverse the effect of DON. - 17 -RESULTS I. Variations in growth pattern of C-4 cel l l ines Treatment of C-4 cultures with trypsin, an endopeptidase, produced suspensions that consisted mainly of clumps. When such cell suspensions were plated, C-4I colonies that grew were round while C-4II colonies were irregular (Figs. 4a and 4b). In the course of this study, i t was observed that the growth pattern of C-4I cel ls could be altered by the combined use of trypsin and EGTA. Such dissociation treatment produced suspensions that consisted mainly of single c e l l s . When these suspensions were plated, C-4I and C-4II colonies were both irregularly-shaped (Figs. 4c and 4d). These observations indicated that the growth pattern of C-4I ce l l s depended on the maintenance of c e l l - c e l l contacts, in contrast to that of C-4II c e l l s . C-4II cel ls grew relatively independently of their c e l l - c e l l contacts. II. DON Stabi l i ty UV absorption of DON, at 274 mm, is thought to be proportional to i t s biological activity (Dion et^  aj_., 1956). After an i n i t i a l 24 hours of incubation at 37°C, the UV absorption of 1 ug/ml of DON in pH 7.0 phosphate buffer decreased by 26-42% (average of 34%) (Fig. 5). Storage of frozen samples for up to 10 days did not change the results. These results were consistent with these of Dion et a l . , (1956), who reported a decrease in stabi l i ty of 32 ug/ml DON of about 30% at 30°C after 18 hours. From days 2 to 7, there was l i t t l e change in UV absorption. However, from days 8 to 11, there was a slight increase of 0.05 to 0.10 ug/ml of DON (Fig. 5). - 18 -C-4I C-4II Figs. 4 a -d . Growth patterns of C-4 cel l l i nes . Cultures were stained with 4% aqueous toluidine blue, x 90. Figs. 4a and 4b. C-4I and C-4II cultures, respectively. These cultures were grown from large clumps. Figs. 4c and 4d. C-4I and C-4II cultures, respectively. These cultures were grown from single c e l l s . Figure 5 STABILITY OF DON WITH TIME, AT 37°C a DON CONCENTRATION (ug/ml) 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 2 S o l 10 Key: 11 DAYS OF -> INCUBATION AT 37°C a I • • As determined by UV absorption of 1 ug/ml DON (diazoketones) at 274 nm. Range of values Expt 1: Measured aliquots each day. Expt 2: Froze aliquots each day. Measurements determined on last day of experiment. - 20 -Table 1 STABILITY OF DON AT 37°Ca (calculated from F ig . 5) TREATMENT*3 DON CONCENTRATIONS (ug/ml ) c day 4 day 7 day 11 Expt #1 single dose 0.62 0.56 0.62 double dose 1.21 1.10 1.24 t r ip le dose 1.78 1.65 1.84 Expt #2 single dose 0.59 0.57 0.59 double dose 1.24 1.12 1.33 t r ip le dose 1.98 1.67 1.93 aComputed from F ig . 5, where duplicate samples of 1 ug/ml DON were incubated in phosphate buffer, pH 7.0, at 37°C. ^Number of doses of 1 ug/ml DON as used in cultures: a single dose would be added on day 1, a double dose on days 1 and 2, and a t r ip le dose on days 1, 2, and 3. The calculations are based on DON samples measured each day in expt #1 or at the end of the expt (day 11) in expt #2 (see F ig . 5). cComputed DON concentrations in cultures of various ages. - 21 -Therefore, i t was assumed throughout this study that double doses of 1 ug/ml of DON added to cultures on the f i r s t and second days after sub-culture were equivalent to approximately 1.2 ug/ml on day 4 and 1.1 ug/ml on day 7 (Table 1). If t r ip le doses of DON were given, concentrations of DON were assumed to be equivalent to approximately 1.9 ug/ml on day 4 and to 1.7 ug/ml on day 7. III. Dose response of C-4 cel ls To find an effective but relatively nontoxic dose of DON, the follow-ing 3 sets of experiments were carried out. 1. Growth inhibit ion In both cel l l ines , there was increased growth inhibit ion with increasing concentrations of DON (Figs. 6a and 6b). In C-4II cultures, no inhibit ion by 0.5 ug/ml DON was visually observed. This dose was not tested in l ine C-4I (Table 2). Inhibition of cell growth using a single dose of 1 ug/ml DON was visually observed in only a minority of cultures (Fig. 6, Table 2). Inhibition by multiple doses of 1 ug/ml DON was greater than by single doses. Inhibition by 5 and 10 ug/ml was clearly evident in both cel l l ines (Fig. 6, Table 2). Even though l i t t l e inhibit ion was observed visually with 1 ug/ml DON treatment (Table 2), actual cel l counts showed that there was growth inhibi t ion. This growth inhibition was proportional to the number of doses of DON applied (Table 3). Thus, DON, a potential anti-tumor drug, decreased cell growth even at a dose of 1 ug/ml. The discrepancy between visual observa-tions and cell counts could be due to DON decreasing st rat i f icat ion of - 22 -cultures, and/or increasing cel l surface area so that there was a lower number of cel ls covering the same amount of substratum. Both of these changes were introduced by DON, as shown below. 2. Morphology Changes in morphology were observed with increasing concentrations of DON. A dose of choice for subsequent experiments was chosen which had minimal effect on growth and yet produced some morphological ef fects , which could be quantitated. In both cel l l ines , DON-treated cel ls showed the following changes: i ) increase in projected cel l surface area, increase in flatness of cel ls and decreased cell density (Figs. 7, 8, 9, 10, 11), i i ) increased irregularity in colony form with higher DON concentrations (Figs. 7, 8, 9, 11), i i i ) increase in numbers of single cel ls which were spherical rather than f lat (Fig. 11), iv) increased cel l debris in the culture medium, v) decreased nuclear/cytoplasmic ratios (Figs. 7, 8, 9, 10, 11), vi) decreased s t rat i f i cat ion in C-4I cultures, with l i t t l e nuclear overlap (Fig. 7, 8), and v i i ) decreased c e l l - c e l l contacts, with appearance of "holes" within colonies (Fig. 8). In certain areas, cel ls pulled away from each other (Figs. 7, 8, 9). - 23 -Figs. 6a-b. Effects of DON on growth. Ethanol f ixat ion, toluidine blue staining, x l . F ig . 6a. C-4I culture (11 day). DON was added to cultures 1 day after plating. F ig . 6b. C-4II culture (12 day). DON was added to cultures at time of plating. - 24 -Table 2 INHIBITION OF CELL GROWTH3 C-4I dose in ug/ml age of cultures (days) no. of dishes (No. of treatments) 4-5 6-9 1 (S.D.b) _ (+ 4/14) _ 2 7 1 (M.D.C) - ,+ + 13 5 (S.D. & M.D.) + + 8 10 (S.D. & M.D.) + ++ 10 Total = 58 C-4II dose in ug/ml age of cultures (days) no. of dishes (No. of treatments) 4-5 6-8 9-12 0.5 (S.D. & M.D.) - 8 1 (S.D.) + (- 8/ 2 7) - - 51 1 (M.D.) + (+ 4/16) + (+ 7/17) + 34 5 (S.D. & M.D.) +,++ +,++ +,++ 12 10 (S.D. & M.D.) ++ +++ +,++ 11 Total = 116 Key: a Dishes were examined visually for growth. C-4I: based on 8 expts. C-4II: based on 17 expts. b Single dose of DON given at various days (0, 1, and 2) after plating. c Multiple doses of DON. Double doses were given on days 1 and 2. Triple doses were given on days 1, 2 and 3. Scale of inh ib i t ion : - . . . n o inhibit ion . .borderl ine +.. .defi nite ++...marked +++...extreme ( l i t t l e growth) Superscripts indicate the number of cultures showing the various degrees of inhibit ion over the total number of cultures. o - 25 -Table 3 EFFECT OF DON ON GROWTH % GROWTH INHIBITION WITH DON TREATMENT5 CELL LINE DON TREATMENT3 EXPT l c EXPT 2 d C-4I single dose double dose 70.9 (68.4, 73.3) 48.8 (28.6, 69.0) 76.2 (64.3, 88.1) C-4II single dose (non-confluent) double dose 57.9 (55.6, 78.7, 39.4) 41.9 (40.4, 43.4) 73.8 (80.8, 66.7) C-4II (confluent) single dose 26 — Key: a Single dose of 1 ug/ml DON was given on day 1. Double dose of 1 ug/ml DON was given on days 1 and 2. D Values are the average of al l experiments; values in brackets are the means of each experiment. c Based on 2 C-4I and 3 C-4II experiments. Final cel l number was determined on 6 day old duplicate cultures, except for confluent C-4II cultures which were day 17 cultures. d Based on 2 separate experiments for each cell l ine . Final cel l number was determined on 4 day old duplicate cultures. - 26 -I F i g s . 7a-c. E f f e c t s of v a r y i n g concen- t r a t i o n s of DON on C-4I"  c u l t u r e s (day 4f~. DON was day a f t e r p l a t i n g , f i x a t i o n , t o l u i d i n e x 55. added 1 Ethanol blue s t a i n i n g , • F i g . 7a. Control c o l o n i e s . Note: Round c o l o n i e s , with uniform c e l l areas. F i g . 7b. Colonies t r e a t e d with 1 ug/ml DON. Note: Small colony with small c e l l s (small arrowhead). Large colony with small s t r a t i f i e d c e l l s at the rim (l a r g e arrowhead) and large f l a t c e l l s i n the c e n t e r , with a low n u c l e a r / c y t o -plasmic r a t i o and decreased nuclear overlap. F i g . 7c. Colonies t r e a t e d with 5 ug/ml DON. Note: I r r e g u l a r l y - s h a p e d c o l o n i e s and the presence of s p i n d l e -shaped c e l l s (arrowheads). - 27 -> % M f $ ^ F 1 g . 8a. te#s'"€fc 1 * ^ 1 Note: Ev F i g s . 8a-c. E f f e c t of m u l t i p l e doses of  1 ug/ml DON on C-4I c u l - t u r e s . 7 day o l d c u l t u r e s . Ethanol f i x a t i o n , t o l u i d i n e blue s t a i n i n g , x 5 5 . Control c o l o n i e s . Evident nuclear over-7f^^^hv'.^  "j lap and s t r a t i f i c a t i o n F i g . 8b. Colonies t r e a t e d with double doses of 1 ug/ml DON (added days 1 and 2 ) . Note: Evident decreased nuclear o v e r l a p , decreased s t r a t i f i c a t i o n , decreased nuclear/cytoplasmic r a t i o , and increased colony f l a t n e s s . F i g . 8c. Colonies t r e a t e d ^ w i t h t r i p l e doses of 1 ug/ml O p DON (added days 1, 2 and 3 ) . Note: I r r e g u l a r l y -shaped c o l o n i e s with holes, l i n e d by r e t r a c t e d edges (arrowhead). - 28 -F i g s . 9a-c. E f f e c t s of vary- ing concentrations of DON on  C-4II c u l t u r e s (day~8"7^ DUN was added 1 day a f t e r p l a t -i n g . Ethanol f i x a t i o n , t o l u i -dine blue s t a i n i n g , x 55. F i g s . 9a. Control c o l o n i e s . Note: I r r e g u l a r l y - s h a p e d c o l -o n i e s , with pavement-like (columnar) c e l l s t h a t have a high nuclear/cytoplasmic r a -t i o . F i g . 9b. Colonies t r e a t e d with 5 ug/ml DON. Note: Irregular-shaped, but f l a t t e r - l o o k i n g c o l o n i e s . C e l l s are l e s s pavement-like ( l e s s columnar) and have a lower nuclear/cytoplasmic ra-t i o . M i t o t i c f i g u r e s are seen (arrowheads). F i g . 9c. Colonies t r e a t e d with 10 ug/ml DON. Note: Colonies are very i r r e g u l a r - s h a p e d , w i t h r e t r a c t e d edges. C e l l s are f l a t and have a low nuclear/cytoplasmic r a t i o . C e l l s are separated from adjacent c e l l s by "holes", but are s t i l l i n contact by t h i n cytoplasmic processes (arrowheads). - 29 -£fe % \1Qb F i g s . lOa-b. E f f e c t s of m u l t i p l e doses of 1  ug/ml DON on C-4II c u l t u r e s . 4 day old c u l t u r e s . Ethanol f i x a t i o n , t o l u i d i n e blue s t a i n i n g , x 55. F i g . 10a. Control c u l t u r e . Note: M i t o t i c f i g u r e s (arrow-heads). F i g . 10b. Cultures t r e a t e d with double doses of 1 ug/ml DON (added days 1 and 2). Note: C e l l s are f l a t t e r , l e s s pavement-like, l e s s columnar, and have a lower nuclear/ cytoplasmic r a t i o . - 30 -11b F i g s , l l a - b . E f f e c t s of m u l t i p l e closes of 1  ug/ml DOn on C-4II c u l t u r e s . 4 day old c u l t u r e s . Ethanol f i x a t i o n , t o l u i d i n e blue s t a i n i n g , x 55. F i g . 11a. Control c u l t u r e . Note: There are few s i n g l e c e l l s on the substratum. F i g . l i b . Cultures tr e a t e d with t r i p l e doses of 1 ug/ml DON (added days 1, 2 and 3). Arrowsheads: Numerous s i n g l e c e l l s , many of which are spindle-shaped ( i r r e g u l a r l y -shaped) or s p h e r i c a l . - 31 -In both cel l l ines , a l l or a combination of these changes were obvious in cultures treated with high concentrations of DON (5 and 10 ug/ml) and multiple doses of 1 ug/ml DON. Single doses of 1 ug/ml DON produced less obvious changes, while 0.5 ug/ml caused no change in any cultures. Cel1-substratum adhesion Effects of increasing DON concentrations on eel 1-substratum interactions, as represented by changes in morphology of single cel ls adhering to the substratum, were quantitated. Single cel ls adhering to the substratum were c lass i f ied into 3 groups (Figs. 12a-c). Cells that were flattened were considered to be most interactive with the substratum (Fig. 12a). Cells that were irregularly-shaped were only less interactive (Fig. 12b), while spherical cel ls were least inter -active with the substratum (Fig. 12c). In both cell l ines , the percentage of single cel ls that were fu l ly spread and flattened on the substratum decreased with increasing DON concentrations (Fig. 13). In the C-4I cel l l ine , the decrease in flattened cel ls was accompanied by increases in the percentage of spherical ce l l s (Fig. 15). In contrast, in the C-4II cell l ine , the decrease in percentage of flattened cel ls was accompanied mainly by an increase in the percentage of irregularly-shaped cel ls (Fig. 14). Stat is t ica l tests using analysis of variance (Petkau and Crapeau, 1983) indicated that DON treatments in both cel l l ines produced s ta t i s t i ca l l y significant differences in the proportions of flattened c e l l s . - 32 -Figs. 12a-c. Morphologies of single cel ls  (indicative of eel 1-substratum interactions) MORPHOLOGY CROSS-SECTION F ig . 12a. Flattened (round and spread) cel l (most cell-substratum interactive) F ig . 12b. Irregularly-shaped cell (partly eel 1-substratum interactive) F ig . 12c. Spherical (round and unspread) cel l (least eel 1-substratum i nteracti ve) Figure 13 EFFECT OF DIFFERENT CONCENTRATIONS OF DON ON THE PERCENTAGE OF FLATTENED CELLS3 % FLATTENED CELLS Key: \-V \: v v V / / / -/ / CONTROL 7\ EL 10 DON CONCENTRATION (ug/ml) a Cultures were 4 days old. Duplicate cultures/treatment. DON was added on day 1 after plating. Total number of cel ls counted was 2,473 C-4I cel ls and 1,139 C-4II c e l l s . • C-4I Expt #1 Q C-4I Expt #2 Q C-4II Expt #1 [=| C-4II Expt #2 Figure 14 EFFECT OF DIFFERENT CONCENTRATIONS OF DON ON THE PERCENTAGE OF IRREGULARLY-SHAPED CELLS3 % IRREGULARLY-SHAPED CELLS \_ O CONTROL Key: DON CONCENTRATION (ug/ml) 10 a Cultures were 4 days old. Duplicate cultures/treatment. DON was added on day 1 after plating. Total number of cel ls counted was 2,473 C-4I cel ls and 1,139 C-4II c e l l s . • C-4I Expt #1 0 C-4I Expt #2 £ | C-4II Expt #1 • C-4II Expt #2 Figure 15 EFFECT OF DIFFERENT CONCENTRATIONS OF DON ON THE PERCENTAGE OF SPHERICAL CELLS3 % SPHERICAL CELLS / / / / 0 CONTROL Key: PI / / 1. \_ / / / / N / i. v A: DON CONCENTRATION (ug/ml) / / / \ H v V / 10 a Cultures were 4 days old. Duplicate cultures/treatment. DON was added on day 1 after plating. Total number of cel ls counted was 2,473 C-4I cel ls and 1,139 C-4II c e l l s . • C-4I Expt #1 0 C-4I Expt #2 [\] C-4II Expt #1 B C-4II Expt #2 - 36 -Conclusion On the basis of the dose response in terms of growth, morphology and eel 1-substratum adhesion, 1.0 ug/ml DON was chosen for al l subsequent experiments, since i ts effects were def in i te , but less severe than those of 5.0 and 10.0 ug/ml. IV. Effects of 1 ug/ml of DON on the growth patterns of C-4I and C-4II 1 i nes  Colony form It was observed that DON at high doses caused increased irregular ity in colony outlines (Figs. 7a, 7c, 8a, 8c, 9o-c and l l a - b ) . These changes in colony form were quantitated by use of a Zeiss M0P-3 image analyzer and by computer s tat is t ica l analysis. Forms of small colonies (between 5 and 35 cel ls per colony) were i n i t i a l l y determined. However, since i t was later observed that small and large colonies differed in their response to DON treatment in cel l area and colony forms, large colonies (more than 50 cel ls per colony) were also quantitated. In C-4I cultures (Table 4), most small colonies were rounder than control colonies i f they were treated with one dose of DON. In contrast, colonies treated with multiple doses tended to be more irregularly-shaped than control colonies (Table 4). The changes were signif icant with a single dose on day 4 and with a double dose on days 6 to 7. In small C-4II colonies (Table 5), there were no consistent changes in response to DON. One day 4 experiment showed increased roundness of colonies while another similar experiment showed signif icantly increased irregularity with DON treatment. On day 6-7, the colonies showed - 37 -increased irregularity but only the change in the colonies treated with t r ip le doses was s ta t i s t i ca l l y s igni f icant . In large C-4I colonies (Table 4), the majority of treated colonies was more irregular than that of controls. These changes were signif icant in cultures treated with multiple doses and for longer culture periods. In most large C-4II cultures (Table 5), the form of treated colonies became signif icantly more irregular. The variances in colony form were determined for DON-treated and control colonies (Appendix I). In large colonies of both cell l ines , variances in colony form of DON-treated cultures were mainly larger than those of control colonies. However, in small colonies, variances in colony form were generally smaller in DON-treated C-4I and more variable in DON-treated C-4II cultures. Considering control colonies only, small colonies in both cel l l ines were more variable in colony form than large colonies. CONCLUSIONS 1. In small C-4I colonies, the response to DON was a biphasic one: a low dose of DON resulted in increased roundness, while a higher dose resulted in increased i r regular i ty . The response of C-4II colonies was variable. 2. In large colonies in both cel l l ines , DON treatment resulted in colonies becoming more irregular (these changes were more significant in l ine C-4II). As a result , the irregular DON-treated C-4I colonies resembled control C-4II colonies. 3. In both cel l l ines , small colonies responded to DON treatment d i f fe r -ently than large colonies. In addition, in both cel l l ines , the form - 38 -Table 4 C-4I: CHANGE IN FORM OF COLONIES TREATED WITH 1 UG/ML OF DON3 Treatment*3 Time of DON Addition 0 (days after plating) Form of Small Colonies d Age of Cultures day 4 days 6-7 Control Single Dose Single Dose Single Dose Double Dose Triple Dose 2 1 0 1 + 2 1, 2 + 3 0.805 0.853* 0.863* 0.885* 0.826 0.782 0.847 0.862 0.868 0.822 0.746* . 0.790 Total number of colonies = 871 Treatment*3 Time of DON Addition 0 (days after plating) Form of Large Colonies^ Age of Cultures day 4 days 6-7 Expt (i) Expt ( i i ) Control Single Dose Single Dose Single Dose Double Dose Triple Dose 2 1 0 1 + 2 1, 2 + 3 0.885 0.807 0.886 0.865 0.866 0.807 0.893 0.881 0.889 0.845 0.878 0.856 0.833* 0.834* Total number of colonies = 381 a Based on. 4 experiments. Colony form was measured on an image analyzer, MOP 3 (Zeiss). A numerical value of colony form of 1.00 represented the form of a perfectly round c i r c l e . Deviations from this roundness ( i . e . i rregularity) were represented by values less than 1.00. Differences between control and treated colonies were tested for significance at a probability level of 0.05, by an analysis of variance and Newman-Keuls test . In each experiment, the variance among and within treatments were analyzed to determine i f differences were s ignif icant . Hence, among experiments, deviations from control values were of different magnitudes for differences to be s igni f icant . b Total number of doses of 1 ug/ml of DON. c Days after plating. Time of plating was defined as day 0. d Small colonies had 5 to 35 c e l l s . Large colonies had more than 50 e e l l s . * Changes were signif icantly di f ferent , at a probability level of 0.05. - 39 -Table 5 C-4II: CHANGE IN FORM OF COLONIES TREATED WITH 1 UG/ML OF DON3 Treatment5 Time of DON Addition 0 (days after plating) Form of Small Colonies^ Age of Cultures day 4 days 6-7 Expt (i) Expt (i i ) Control Single Dose Single Dose Single Dose Double Dose Triple Dose 2 ' 1 0 1 + 2 1, 2 + 3 0.669 0.688 0.721 0.581* 0.715 0.615 0.714 0.585* 0.580* 0.79 0.77 0.77 0.76 0.75 0.68* Total number of colonies = 1,110 Treatment5 Time of DON Addition 0 (days after plating) Form of Large Colonies d Age of Cultures day 4 days 6-7 Expt (i) Expt (i) Expt ( i i ) Control Single Dose Single Dose Single Dose Double Dose Triple Dose 2 1 0 1 + 2 1, 2 + 3 0.469 0.600 0.478 0.540* 0.492 0.454* 0.430 0.627 0.493*-0.776 0.739* 0.709* 0.713* 0.641* 0.541* Total number of colonies = 591 For legend, see Table 4. - 40 -of small control colonies was more variable than the form of large control colonies, and DON-treated large colonies were more variable than corresponding control colonies. These results indicated that DON altered the growth pattern of the two cel l l ines differently and in a complex fashion. The following experiments were undertaken to determine whether these alterations in growth pattern were due to effects of DON on c e l l - c e l l and/or on eel 1-substratum adhesion. A. Cell shedding It was previously shown that confluent C-4II cultures shed more c e l l s , and a higher proportion of l ive c e l l s , into the culture medium than do C-4I cultures (Auersperg, 1969a). It was postulated that secretion of ECM may fac i l i ta te cel l shedding in the less cohesive C-4II cel l l i n e , but not in the cohesive C-4I l i n e . The following experiment was to determine i f DON had any effect on cell shedding. In C-4I cultures, DON increased cel l shedding, and in contrast to control cultures, most of the shed cel ls were l ive (66-89%, as compar-ed to 45-54% for controls) (Table 6). This may have accounted for the decreased s t rat i f i cat ion in DON-treated cultures (Figs. 7a-b and 8a-b). In non-confluent C-4II cultures, DON caused no increase in the absolute number of cel ls shed and approximately a 2-fold increase in relative numbers. In contrast, the drug caused an increase in shed-ding in a l l confluent C-4II cultures, both in absolute and relative numbers, with the relative increase being about 5-fold (2.0 vs. 10.7 on day 0-6, and 0.8 vs. 4.1 on day 5-6) (Table 6). In addition, the - 41 -proportion of l ive cel ls from DON-treated cultures was high (87-88%), comparable to that of control cultures (87%). These results indicated that DON induced cell shedding in C-4I cultures and also in C-4II cultures, particularly when they were confluent. DON did not cause increased cel l shedding by causing cel l death, since the proportions of shed cel ls that were l ive in most treated cultures was as high or higher than in control cultures. Thus, DON l ike ly increased cel l shedding by decreasing c e l l - c e l l adhesion. B. Ce l l - ce l l aggregation Since DON probably increased cell shedding by decreasing c e l l -cel l adhesion, i t seemed possible that i t would also decrease cell aggregation. The following experiments were carried out to determine what effects DON had on cell aggregation in the two cell l ines . 1. Aggregation of cel ls pretreated with DON prior to trypsin/EGTA dissociation  C-4I: After rotation for 1 day at 70 rpm in the absence of DON, the aggregation of cel ls that were pretreated with DON for 5 days was greatly inhibited (84% single ce l ls ) in contrast to that of controls (46% single cel ls ) (Table 7). Aggregation of cel ls that were pre-treated with DON for 1 day was also inhibited but less so (67% single c e l l s ) . In addition, control clumps that resulted were round and com-pact with f lat cel ls on the surface (Fig. 16a), while treated clumps - 42 -Table 6 ABSOLUTE AND RELATIVE NUMBER AND VIABILITY OF SHED CELLS PER CULTURE3 C-4I C- 411 C-4II (non-conf1uent) (confluent) Control DON Control . DON . -Control DON Day 0-6 Number of shed cel ls (x 10 4 ) : absolute 6.4 20.8 13.4 9.4 30.4 48.0 relat i ve*3 0.5 3.1 1.2 2.4 2.0 10.7 % v i a b i l i t y 0 of shed cel ls 54 66 82 73 87 88 Day 5-6 Number of shed cel ls (x 10 4 ) : absolute 2.0 5.8 6.4 3.7 13.2 20.6 relative 0.1 0.9 0.6 0.9 0.8 4.1 % v iab i l i t y of shed cel ls . 45 89 87 81 87 87 a Average total shed cel ls (x lo 4) per culture; based on 2 C-4I, 3 non-confluent C-4II and 2 confluent C-4II experiments. In each experiment, treatments were duplicated. b Absolute number of shed ce l ls relative to the number of cel ls growing in the culture f lasks. c Percentage of total cel ls shed into the culture medium that were l ive (determined by dye exclusion test ) . - 43 -were smaller, less compact, irregularly-shaped and al l cel ls remained more or less spherical (Figs. 16b and 16c). After rotation for 2 days at 70 rpm, DON-pretreated cultures had more clumps than after 1 day of rotation, but s t i l l fewer than control cultures. C-4II: In 2 separate experiments, after either 1 or 2 days of rotation, there were only sl ight differences between control and DON-pretreated cultures, except for a limited decrease in aggregation of one set of cultures pretreated with DON for 5 days (Table 8, experiment 1). The clumps that formed from both control and DON-pretreated cel ls were mainly irregular and small (Figs. 17a and 17b). Summary of results and conclusion: In the C-4I cel l l ine , DON pretreatment, whether for 1 or 5 days, caused a definite decrease in c e l l - c e l l aggregation and in cell spreading or cell deformation. Pretreatment with DON for 5 days caused a greater inhibit ion of aggregation than pretreatment for 1 day. In the C-4II cel l l i n e , DON pretreatment, whether for 1 or 5 days, caused only an inconstant decrease in c e l l - c e l l aggregation i f cultures were rotated for 2 days. There was no difference in morphology of clumps formed from control and DON-pretreated c e l l s . In both cases, the clumps were irregularly-shaped and small. To conclude, DON clearly decreases cell adhesion in C-4I but not in C-4II clumps. - 44 -Table 7 C-4I: AGGREGATION OF CELLS PRETREATED WITH DON3 Treatment0 % of Cells and Clumpsc Total Number % Single Cells % Clumps Counted Control 46 54 1,232 1 day DON pretreatment 67* 33* 1,923 5 day DON pretreatment 84* 16* 1,326 a Based on 2 experiments; results presented here are representative of 1 experiment only. All treatments were duplicated. D DON (1 ug/ml) was added to cultures for 1 or 5 days prior to dissociation into single c e l l s . No DON was added while cultures were rotated for 1 day at 70 rpm. c Samples were sized (hemocytometer sl ide) and c lass i f ied either as single ce l ls or as clumps (with 2 or more c e l l s ) . * Controls values were signif icantly different from treated values, as determined by analysis of variance and Newman-Keuls tests , at a probability level of 0.05. C-4I: Aggregation of c e l l s p r e t r e a t e d w i t h DON (1 ug/ml). Fixed 1 day a f t e r shaking at 70 rpm. No DON was added during shaking, x 90. Control c u l t u r e . 1 day DON-pretreated c u l t u r e . 5 day DON-pretreated c u l t u r e . F i g s . 16a-c. F i g . 16a F i g . 16b F i g . 16c - 46 -Table 8 C-4II: AGGREGATION OF CELLS PRETREATED WITH DON3 Treatment13 %'of Cells and Clumpsc Total Number % Single Cells % Clumps Counted Expt #1 1 day of shaking: control 78.9 21.2 586 1 day DON pretreatment 79.6 20.4 567 5 day DON pretreatment 68.4 31.7 451 2 days of shaking: control 5.4 94.6 813 1 day DON pretreatent 10.6 89.5 1,008 5 day DON pretreatment 23.0 77.1 789 Expt #2 1 day of shaking: control 49.0 51.0 635 1 day DON pretreatment 42.3 57.7 442 5 day DON pretreatment 46.5 53.5 1,752 2 days of shaking: control 27.9 71.9 583 1 day DON pretreatment 19.2 80.7 666 5 day DON pretreatment 20.6 79.5 1,446 a Based on 2 experiments. In expt #2 only, the dissociated cel ls were syringed with a 21 gauge needle in an attempt to dissociate the clumps more completely prior to shaking. D DON (1 ug/ml) was added to cultures for 1 or 5 days prior to dissociation into single c e l l s . No DON was added while cultures were rotated for up to 2 days at 70 rpm. c Samples were sized (hemocytometer sl ide) and c lass i f ied either as single cel ls or as clumps (with 2 or more c e l l s ) . F i g s . 17a-b C -4II : Aggregation of c e l l s p r e t r e a t e d w i t h DON (1 ug/ml). Fixed 2 days a f t e r shaking at 70 rpm. No DON was added during shaking, x 90. F i g . 17a Control c u l t u r e . F i g . 17b 5 day DON-pretreated c u l t u r e . - 48 -2. Aggregation of ce l ls treated with DON after trypsin/EGTA dissociation  (Cells not pretreated with DON prior to dissociat ion) . Any DON-induced inhibit ion of aggregation in the DON-pretreated shaker cultures could have resulted from a slower rate of recovery of adhesive cell surface components following trypsinization, due to depletion of glycosaminoglycan precursors stored during DON-pretreat-ment. It could also have been influenced by cell damage due to long-term DON pretreatment. To reduce the influence of these poss ib i l -i t i e s , the aggregation experiment was modified so that freshly trypsinized cel ls had a recovery period, in which trypsin-sensit ive cel l surface components were regenerated (see flow diagram, p. 15). During this recovery period, shaker cultures were maintained at a relatively fast speed (200 rpm) so that there was minimal c e l l - c e l l aggregation while cel l surfaces were being repaired (Takeichi, 1977). The recovery period was assumed to be 24 hours long since recent l i terature on cel l surface regeneration indicated that cel ls were fu l ly adhesive and had regenerated their cel l surface components with-in 24 hours after trypsinization (Steinberg ejt al_., 1973; Dembitzer et a l . , 1980b). After this recovery period at a rapid speed, the cel ls were allowed to aggregate at the relatively slow speed of 70 rpm. In addition to the above modification, cultures were not treated with DON prior to trypsinizat ion. This eliminated the possibl ity that decreased aggregation was due to the impaired ab i l i t ies of damaged cel ls to aggregate. Instead, cultures were only treated with DON for a shorter period of time after t rypsinizat ion, during the ce l l s ' recovery from the effects of t rypsin. It was previously observed that - 49 -there was a lag period (of about 1 day) between the time of addition of DON treatment and the time that changes were visually observed. Hence, DON was added to cel ls in shaker cultures during their recovery from trypsinization, so that DON-induced effects would be evident soon after the onset of maximal aggregation at 70 rpm. Hence, ideal ly , at 24 hours of recovery with minimal aggregation at 200 rpm, i . e . at the onset of maximal aggregation at 70 rpm, control and DON-treated cel ls in shaker cultures would be maximally adhesive but s t i l l equally as unaggregated. The effects of DON should become apparent subsequently. The differences in aggregation of control and DON-treated trypsin-recovered cel ls were quantitated 24 hours after the onset of maximal aggregation (70 rpm), i . e . 48 hours after trypsinizat ion. The optimal time of DON addition was worked out i n i t i a l l y with the C-4II cel l l i ne , as shown below, and later applied to the C-4I l ine . C-4II: In three experiments, DON was added to shaker cultures immediately after dissociat ion. In al l three experiments, there was some indication that DON-treated cultures were less aggregated than control cultures already after 24 hours of rapid shaking and also after a further 24 hours of shaking at 70 rpm (Fig. 18; Table 9, part i ) . In one experiment, DON was added to cultures 6 or 12 hours after d i s -sociation. DON-treated cel ls were again less aggregated than control cel ls at 24 hours after rapid shaking, and also after 48 hours of fast and - 50 -slow shaking (Table 9, part i i ) . Hence, DON decreased cel l aggregation after an incubation period of only 18 hours, and possibly 12 hours. In f ive experiments, DON was added 18 hours after dissociat ion. Four experiments indicated that treated cel ls (shaken at 200 rpm in the presence of DON for 6 hours) were as aggregated as control cel ls (Fig. 19). However, at the end of a total 48 hours of shaking, there was some indication that control cel ls were sl ightly more aggregated than treated ce l ls (Fig. 19; Table 9, part i i i ) . In one experiment, DON was added to shaker culture after 24 hours of rapid shaking. At the end of 24 hours of slow shaking, there was l i t t l e difference between treated and control cultures (Table 9, part iv ) . The clumps found in control and DON-treated cultures were i r regular ly -shaped. To summarize, when DON was added to cultures which were recover-ing from effects of trypsinization, there were only borderline differences detected between control and treated cultures. C-4I: In al l experiments, DON was only added to cultures 18 hours after t ry -psinizat ion, while the cultures were recovering from the effects of try-psinization as they were rotating at 200 rpm. Results generally indicated that DON caused a small, insignif icant inhibit ion of c e l l - c e l l aggregation into clumps, with diameters which ranged from 10 to 20 um (Table 10) and with diameters greater than 20 um (Table 11). There was no difference detected between the morphologies of control and treated clumps. Table 9 C-4I I : AGGREGATION OF CELLS TREATED WITH DON AFTER TRYPSIN/EGTA DISSOCIATION3 % of Cells and Clumps with Diameter Time of DON Addition 5 Incubation Time (Hours) Range (um)d (Hrs after Dissociation) with DON1- 12-14 M4-16 >16-18 >18-20 >20-25 >25 Part i control 0 11.9 34.6 30.9 14.9 6.0 1.7 DON, 0 hours 24 19.1 36.7 25.4 12.0 5.6 1.2 Part i i control 0 20.9 38.1 28.9 12.2 DON, 6 hours 18 31.6 39.9 20.1 8.4 DON, 12 hours 12 37.0 37.7 18.2 7.1 Part i i i control 0 17.4 28.6 29.5 16.5 6.0 2.2 DON, 18 hours 6 14.9 33.0 31.2 14.9 5.1 0.8 Part iv control 0 14.8 33.2 29.5 16.6 5.2 0.6 DON, 24 hours 0 22.1 38.1 25.9 13.8 6.7 0.9 a Single cel ls were shaken for 24 hours at 200 rpm, and then for another 24 hours at 70 rpm. 5 Treatments (1 ug/ml DON) were added at various times after subculture (trypsin/EGTA d is -sociation), while cultures were shaken at 200 rpm. c Incubation time (hours) in the presence of DON, at the end of 24 hours of shaking at 200 rpm. d Al l samples were sized l ive on a Coulter counter at the end of the experiment ( i . e . 24 hours at 200 rpm followed by another 24 hours at 70 rpm). Parts: i . . . representative of 2 additional experiments i i , i v . . . t h i s experiment was not duplicated i i i . . . representat ive of 4, out of 5, additional experiments - 52 -F ig . 18 C-4II: AGGREGATION OF CELLS TREATED WITH DON AFTER TRYPSIN/EGTA DISSOCIATION3 (after 24 hours shaking at 200 rpm, and another 24 hours at 70 rpm.) (Tracing of coulter counter sizing) NUMBER OF CELLS DIAMETER (um) Legend: a 1 ug/ml of DON was added immediately after dissociation (see Table 9, part i ) . — Control sample 1 Control sample 2 — Control sample 3 DON-treated sample 1 DON-treated sample 2 — DON-treated sample 3 - 53 -F i g . 19 C-4II: AGGREGATION OF CELLS TREATED WITH DON OR DON PLUS GSA AFTER TRYPSIN/EGTA DISSOCIATION3 (Tracing of c o u l t e r counter s i z i n g ) NUMBER OF CELLS AND AGGREGATES DIAMETER (um) Legend: a 1 ug/ml of DON (see Table 9, part i i i ) and/or 100 ug/ml GSA was added 18 hours a f t e r d i s s o c i a t i o n . A f t e r 24 hours shaking at 200 rpm: Control sample DON-treated sample DON + GSA sample A f t e r 24 hours shaking at 200 rpm, followed by another 24 hours at 70 rpm: Control sample DON-treated sample DON + GSA sample - 54 -Table 10 C-4I : AGGREGATION OF CELLS TREATED WITH DON AND/OR GSA AFTER TRYPSIN/EGTA DISSOCIATION3 Treatment0 % of Cells and Clumps with diameter range (um)c Expt #1 10-12 >12-14 >14-16 >16-18 >18-20 control 28.6 32.4 26.3 12.6 DON 32.9 38.1 20.1 8.9 DON + GSA 30.9 39.5 20.5 9.1 Expt #2 control 8.3 29.3 25.9 22.7 13.9 DON 7.5 31.8 26.8 22.6 11.4 DON + GSA 10.6 40.0 25.6 16.3 7.5 Expt #3 control 7.9 30.9 30.2 21.9 9.1 DON 7.9 32.8 31.4 18.9 8.9 DON + GSA 5.7 26.6 33.4 23.2 11.0 a Based on 3 experiments. b 1 ug/ml DON with or without 100 ug/ml glucosamine (GSA) added 18 hours after subculture while shaking at 200 rpm. c Samples sized l ive on a Coulter counter, at the end of 24 hours at 200 rpm and another 24 hours at 70 rpm. Diameters (12-16)um represent mainly single c e l l s . - 55 -Table 11 C-4I: EFFECT OF GSA ON AGGREGATION OF CELLS TREATED WITH DON AFTER TRYPSIN/EGTA DISSOCIATION (% clumps of more than 20 um diameter) Treatment % Sized in the Diameter Range (um)a (20)-(<43) um (43)-(<78) um 778 um Expt. A control 76.2 22.2 1.6 DON0 77.5 21.3 1.3 DON + 100 GSAC 72.7 24.9 2.5 DON + 200 GSA 70.4 26.4 3.3 <20 um (20)-(<43) um (43)-(<78) um >78 um Expt. B control 67.5 24.9 4.1 3.5 DON 76.6 20.8 1.8 0.9 DON + 100 GSA 46.5 47.6 3.6 2.3 a Based on 2 experiments, duplicate or t r ip l i cate samples per treatment. Cells and clumps were sized on an Artek counter; area setting = 482, sensit iv i ty of 730 and 590, at least 8 f ie lds of 4 mm^  each were counted. Samples were sized fixed (2% glutaraldehyde in Mil lonig's buffer) and stained (4% eosin). Diameter range settings were set using standard calibration particles (Coulter Electronics) . 0 1 ug/ml DON was added 18 hours after trypsinizat ion. c ug/ml GSA (glucosamine) added, 18 hours after trypsinizat ion. - 56 -Conclusion: With DON present only during recovery from trypsinization, there was a borderline effect on cell aggregation after 24 hours of slow shaking in both cell l ines , irregardless of the duration of DON treatment. 3. Effects of glusosamine on aggregation of eel 1s "treated with DON after  trypsin/EGTA dissociation In v i t ro , DON had been shown to inhibit adhesion of rat palatal shelves (Greene and Pratt, 1977), to decrease the synthesis of sul fat -ed glycosaminoglycans by embryonic chick heart f ibroblasts (Spooner and Conrad, 1977), and to decrease migration of embryonic heart cel ls (Funderburg and Markwald, 1981). These authors also used glucosamine (GSA) to counteract these inhibitory effects of DON, since DON i n -hibits the synthesis of glucosamine-6-phosphate. Their results indicated that glucosamine either part ia l ly or fu l ly reversed the effects of DON. In this study, i t was previously concluded that in both C-4 cel l l ines , DON had a borderline inhibitory effect on the aggregation of trypsin-recovered cel ls (Figs. 18 and 19 showed that in C-4II c e l l s , this inhibitory effect was small but consistent). Hence, in this study, GSA was added simultaneously with DON to shaker cultures to determine what ef fects , i f any, GSA had on cell aggregation in the absence or presence of DON. C-4I: Shaker cultures were sized l ive at the end of each experiment on a Coulter counter (Tables 10 and 11). These results showed that for clumps less - 57 -than 20 um in diameter, 100 ug/ml of GSA had no effect in 2 experiments, but increased aggregation beyond that of control ce l ls or DON-treated ce l ls in experiment #3 (Table 10; F - r t ^ — S i n c e many C-4I clumps were as large as 78 um in diameter, and the Coulter counter best sized particles less than 40 um in diameter, the large clumps of the cultures were sized on an Artek counter after they were fixed and stained. Of a total of 3 experiments, clump sizes in only two experiments were determined (Table 11), as one experiment contained too much debris. These results indicated that there was either partial reversal (Table 11, experiment B) of inhibit ion of aggregation in DON + GSA cultures, or that the addition of GSA increased aggregation of cel ls beyond that observed in control or DON-treated cultures (Table 11, experiment A). In other experiments where 10, 50 and 200 ug/ml of GSA were used, there was no consistent trend indicating that GSA increased aggregation. However, i t was observed that al l clumps, irregardless of treatment, were round and large (Figs. 20a-e), with the possible exception of those treated with DON only, which appeared to have a more irregular outl ine. C-4II: In six experiments in which 100 ug/ml GSA was added concurrently with DON, there was no clearcut indication that GSA increased aggregation (Fig. 19). Of three experiments in which control cultures were compared to GSA-treated cultures (10, 100 and 200 ug/ml), only one experiment indicated that at the end of 24 hours of rapid shaking in which cultures were treat-ed with GSA for 6 hours, the GSA-treated cultures were more aggregated than the control cultures. mmm Figs . 20a-e. C-4I: Aggregation of c e l l s Drugs were added 18 hours a glutaldehyde i n M i l l o n i g ' s F i g . 20a Control c u l t u r e F i g . 20b 1 ug/ml DON F i g . 20c 1 ug/ml DON + 10 ug/ml GSA F i g . 20d 1 ug/ml DON + 50 ug/ml GSA F i g . 20e 1 ug/ml DON + 100 ug/ml GSA 0 0 - 59 -Conclusion: There was no clear cut indication that GSA, when added with DON to C-4I and C4II cultures, increased aggregation of cel ls beyond that which occured in cultures treated with DON only except, possibly, for large C-4I clumps; however, GSA reverted the effect of DON on the shape of C-4I aggregates and thus, presumably, on the deformabi1ity and/or abi l i ty to spread of C-4I c e l l s . V. Morphology of single adherent ce l ls treated with 1 ug/ml DON Among controls in both cel l l ines , the C-4II cel l l ine had a higher percentage of single fu l ly spread c e l l s , which indicated that C-4II ce l ls were more interactive with the substratum (Auersperg, 1969a). To determine the effect of DON on eel 1-substratum interactions, the morphologies of single cel ls adhering to the plastic substratum were quantitated; since cel l morphology was indicative of substratum adhesive-ness and of eel 1-substratum interactions (Auersperg, 1969a; Harris, 1973). Single cel ls were c lass i f ied into three types of morphologies (Fig. 12), of which flattened cel ls were most substratum interactive, irregularly-shaped cel ls were less interactive, and spherical cel ls were least interactive. In both cel l l ines , increasing concentrations of DON caused propor-tional decreases in the percentage of cel ls that were flattened (Fig. 21). ANOVA analysis (Petkau and Crapeau, 1983) showed that these decreases were s ta t i s t i ca l l y s igni f icant . The decrease in percentage of cel ls that were flattened in response to 1 dose of 1 ug/ml DON treatment was greater in C-4II cultures than in C-4I cultures (Fig. 21). Most of the DON-treated C-4II ce l ls remained partly - 60 -Fig . 21' EFFECT OF DON ON THE MORPHOLOGY OF SINGLE CELLS % OF FLATTENED CELLS 17 „ 16 _ 15 _ 14 _ 13 _ 12 _ C 11 _ _ 10 _ _ 9 _ _ 8 _ _ 0 CONTROL SINGLE DOUBLE TRIPLE TOTAL CUMULATIVE DOSES OF DON Key: • C-4I EXPT. 1 0 C-4II EXPT. 1 [21 C-4I EXPT. 2 • C-4II EXPT. 2 (3 C-4I EXPT. 3 Each dose was equivalent to 2 ug of DON ( i . e . 1 ug/ml). The f i r s t dose was added on day 1 after plating. The second dose was added on day 2, while the third dose was added on day 3. Cultures were fixed on days 4-5. - 61 -F ig . 22 EFFECT OF DON ON THE MORPHOLOGY OF SINGLE CELLS % OF IRREGULARLY SHAPED CELLS 100 • C-4I EXPT. 1 M c"4 1 1 E X P T- 1 0 C-4I EXPT. 2 H C-4II EXPT. 2 Q C-4I EXPT. 3 Each dose was equivalent to 2 ug of DON ( i . e . 1 ug/ml). The f i r s t dose was added on day 1 after plating. The second dose was added on day 2, while the third dose was added on day 3. Cultures were fixed on day 4-5. - 62 -F ig . 23 EFFECT OF DON ON THE MORPHOLOGY OF SINGLE CELLS % OF SPHERICAL CELLs 100 0 CONTROL SINGLE DOUBLE TRIPLE TOTAL CUMULATIVE DOSES OF DON Key: • C-4I EXPT. 1 0 C-4II EXPT. 1 Q C-4I EXPT. 2 B C " 4 1 1 E X P T - 2 Q C-4I EXPT. 3 Each dose was equivalent to 2 ug of DON ( i . e . 1 ug/ml). The f i r s t dose was added on day 1 after plating. The second dose was added on day 2, while the third dose was added on day 3. Cultures were fixed on days 4-5. - 63 -adherent or i r r e g u l a r l y - s h a p e d ( F i g . 22) and d i d not round up completely ( F i g . 23). In c o n t r a s t , the decrease i n percentage of f l a t t e n e d C-4I c e l l s was accompanied by an increase i n percentage of s p h e r i c a l c e l l s ( F i g . 23). Conclusion: DON s i g n i f i c a n t l y decreased the spreading and adhesion of c e l l s i n both c e l l l i n e s . However, C-4II c e l l s showed a greater decrease i n percentage of f l a t t e n e d c e l l s . I f DON decreased eel 1-substratum adhesion and i f C-4II c e l l s were more eel 1-substratum i n t e r a c t i v e , then DON should a f f e c t adhesion of C-4II c e l l s more than C-4I c e l l s . The r e s u l t s of these experiments support t h i s c o n c l u s i o n . VI. C e l l area In a d d i t i o n to changes i n colony form, there were a l s o changes i n projected c e l l areas (projected two dimensionally) i n c u l t u r e s t r e a t e d w i t h DON ( F i g s . 7a-b, 8a-b, 9a-c, lOa-b and l l a - b ) . In small C-4I and C-4II c o l o n i e s , the changes i n areas of DON-treated c e l l s were v a r i a b l e , although i n general, DON-treated c e l l s tended to be l a r g e r than c o n t r o l c e l l s (Tables 12 and 13). DON-treated l a r g e C-4I c o l o n i e s tended to have s t r a t i f i e d rims or edges ( F i g s . 7a-b) and f l a t t e r colony centers ( F i g s . 7a-b and 8a-b). Therefore, the average c e l l area of large c o l o n i e s was c a l c u l a t e d by determining the area of the center of the colony only and d i v i d i n g t h i s area by the number of n u c l e i ( c e l l s ) i n that area. Table 12 C-4I: CHANGE IN CELL AREA3 Time of DON addition 1 3 Average cel l area in small colonies Total number of doses (xlO- 3 mm2) of 1 ug/ml DON added (days after plating) day 4 cultures day 6-7 cultures control _ 2.51 2.20 1 2 2.37 2.16 1 1 2.48 2.46 1 0 2.48 2.50 2 1 + 2 2.60 1.94 3 1, 2, 3 2.64 2.46 Time of DON addit ion 0 Average cell area in large colonies Total number of doses (xlO- 3 mm2) of 1 ug/ml DON added (days after plating) day 4 cultures day 6-7 cultures control _ 2.19 1.93 1 2 3.22* 2.69* 1 1 2.74* 2.59* 1 0 2.70* 2.49* 2 1 + 2 3.42* 3.02* 3 1, 2, 3 3.65* 3.34* a Projected cell areas (xl0~3 mm2). Using a camera lucida, colonies were projected and analyzed by a digital image analyzer (Zeiss MOP 3). In small colonies (5-35 c e l l s ) , the entire colony area was projected and the number of cel ls (nuclei) counted. In large colo-nies (more than 50 ce l l s ) , the cell area was calculated by dividing the area of centers of colonies by the number of ce l ls (nuclei) in the center (since some colonies had a s t ra t i f ied edge or rim, the cel ls in the colony edge or rim were omitted). D Doses of 1 ug/ml of DON were added at various days after plating or subculture; day 0 was the day of plating. * The difference between the average cell area of treated cel ls and that of control ce l ls was s ta t i s t i ca l l y s ignif icant. The data was analyzed using analysis of variance and Newman-Keuls tests , at probability levels of 0.05. Table 13 C-4II: CHANGE IN CELL AREA3 Time of DON addit ion 0 Average cel l area in smal 1 colonies Total number of doses (xlO~ J mm2) of 1 ug/ml DON added (days after plating) day 4 cultures day 6-7 cultures Expt. (i) Expt. ( i i ) control - 2.04 0.99 1.46 1 2 2.73* 1.33 1 1 1.67* 1.44 1 0 2.31* 1.18 1.47 2 1 + .2 3.17* 2.19* 1.70* 3 1, 2, 3 1.88* 2.00* Time of DON addit ion b Average cel l area in large colonies Total number of doses (xlO -- 3 mmO of 1 ug/ml DON added (days after plating) day 4 cultures day 6- 7 cultures Expt. (i) Expt. ( i i ) control - 1.76 1.29 1.17 1 2 2.87* 1.28 1 1 2.10* 1.36 1 0 2.07* 1.40 1.42 2 1 + 2 3.99* 2.84* 1.72* 3 1, 2, 3 2.74* 1.95* a Projected cel l areas (xl0~ J mm )^. Using a camera lucida, colonies were projected and analyzed by a digital image analyzer (Zeiss MOP 3). In small colonies (5-35 c e l l s ) , the entire colony area was projected and the number of cel ls (nuclei) counted. In large colo-nies (more than 50 c e l l s ) , the cell area was calculated by dividing the area of centers of colonies by the number of cel ls (nuclei) in the center (since some colonies had a s t rat i f ied edge or rim, the cel ls in the colony edge or rim were omitted). 0 Doses of 1 ug/ml of DON were added at various days after plating or subculture; day 0 was the day of plating. * The difference between the average cell area of treated cel ls and that of control ce l ls was s tat i s t i ca l l y s ignif icant. The data was analyzed using analysis of variance and Newman-Keuls tests , at probability levels of 0.05. - 66 -Results indicated that in the centers of large C-4I colonies, a l l DON-treated cel ls were s ignif icant ly larger than control c e l l s , i rregard-less of length of culture or treatment (Table 12). In large C-4II c o l -onies, DON-treated cel ls in a l l cultures were also larger than control c e l l s , but the changes were signif icant mainly in cultures treated with multiple doses of DON (Table 13). VII. Cell volume Previous results on cel l area indicated that DON, when added to C-4 cultures, increased the projected surface area of c e l l s . Most of these changes were s ta t i s t i ca l l y signif icant in large colonies. To determine i f DON increased the surface area of cel ls by increasing cel l volumes, cel ls were sized on a Coulter counter. C-4 cultures were treated with DON one day after plating. On the fourth day after plat ing, the cultures were dissociated using trypsin/EGTA. Previous results, as well as the results of Dembitzer et al_. (1980a), indicated that such treatments produced mainly single c e l l s . In this study, only 1.3% of control C-4I particles sized were greater than 20 um in diameter (Table 14), and only approximately 1% of control C-4II particles counted were of that size (Table 15). In both cell l ines , DON increased cel l volume. This increase was generally proportional to the number of doses of DON given (Tables 14 and 15: see modal diameters and volumes). Also in both cell l ines , ce l ls in DON-treated cultures were not normally distr ibuted, but instead, they were posit ively skewed. Coulter counter plots of dissociated C-4 cel ls not only indicated the shapes of the cell d istr ibut ions, but also indicated the modal diameters % counts greater than 20um C-4I: Table 14 EFFECT OF DON ON CELL VOLUME Percentage of a l l ce l l s s ized 9 Diameter range Control DON treatment0 (um) 1 dose 2 doses (i) ( i i ) (i) ( i i ) (i) ( i i ) <8-10 3.6 2.0 3.0 2.5 3.6 4.4 >10-12 11.9 9.5 4.9 6.6 4.5 6.4 >12-14 23.3 28.6 16.2 21.4 15.0 13.9 >14-16 33.4 31.6 33.7 31.2 32.4 23.9 >16-18 19.6 20.5 26.2 27.0 27.3 30.9 >18-20 8.3 7.7 15.9 11.4 17.1 20.5 Modal diameter c (um)(+ S.D.) 13.60 (+0.57) 13.80 (+0.36) 14.65 (+0.22) 14.60 (+0.28) 14.65 (+0.22) 16.10 (+0.92) Modal volume (um )^ 1308 1367 1652 1618 1652 2170 1.3 1.9 4.3 a Duplicate cultures per treatment (except for DON treatment, 2 doses, experiment ( i i ) . Four day cultures were sized on a Coulter counter, after being dissociated (with trypsin/EGTA) into single ce l l s . 0 Cultures were treated with DON at a concentration of 1 ug/ml. 1 dose-treated cultures were treated with DON 1 day after plating; while 2 dose-treated cultures were treated with 1 dose of DON 1 day after plating and with a second dose of DON 2 days after plating. c Modal diameter as determined from plots of cell s izes, on a Coulter counter. +_ S.D. or +_ standard deviation. Modal volumes were determined from modal diameters. ( i ) , ( i i ) . . . Two separate experiments. Table 15 C-4II: EFFECT OF DON ON CELL VOLUME Percentage of al l ce l ls s i zed 3 Diameter range Control DON treatment0 (um) 1 dose 2 doses (i) ( i i ) (D ( i i ) (i) ( i i ) <8-10 2.7 4.4 5.6 4.2 6.0 6.2 >10-12 4.4 8.7 4.2 4.9 3.0 3.7 >12-14 27.0 34.7 20.6 26.6 9.5 9.4 >14-16 38.7 32.9 38.7 36.6 26.7 26.5 >16-18 21.1 14.9 19.9 21.0 33.5 32.7 >18-20 6.1 4.4 11.0 6.7 21.3 21.5 Modal diameter c 14.0 13.7 14.8 14.1 15.9 16.0 (um)(+ S.D.) (+0.18) (+0.02) (+0.02) (+0.06) (+0.02) (+0) Modal volume (um )^ 1,440 1,330 1,700 1,470 2,120 2,130 % counts greater than 20um 0.9% 1.0% 1.3% 1.5% 2.5% 2.7% a Duplicate cultures per treatment. Four day cultures were sized on a Coulter counter, after being dissociated (with trypsin/EGTA) into single c e l l s . 0 Cultures were treated with DON at a concentration of 1 ug/ml. 1 dose-treated cultures were treated with DON 1 day after plating; while 2 dose-treated cultures were treated with 1 dose of DON 1 day after plating and with a second dose of DON 2 days after plating. c Modal diameter as determined from plots of cell s izes, on a Coulter counter. +_ S.D. or +_ standard deviation. Modal volumes were determined from modal diameters. ( i ) , ( i i ) . . . Two separate experiments. - 69 -as represented by the peaks of graphical plots. In C-4I cultures (Table 14), the average modal diameter of control cel ls was approximately 13.70 um. However, in C-4II cultures (Table 15), the average modal diameter of control ce l ls was approximately 13.85. Thus, the results indicated that the majority of C-4II ce l l s were sl ight ly larger and less variable than C-4I c e l l s . Comparing modal diameters of single-dose treated c e l l s , C-4I treated cel ls had a diameter of approximately 14.63 um, which was similar to that of C-4II ce l ls of 14.45 um. However, when cel ls were treated with 2 doses, C-4II cel ls had a diameter of approximately 15.95 um, while C-4I cel ls had a smaller diameter of about 15.38 um. VIII. Summary of the properties of control C-4 cel ls used in this study The properties of control C-4 cel ls are summarized and presented in Table 16. 1. The growth pattern of C-4I cultures depended on the dissociation method, or on the maintenance of c e l l - c e l l contacts. 2. The growth patterns of C-4 cel ls were substratum-independent. If C-4I cel l sheets were suspended in culture medium after t rypsinizat ion, they tended to retract into round and cohesive clumps (Figs. 24 a -c ) . In contrast, C-4II cel l sheets remained generally irregular and only retracted to a limited extent (Figs. 25 a -c ) . 3. C-4II colonies, irregardless of s ize, were always more irregular than C-4I colonies. In general, large C-4II colonies were more irregular than small colonies. In contrast, large and small C-4I colonies were equally round. - 70 -4. C-4I cultures shed fewer cel ls (50% were l ive) than C-4II cultures (over 80% were l i ve ) . Among C-4II cultures, confluent cultures shed more than non-confluent ones. 5. Shed C-4I cel ls were less prol i ferat ive than shed C-4II ce l ls (Auersperg, 1969a). 6. Aggregated C-4II clumps were small and irregular (Fig. 17a), while C-4I clumps were larger, round and compact (Fig. 16a). 7. Single C-4I cel ls were less flattened on a plast ic substratum than C-4II c e l l s . In contrast, in the C-4II cel l l ine , there were fewer spherical , more-irregularly shaped and more flattened c e l l s . Hence, C-4II cel ls were more interactive with the substratum than C-4I c e l l s . 8. In both cel l l ines , ce l ls in small colonies had larger projected cel l surface areas than those in large colonies. 9. Cell diameter and volume of C-4I cel ls were sl ightly smaller than that of C-4II c e l l s . IX. Summary of the effects of DON treatment on C-4I and C-4II cel ls The effects of DON treatment on C-4I and C-4II cel ls are summarized and presented on Tables 17 and 18 respectively. Br ie f ly , both C-4 ce l ls showed qualitatively similar effects on treatment with DON. For example: 1. Colonies were generally more i rregular , 2. More cel ls were shed into culture medium, 3. Cells were less cohesive as shown by "holes" within colonies, 4. Single cel ls were less spread/interactive on plastic substratum, and 5. Cells were larger, both in projected cell surface area and volume. - 71 -In addition, properties of DON-treated C-4I ce l ls resembled those of C-4II c e l l s . For example: 1. C-4I colonies grew irregular ly , 2. C-4I cultures shed more viable c e l l s , 3. C-4I cel l in shaker cultures aggregated less, and 4. Aggregated C-4I clumps (when treated prior to dissociation) were irregular and smal1. - 72 -Table 16 SUMMARY OF PROPERTIES OF CONTROL C-4I AND C-4II CELLS 1. Colony form i f seeded as: (i) clumps ( i i ) single cel ls 2. Cell sheets (clumps) hours after trypsinization 3. Colony form* of: (i) smal1 colonies ( i i ) large colonies 4. Cell shedding 5. Shed cell prol i feration 6. Ce l l - ce l l aggregates Morphology** of single cel ls (percentage of total c e l l s ) : (i) spherical ( i i ) irregularly-shaped ( i i i ) flattened Cell area* (x lO - 3 mm2) i n : (i) small colonies ( i i ) large colonies Cell diameter (um) (+_standard deviation) Cell volume (um3) C-4I round i rregular round-up 0.85 (.81-.91) 0.85 (.81-.89) 0.5% shed (50% l ive) l i t t l e round, large, compact 50.2-72.8 19.6-43.3 5.4-7.6 2.7 (2.2-3.1) 2.4 (1.9-3.1) 13.6-13.8 (+0.57-+0.36) 1308-1367 C-4II i rregular i rregular remain irregular 0.74 (.67-.79) 0.66 (.47-.78) non-confluent: 1.2% shed (82% l ive) conf1uent: 2.0% shed (87% l ive) more i rregular, smaller, less compact 34.1- 51.7 31.2- 60.3 5.6-17.1 1.6 (1.0-2.0) 1.4 (1.2-1.8) 13.7-14.0 (+0.02-+0.18) 1330-1440 * Mean of MOP data of al l experiments. Range of values are presented within brackets. ** Values are range of percentage of separate experiments (Figs. 21-23). - 73 -Figs. 24 and 25. C-4I and C-4II cel l clumps, hours after t rypsinizat ion,  respectively: Figs. a. 0 hour after trypsinizat ion, x 135 Figs. b. 1 hour after trypsinizat ion, x 225 Figs. c. 3 hours after trypsinizat ion, x 225 - 75 -Table 17 SUMMARY OF THE EFFECTS OF DON TREATMENT ON C-4I CELLS CONTROL DON LOW DOSE" Growth inhibit ion Colony form* of: (i) smal1 colonies ( i i ) large colonies Cell shedding Aggregation of cel ls treated with DON: (i) prior to dissociation ( i i ) after dissociation round (.81-.91) round (.81-.89) l i t t l e ; 50% l ive aggregation; round clumps aggregation; round clumps Morphology** of single cel ls (Percentage of total number of c e l l s ) : (i) spherical 61% (50.2-72.8) ( i i ) i r regular ly -shaped 33% (19.6-43.3) ( i i i ) flattened 6% (5.4-7.6) Cell area* ( x lO - 3 mm2) i n : (i) small colonies 2.7 (2.2-3.1) ( i i ) large colonies 2.4 (1.9-3.1) Cell volume (um3) 1,308-1,367 rounder (short-term)/ i rregular (long-term) more irregular 6 fold increase; 78% l ive less aggregation; irregular clumps less aggregation; round clumps HIGH DOSE ++ irregular most irregular more (57.6-77.6) most (54.5-84.3) less (17.9-39.8) less (0-4.5) less (15.7-41.5) least (0-4.1) generally larger variable, generally larger al l s ignif icantly al l s ignif icantly 1arger 22% larger larger 43% larger * Mean of MOP data of al l experiments. Range of values are presented within brackets. ** Range of values of separate experiments are presented within brackets. - 76 -Table 18 SUMMARY OF THE EFFECTS OF DON TREATMENT ON C-4II CELLS CONTROL DON LOW DOSE" HIGH DOSE Growth inhibit ion Colony form* of: (i) smal1 colonies ( i i ) large colonies Cell shedding of: (i) non-conf1uent cultures ( i i ) confluent cultures Aggregation of ce l ls treated with DON: (i) prior to dissociation (i i ) after dissociation ++ i rregular (.67-.79) irregular (.47-.78) 1.2% shed; most l ive 2.0% shed; most l ive aggregation; irregular clumps aggregation; irregular clumps Morphology** of single cel ls (Percentage of total number of c e l l s ) : ( i ) spherical 43% (34.1-51.7) (i i ) i rregularly-shaped 46% (31.2-60.3) ( i i i ) flattened 11% (5.6-17.1) Cell area* (x lO - 3 mm2) i n : (i) small colonies 1.6 (1.0-2.0) ( i i ) large colonies 1.4 (1.2-1.8) Cell volume (um3) 1,330-1,440 inconclusive more irregular 2 fold increase; most l ive 5 fold increase; most l ive most irregular most irregular aggregation; irregular clumps less aggregation (borderli ne); irregular clumps more (28.5-65.1) less (19.0-51.9) more (27.7-71.6) less (0-12.4) variable, generally larger al l larger 13% larger most (45.8-80.7) least (0.9-2.3) al l s ignif icant ly larger al l s ignif icant ly larger 51% larger *, ** See legend of Table 17. - 77 -DISCUSSION Since i ts discovery in 1956 (Ehrlich et al_., 1956), DON has been characterized and its biological act iv i t ies analyzed (Dion e_t al_., 1956; Maxwell and Nickel , 1957). However, there have been few reports on the effects of DON on the morphology of cultured c e l l s . This study examines the effects DON has on two morphologically different types of cultured human cervical cancer c e l l s , in an attempt to understand the basis for their different growth patterns. However, prior to examining the effects of DON, the properties of the control C-4I and C-4II cel ls wil l be discussed. I. Control C-4I and C-4II ce l ls (Table 16) The growth patterns of certain cell types depend on the presence of calcium in the culture medium (Auersperg, 1969b; Hennings and Holbrook, 1983). In C-4I cultures, the maintenance of Ca+ +-dependent c e l l - c e l l contacts greatly influenced their growth patterns. In contrast, C-4II cultures grew relatively independently of their c e l l - c e l l contacts. The cohesiveness of C-4I cel ls could be due to the presence of numerous desmosomes and microvi l l i (Auersperg, 1969a), which were less numerous in the less cohesive C-4II cel l l ine . The growth patterns of these cell lines were substratum-independent. The greater abi l i ty of C-4I cel l sheets to retract into round clumps could, again, be due to the more numerous intercel lular contacts, while the relative absence of intercel lu lar contacts in C-4II cel ls could account for their relative inabi l i ty to retract as cohesive units (Figs. 24 and 25). The forms of small and large colonies suggest that C-4II cel ls tend to exist relatively independently of each other as the colonies - 78 -grow, since large C-4II colonies were more irregular than small ones. In contrast, both small and large C-4I colonies tend to grow cohesively, since small colonies were as round as large ones. Shedding patterns were different between the C-4 cell l ines . Shedding of C-4I cel ls might be limited by their numerous c e l l - c e l l contacts. In contrast, C-4II cel ls have fewer intercel lular contacts and this may be the reason why they shed more readily into the culture medium. C-4II cel ls were more eel 1-substratum interactive, which might have accounted for the greater abi l i ty of shed C-4II ce l ls to reattach and to prol i ferate. Many factors, such as serum factors and serum concentration, low molecular weight nutrients, plating density and anchorage dependence, regulate cell prol i feration in culture (Lanks and Kasambalides, 1980). It has been documented that most cel ls in culture do not proliferate unless they can attach and spread on a surface (Stoker et al_., 1968; Maroudas, 1973). In both cel l l ines , ce l ls in small colonies generally had larger projected cel l surface areas (computed as area of colony/number of nuclei) than those in large colonies (Tables 12 and 13). This could be explained by the fact that in large C-4I colonies, cultures were more s t rat i f ied (with more overlap of nuclei) while in C-4II colonies, ce l ls in large colonies were more columnar with genuinely smaller apical cel l surface areas. Since C-4I is a highly s t rat i f ied cell l ine , the cel l surface area was greatly underestimated because of multi-1ayering and overlapping of nuclei . In contrast, the estimated cel l surface area in C-4II cultures was more valid since st rat i f icat ion in these cultures is l imited. In small and large colonies, C-4I projected cell surface areas were always - 79 -larger than C-4II cel l areas, suggesting that C-4I ce l ls were also f lat ter than C-4II c e l l s . This would relate well with previous observations (from vertical sections) that the morphologies of confluent C-4I cel ls ranged from flattened to polygonal, while the morphologies of confluent C-4II ce l ls ranged from cuboidal to columnar (Auersperg, 1969a). The volume of C-4I cel ls was sl ight ly smaller than that of C-4II c e l l s . Since the projected cel l area of C-4I cel ls was larger than that of C-4II c e l l s , C-4I cel ls also must have been f la t te r . To summarize, the growth patterns of the two cel l lines are influenc-ed by both c e l l - c e l l and eel 1-substratum adhesions. However, the C-4I cel l l ine is more influenced by c e l l - c e l l than by eel 1-substratum interactions, while the reverse is true for the C-4II cell l ine . Multiple mechanisms of adhesion have been documented in many types of normal, embryonic and tumor cel ls and cell l ines (Takeichi, 1977; Magnani et aj_., 1981; Hayashi and Ishimaru, 1981; Fischer and Schachner, 1982). Many cel l types display different mechanisms of adhesion depending on the method of dissociat ion. Takeichi (1977) showed that cel ls with a calcium independent mechanism of adhesion formed less t ightly adhering clumps with decreased intercel lu lar contacts. Hayashi and Ishimaru (1981) isolated a cel l adhesive factor from island-forming rat ascites hepatoma c e l l s , but not from hepatoma cel ls that grew singly and did not form islands. When the latter cel ls were incubated with this adhesive factor, stable clumps with junctional complexes formed. Dembitzer (1980a) and Auersperg (1969b) showed that c e l l - c e l l and eel 1-substratum adhesions in C-4I cultures were qualitatively di f ferent ; the former was EDTA (ethylene dinitr i1otetraace-t i c acid, a cation chelator) sensitive and trypsin resistant, while the latter was trypsin sensitive and EDTA resistant. - 80 -II. DON Stabi l i ty In this study, UV absorption of 1 ug/ml DON in pH 7 phosphate buffer at 274 nm decreased by about 20% over 18 hours or 26% over 24 hours and by 25% over 18 to 90 hours at 37°C (Fig. 5, experiment 1). These results were not inconsistent with the results of Dion et a l . (1956). He reported that UV absorption of 32 ug/ml DON in pH7 phosphate buffer decreased by about 26% over 18 hours, and by 46% over 18 to 90 hours at a lower temper-ature of 30°C. There was a sl ight increase of absorption (about 3%) over 90 to 168 hours in this study (Fig. 5), while Dion reported a small decrease of 5% over the same time period (unfortunately, he did not measure beyond 168 hours). In the present study, the slight increase in UV absorption by DON could possibly be due to formation of DON breakdown products, which absorb UV rays. According to Dion (1982), there had never been any i n -vestigation of DON breakdown products. However, he assumed that DON degradation resulted in loss of the diazo group with subsequent replace-ment by a hydroxy1 group, which did not absorb UV radiation between 220-320 nm. Dion et a_l_. (1956) reported that the decrease of UV absorption by DON in 0.1N alkal i was accompanied by a decrease in act iv i ty against Torulop- sis albida. Hence, i t was assumed by Dion and in this study that UV absorption was equivalent to DON biological act iv i ty . III. DON-induced changes in growth'pattern'of"C-4"ce1Is,"as "represented  by changes in colony form. With DON treatment, small C-4I colonies showed a biphasic response: single doses produced rounder colonies while multiple doses produced more - 81 -irregular ones. In contrast, the results with small C-4II colonies were inconsistent. Large colonies in both cell lines became more i rregular , with more signif icant changes in C-4II colonies. Increase in the roundness of colonies could be due to factors such as: (i) a uniform inabi l i ty of colonies to spread so that colonies remained round and unspread (possibly caused by DON decreasing eel 1 -substra-tum adhesion), or ( i i ) increased intercel lu lar cohesion within the colony. In this study, since DON probably decreased cel l cohesion, factor ( i i ) is unlike-ly . Hence, i t is possible that colonies, especially small ones, have d i f f i cu l ty adhering to and spreading onto the substratum; this d i f f i cu l ty may be even more enhanced in the presence of DON. Increase in i rregularity of colonies could be due to: (i) cel ls in a colony spreading non-uniformly, ( i i ) an uneven retraction of colony edges due to an inabi l i ty of the cel ls to adhere, or ( i i i ) decreased intercel lu lar cohesion so that cel ls in a colony do not act as part of a cohesive unit. Changes in colony form were complex since they were influenced by many factors such as colony sizes, c e l l - c e l l and eel 1-substratum inter -actions, and the number of doses of DON added. Small control colonies in both cel l lines were always more variable in form than large colonies (Appendix I). This, again, suggests that small colonies adhered and spread less readily than large colonies since small colonies had smaller substratum adhesion areas than large colonies. In addition, small colonies, unlike large colonies, had a higher - 82 -proportion of cel ls around the colony edge compared to cel ls in the center of the colony. In addition, since small C-4I colonies were less s t r a t i -f i e d , they were probably less influenced by c e l l - c e l l , than by ce l l - sub -stratum, interactions. Unlike C-4I colonies, C-4II colonies were less cohesive as a unit, the cel ls grew more independently of each other as colonies enlarged and there were no morphologic distinctions observed between rim/edge and center c e l l s . IV. Effect of DON on cel l shedding. In this study, i t was shown that DON decreased c e l l - c e l l adhesion in C-4I and in confluent C-4II cultures. This resulted in increased numbers of shed c e l l s , of which the majority were l i ve , and in a change to less s t rat i f ied C-4I and to less columnar C-4II cultures. Cell shedding or cel l detachment is a phenomenon associated with tumor necrosis (Weiss, 1977a and 1978). Lysosomal enzymes from necrotic sites have been shown to fac i l i ta te cel l detachment from tumors. Thus, i t is possible that metastasis is fac i l i tated by lysosomal enzymes acting on tumors (Weiss, 1977a and 1978) and/or host tissues such as basement membranes (Liotta et a_l_., 1980). Such enzymes have been thought to be of both tumor and non-tumor (e.g. host macrophage) or ig in . In this study, one suggestion was that necrotic materials and degradative enzymes produc-ed by damaged DON-treated cel ls could have increased cel l shedding. This possib i l i ty was not very l i ke l y , since in vivo, the presence of necrotic centers in C-4I tumors grown in hamsters was not associated with increased cel l detachment, or with a dispersive or in f i l t ra t i ve growth pattern. Similar ly , C-4II tumors grown in hamsters had an in f i l t ra t i ve growth pattern even though they did not have necrotic centers (Auersperg, 1969a). - 83 -Even though DON increased cel l shedding in both C-4 cultures, i t was not determined i f these DON-treated shed cel ls were prol i ferat ive . Even i f they were less prol i ferat ive in culture, they might s t i l l be able to form metastases in vivo. Tumor cel ls from a single tumor have heterogenous prol i ferat ive and metastatic potentials (Weiss, 1977b; F id le r , 1978; Liotta et aj_., 1980; Fidler and Hart, 1981; Easty et al_., 1981). Hence, metastatic potential is not necessarily directly related to the number of cel ls detached or to the c e l l ' s prol i ferat ive capacity in  v i t ro . Instead, metastatic potential has been correlated with enzymatic degradation of collagen in basement membranes (Liotta et al_., 1980; DeVore et al_., 1980; Alhadeff and Holzinger, 1981), with cel l surface properties (Yogeeswaran and Salk, 1981; Alhadeff and Holzinger, 1981), with increased GAG content in tumor parenchyma and capsule (Toole et aj_., 1979) and with abi l i ty to inhibit embryonic cell aggregation (Mas!ow et al_., 1976; Weiss and Maslow, 1980). DON is currently being investigated as a potential anti-tumor drug. This study showed that DON increased cell shedding in v i t ro . However, further experiments wil l have to be conducted to determine i f DON does increase cell shedding in vivo, causing increased metastasis. Obviously, the treatment of cancers with such chemotherapeutic drugs, which increase metastasis, would be detrimental to the cancer patient. V. Cell Aggregation. DON pretreatment caused a signif icant decrease in c e l l - c e l l aggregation and a decrease in cell spreading or cell deformation within C-4I clumps in suspension culture. However, similar pretreatment in C-4II cultures did not produce consistent decreases in c e l l - c e l l aggregation. - 84 -Control and pretreated C-4II aggregates were similar morphologically. In C-4I cultures, the DON-induced decrease in aggregation could have been due to several factors, such as: ( i) a decrease or alteration of cel l surface adhesive components, ( i i ) a decreased rate of recovery of adhesive trypsin-sensit ive and DON-sensitive cell surface components, or ( i i i ) cel l damage, due to long-term DON pretreatment. Since pretreated cultures after 48 hours of shaking had more clumps than cultures after 24 hours of shaking, the f i r s t two factors could possibly be true. If DON pretreatment caused a depletion of glycosylated stores in t race l lu lar ly , then: (i) the cell surfaces would be depleted of glycosylated adhesive cel l surface components (which would be consistent with the observation that DON-treated cultures were always more easily dissociated with trypsinthan control cultures), and ( i i ) the regeneration of glycosylated trypsin-sensit ive and DON-sensitive components would be slower in DON-pretreated than in control cultures. The irregularity and decreased compactness of DON-treated clumps could be due to a decrease in adhesion, in cel l deformabi 1 ity or cel l re-arrangement between DON-pretreated c e l l s . Cell re-arrangement within an aggregate occurs in cel l sort ing, a phenomenon that occurs after heterogenous cel ls have adhered to form mixed aggregates. Cells within mixed aggregates tend to sort out, so that l ike cel ls that form the inner sphere are different from those that form the outer sphere. Many theories attempt to explain this sorting-out phenomenon. One is that desmosomes influence eel 1-sorting. Cells which form more desmosomes tend to sort out internal ly , in contrast to cel ls of the exterior that have fewer - 85 -desmosomes (Overton, 1977; Wiseman and St r ick ler , 1981).. Desmosomes are adherens-type junctions (Staehelin and Hul l , 1978) that rapidly form in culture. Overton (1977) showed that in embryonic c e l l s , desmosomes formed as early as 3 hours. Dembitzer et al_. (1980b) demonstrated that C-4I cel ls with repaired cel l surfaces reformed desmosomes within 90 minutes. In this study, C-4I re-arrangement (l ike desmosome formation) was also demonstrated to be a rapid process. F ig . 24b showed that cel l re-arrangement had already begun within 1 hour, while f i g . 24c showed that cell re-arrangement was complete within 3 hours. It is l ikely therefore, that desmosomes play a role in cell re-arrangement in C-4I cultures. Unlike the C-4I cel l l ine , the C-4II l ine is a relatively desmosome-free one. Hence, even with DON pretreatment of C-4II c e l l s , the clumps that resulted remained irregular (like control clumps, with no cell re-arrangement), and the DON-induced inhibit ion of aggregation was inconstant and ins igni f icant . Therefore, the results suggest that desmosomes are one factor affected by DON pretreatment. Intercellular glycoproteins of desmosomes have been isolated (Gorbsky and Steinberg, 1981). It was found that desmosome cores were rich in glycoproteins of specif ic molecular weights (100,000; 115,000; 150,000 daltons). It is possible that DON, which inhibited glycosylation, also inhibited desmosomal glycoprotein synthesis. In both cel l l ines , DON which was added 18 hours after trypsinization decreased c e l l - c e l l aggregation only s l ight ly . This could be due to the fact that during the 18 hour recovery period, glycosylated stores and cel l surface components had already been restored. It is also possible that cultures had not been treated with DON for a suff icient period necessary - 86 -to produce clearly observable differences between control and treated cultures. In the C-4II cel l l ine , 6 hours of DON treatment was insuff ic ient for differences in aggregation between control and treated cultures to be detected. However, longer periods of DON treatment (18 and possibly 12 hours) produced a small inhibit ion of aggregation. Thus, there was a lag period between the time DON was added to cultures and the time DON-induced effects were suff icient to be quantitated. However, the lag period, which was more than 6 hours but less than 18 hours, was less than that previously assumed on the basis of visually detected differences in colony and cel l form and growth which were observed on the second day after DON addition. The lag period l ikely represents the time taken to deplete intracel lu lar glycosylated stores and/or the time of turnover of DON-sensitive cel l surface materials. Considering al l experiments in both cel l l ines , the effects of GSA on aggregation of DON-treated cel ls were not entirely clear. However, in some experiments, there were indications that DON plus GSA cultures were more aggregated than DON-treated cultures, and similar to control c u l -tures. Similarly , the addition of GSA alone to C-4II cultures produced inconclusive results. Of 3 experiments, only 1 experiment indicated that addition of GSA to shaking cultures increased c e l l - c e l l aggregation. Since the effects of DON on trypsin-recovered cel ls were small, i t was d i f f i cu l t to determine i f these small DON-induced effects could be prevented or diminished by the concurrent addition of GSA. Perhaps, the addition of GSA to DON-pretreated cel ls would have been preferable as results in those experiments were more evident and any changes due to GSA might have been more easily detected. - 87 -Previous autoradiography studies by Lee (1978) showed that over 90% of labelled GSA in C-4II cultures was extracel lular , irregardless of whether label l ing was for 3 hours or 8 days after cultures were sub-cultured or after they reached confluency. In al l cultures, most extra-ce l lu lar label was found between c e l l s . More label was basally- than apical ly - located. In addition, cultures labelled after they were con-fluent had a higher total number of grains per cell and a higher percen-tage of grains labelled in terce l lu lar ly , than cultures labelled after they were subcultured. This observation lends further support to the original hypothesis that the secretion of GSA-containing extracellular materials might influence the shedding of cel ls into culture medium; since both phenomena were more prominent in confluent cultures. My studies did not prove conclusively that DON-treated cel ls aggregated more in the presence of GSA or that DON decreased GAG synthesis. However, other researchers have shown that DON decreased labelled GSA incorporation into cel ls (Greene and Pratt, 1977) and labelled sulfate incorporation into GAGs (Pratt et al_., 1976; Spooner and Conrad, 1977; Green and Pratt, 1977; Hurmerinta and Thesleff, 1982). Since GSA-containing macromolecules have adhesive intercel lu lar functions (Pessac and Defendi, 1972; Yamada and Olden, 1978; Mosher and Furcht, 1981), their depletion (presumably due to DON) would be expected. to cause increased cell shedding from confluent C-4I and C-4II cultures and less from non-confluent C-4II cultures [since the latter have less GSA-label (Lee, 1978) and hence, less GSA-containing components in terce l lu lar l y ] . This could well explain this project's results on cel l shedding. With regards to Lee's work on C-4II cultures, one could attempt to explain his observations, that cultures labelled after reaching confluency had more label intercel lu lar ly than - 88 -cultures labelled after subculture, by considering the changes in morphology of C-4II cel ls as they grew from a non-confluent to a confluent state. Non-confluent cel ls were usually flattened against the substratum; hence, the number and percentage of grains located intercel1ularly would be low. In contrast, confluent c e l l s , being cuboidal or columnar, might have more grains located interce l lu lar ly . Hence, the percentage of grains located intercel lu lar ly over the total number of extracellular grains would also be higher. VI. Morphology of single adherent c e l l s . Cell morphology was indicative of eel 1-substratum adhesiveness and interactions (Harris, 1973). According to Harris, cel ls accumulated on substrata according to a hierarchy of eel 1-substratum af f in i ty which corresponded to the order of relative wettability of the substratum. In addition, the morphology of cel ls altered with changes in the substratum. Cells were flattened on more wettable substratum, but were rounder and more easily detached on less wettable substratum. The results of this study indicated that in both cel l l ines , DON signif icantly decreased the spreading and adhesion of cel ls to plastic substratum, as indicated by the decrease in the proportion of f 1 attened  single c e l l s . However, DON-treated C-4II ce l ls showed a larger decrease in percentage of fu l ly spread or flattened cel ls than DON-treated C-4I c e l l s . This could be due to the fact that the C-4II cel l l ine was the more eel 1-substratum interactive cel l l ine of the two. These treated C-4II cel ls detached incompletely, became partly adherent and i r regular ly -shaped. In contrast, since C-4I cel ls were less adhesive to the substra-tum, C-4I ce l ls detached more readily and completely with DON treatment - 89 -and became spherical or minimally spread. The percentage of least adhesive spherical C-4I and C-4II cel ls might even have been higher since some of these cel ls might have detached and floated off into the medium. In addition to results on cel l shedding in this study, a review of past l i terature has shown that DON also increased absolute numbers and the percentage of detached cel ls in fibroblast cultures (Spooner and Conrad, 1975). The observations that C-4II ce l ls were more cell-substratum interactive were also supported by studies by Auersperg (1969a and 1969b) who showed that C-4I clumps adhered preferentially to C-4I colonies rather than to the substratum, in contrast to C-4II clumps which adhered pre-ferential ly to the substratum rather than to C-4II colonies. VII. Effect of DON on projected surface areas of cel ls within colonies. In both cel l l ines , there was a trend towards an increase in project-ed cel l surface areas with DON treatment. This increase was signif icant in a l l large C-4I and in most large C-4II colonies. The increase in cel l area in both cel l lines was due, at least in part, to an increase in cel l volume and an observed increase in colony flatness (Figs. 8a-b, 9a-b, and lOa-b), but other factors may have been involved. A decrease in s t rat i f icat ion in the C-4I cel l l ine might account partly for an apparent increase in cell area. However, this decrease in st rat i f icat ion would not account for the increase in cell area of C-4II cel ls since s t rat i f i cat ion of C-4II cultures was l imited. Instead, the increase in cel l area of DON-treated C-4II cultures might have been due to an increase in colony flatness associated with a decrease in cel l density. - 90 -VIII. Effect of DON on cel l volume. This study conclusively showed that in both cell l ines, the increase in cel l area with DON treatment was part ial ly due to an increase in cell volume. This increase in cell volume could be due to several factors, one of which was damage to the c e l l , resulting in cell swelling. Another factor could be an inhibit ion of cel l d iv is ion. DON has been shown to inhibit DNA synthesis by inhibit ing glycine and formate incorporation into nucleic acids (Maxwell and Nickel, 1957). Hence, inhibit ion of DNA synthesis by DON treatment might have inhibited cel l division and growth in the C-4 cel l l ines. In this study, even though mitotic figures were occasionally observed in DON-treated cultures, the possibi l i ty that inhibit ion of DNA synthesis resulted in an increase in cell volume cannot be ruled out. Erlinger and Saier (1982) found that kidney epithelial cel ls from a less dense culture had larger volumes than those from dense cultures. Hence, i t is also possible that C-4 cel ls might display similar properties. Further experiments would determine i f the volume of C-4 cel ls is inversely proportional to cell density. IX. Relationship between colony form and s ize , cel l s i ze , and dose of DON. C-4I Low doses of DON increased the roundness of small colonies. This might be due to an inabi l i ty of colonies to spread, as a result of a de-crease in eel 1-substratum adhesion. When small colonies were treated with higher doses, the decrease in eel 1-substratum adhesion caused not only an inabi l i ty of colonies to spread, but also an uneven retraction of colony edges that had i n i t i a l l y been adhering to the substratum. Decreased - 91 -intercel lu lar cohesion might also have caused cel ls to spread as part of a less cohesive unit (forming irregular colonies) and to shed in increased numbers (forming less s t r a t i f i e d , f lat ter colonies and giving the impression that cel ls had larger projected surface areas). These reasons might also account for the increase in i rregularity and flatness of large DON-treated colonies. C-4II Low doses of DON decreased both types of adhesions in this less co-hesive cell l ine . Thus, DON-treated cel ls in small colonies became more l ike DON-treated single cel ls which were less adherent and either became more irregular or rounder. A high dose also decreased eel 1-substratum adhesion since treated single cel ls and treated colonies were mainly irregular (partly adher-ent). Cells that rounded up might have detached from the substratum, causing increased shedding. A high dose also decreased c e l l - c e l l adhesion causing cel ls in a colony to be less cohesive and more independent, holes to appear between cel ls within colonies, and the colonies to be s i g n i f i -cantly more irregular. X. Conclusion. DON not only inhibited the growth of C-4I and C-4II c e l l s , but i t also changed their colony morphology, degree of s t ra t i f i ca t ion , shedding patterns, cell areas and volumes, and spreading on plastic substratum. In addition, treatment of C-4I ce l ls with DON prior to cel l dissociation resulted in their decreased aggregation in shaker cultures and in an altered morphology of aggregates, resembling that of C-4II clumps. - 92 -This study and that of Conrad and Spooner (1975) indicate that DON increased the shedding of cel ls that were grown in v i t ro . It is presently unknown i f DON would increase cell shedding in vivo, possibly resulting in increased metastasis. Recent l i terature has reported that some drugs, with anti-tumor act iv i ty , also enhance metastasis (Heppner et aj_., 1978). Hence, i f DON has such properties, i t may prove disadvantageous to treat tumors in patients with DON. Further experiments would resolve this question. Results of this study suggest that DON decreased synthesis of DON-sensitive extracellular materials (ECM) that were responsible for the cohesiveness of C-4I c e l l s . Cohesiveness of C-4II cel ls was also decreased, but to a lesser extent. From the results of this study, i t is unlikely that cel l shedding or dispersion of C-4II ce l l s in vitro is a result of secretion of ECM. If this was the case, then treatment of C-4II cel ls with DON would presumably decrease ECM synthesis resulting in a decrease in cel l shedding. The opposite effect of DON was observed. In the course of this study, i t was observed that C-4I c e l l s , with DON treatment, came to resemble C-4II cel ls with regards to their colony form, shedding pattern, decreased aggregation in shaker cultures and clump morphology. An interpretation of these observations is that C-4II ce l ls may be less cohesive because they produce less DON-sensitive, cohesive ECM. In contrast, C-4I cel ls may be producing more of such ECM, resulting in cultures that grow cohesively and which do not shed much. Hence, with DON treatment, C-4I cel ls would be less cohesive—they would tend to produce more viable shed c e l l s , fewer aggregated clumps which are also smaller and more irregular, and also, irregular colonies. - 93 -SUMMARY 1. DON, a potential chemotherapeutic drug, was shown in vitro to inhibit the growth of C-4I and C-4II c e l l s , two cell lines that were or ig ina l -ly derived from one human cervical carcinoma biopsy specimen. How-ever, in addition to i ts growth inhibitory act iv i ty , DON also had numerous other effects on their growth and shedding patterns, and morphology. 2. On visual observation, DON-treated C-4 cultures showed changes in colony form, cel l s i ze , degree of s t rat i f icat ion and degree of cel l f lattening, c e l l - c e l l contacts, nuclear/cytoplasmic rat ios, and number and morphology of single adhering c e l l s . Some of these changes were quantitated. 3. By quantitating projected colony form and cel l s izes, i t was found that in general, large colonies in both cel l lines became s igni f icant -ly more irregular and cel ls were signif icantly larger in surface areas. However, results from small colonies were less prominent and more variable. Changes in growth pattern, or changes in colony forms, could have resulted from changes in c e l l - c e l l and/or eel 1-substratum adhesions. Increase in cel l areas was determined to be due to an increase in cel l volume and, possibly, an increase in colony f latness. 4. Shedding patterns of DON-treated C-4I and confluent C-4II cultures indicated that DON induced shedding in C-4I cultures, while shedding was enhanced in C-4II c e l l s . Most DON-treated shed cel ls were l ive as determined by dye exclusion tests . Increased shedding was probably due to decreased c e l l - c e l l cohesion, which probably also - 94 -accounted for the observed "holes" (or areas of decreased c e l l - c e l l contact) within colonies. 5. When C-4I ce l l s (from cultures treated with DON prior to dissociation into single ce l ls ) were shaken in gyratory shakers, the clumps that formed after 24 hours were small, irregularly-shaped (resembling C-4II clumps) and signif icantly fewer than those from control cultures. It was clear that c e l l - c e l l cohesion was greatly decreased in C-4I c u l -tures. In contrast, C-4II cel ls from DON-treated cultures did not show a signif icant decrease in aggregation. 6. In both cel l l ines , i f single cel ls were treated with DON while they were recovering from the effects of dissociat ion, an incubation period with DON of 18 hours was suff ic ient to inhibit cell aggregation. An incubation period of 6 hours had no effect . However, i f cel ls were slowly shaken in the presence of DON for a further 24 hours, a small but consistent decrease in aggregation was noted in treated cultures. Attempts to reverse DON effects by glucosamine addition gave inconclu-sive results. 7. DON also decreased cell-substratum adhesion, by decreasing the spread-ing of single cel ls on the substratum in both cell l ines . 8. To conclude, since DON did not decrease cell dispersion or shedding in  v i t ro , the process of cel l shedding did not seem to result from syn-thesis and secretion of ECM. On the contrary i t suggests that ECM is necessary for adhesion. The basis for the difference in growth patterns between the two cel l lines may be that c e l l - c e l l cohesion was dependent on the synthesis and secretion of DON-sensitive cell sur-face-associated ECM. If C-4I cel ls (being more cohesive) produced - 95 -more of this cohesive ECM than less cohesive C-4II c e l l s , then DON-treated C-4I cel ls would resemble C-4II c e l l s . Therefore, the results of this study tend to support the alternate hypothesis that the syn-thesis and secretion of DON-sensitive, cohesive cell surface-associated extracellular material is associated with a compact growth pattern of tumors, and that a decrease in the production of such ECM may lead to a dispersed or in f i l t ra t i ve pattern of growth. Examples of such extracellular macromolecules include hyaluronate and f ibro -nectin, both of which bind to eel 1-surfaces and have cohesive func-t ions. 9. The investigation of the tumor-inhibitory activity of potential chemo-therapeutic agents should also include a study of other effects of the agent on the tumor. For example, the effects of the agent on the tumor's growth pattern should also be considered. This study showed that DON increased cel l shedding from cultured tumor c e l l s . The pertinent question is whether i t would have similar cell dispersion -enhancing effects in vivo. - 96 -BIBLIOGRAPHY Alhadeff, J . A . , and Holzinger, R.T. (1981). Sialyltransferase, s i a l i c acid and sialoglycoconjugates in metastatic tumor and human l iver t issue. Int. J . Biochem. 14:119-126. Auersperg, N. (1969a). Histogenetic behavior of tumors. I. Morphologic variations in vitro and in vivo of two related human carcinoma cell l ines . J . Nat. Cancer Inst. 43:151-173. Auersperg, N. (1969b). Histogenetic behavior of tumors. II. Roles of cel lu lar and environmental factors in the in vitro growth of carcinoma c e l l s . J . Nat. Cancer INst. 43:175-190. Auersperg, N. (1972). Histogenetic behavior of tumors. III. Possible relationships to patterns of glycolysis . J . Nat. Cancer Inst. 48:1589-1596. Auersperg, N., and Worth, A. (1966). Growth patterns in vitro of invasive squamous carcinomas of the cervix - A correlation of cu l tura l , h istologic , cytogenetic and c l in ica l features. Int. J . Cancer J L :219-238. Auersperg, N., Erber, H., and Worth, A. (1973). Histologic variation among poorly differentiated invasive carcinomas of the human uterine cervix. J . Nat. Cancer Inst. 51:1461-1477. Auersperg, N., and Erber, H. (1976). Effects of tissue culture environ-ment on growth patterns and ultrastructure of poorly differentiated human cervical carcinomas. J . Nat. Cancer Inst. 57:981-994. Burchenal, J .H . (1979). Antitumor effects of azaserine and DON. Cancer Treat. Rep. 63:1031-1032. Cabanillas, F. (1979). DON. Drugs of the Future 6^:572-576. Catane, R., Daniel, D.V.H., Glaubiger, D.L. , and Huggia, F.M. (1979). Azaserine, DON, and Azotomycin: Three diazo analogs of L-glutamine with c l in ica l antitumor act iv i ty . Cancer Treat. Rep. 63:1033-1038. Cooney, D.A., Hiremagalur, N., Jayaram, Milman, H.A., Homan, E.R., P i t t i l o , R., Geran, R.I. , Ryan, J . , and Rosenbluth, R.J. (1976). DON, C0NV and D0NV - III. Pharmacologic and toxic studies. Bioc. Pharm. 25:1859-1870. Culp, L.A. , Ansbacher, R., and Domen, C. (1980). Adhesion sites of neural tumor c e l l s : Biochemical Composition. Biochem. 19:5899-5907. Dembitzer, H.M., Schermer, A . , Herz, F. , and Koss, L.G. (1980a). A pre-liminary fine structural analysis of c e l l : c e l l and eel 1:substrate adhesion in C4I. 38th Ann. Proc. Electron Microscopy Soc. Amer.: 770-771. - 97 -Dembitzer, H.M., Herz, F. , Schermer, A . , Wolley, R.C., and Koss, L.G. (1980b). Desmosome development in an in vitro model. J . Cell B io l . 85:695-702. DeVore, D.P. , Houchens, D.P. , Ovejera, A .A. , D i l l , J r . , G.S. , and Hutson, T.B. (1980). Collagenase inhibitors retarding invasion of a human tumor in nude mice. Expl. Cell B io l . 48:367-373. Dion, H.W. 1982. Personal Communication. Dion, H.W., Fusari , S .A . , Jakubowski, Z . L . , Zora, J . G . , and Bartz, Q.R. (1956). 6-diazo-5-oxo-L-norleucine, a new tumor-inhibitory substance. IV. Isolation and characterization. J . Am. Chem. Soc. 78: 3075-3077. Duvall, L.R. (1960). 6-dizao-5-oxo-L-norleucine. Cancer Chemo. Rep. ^ :86-98. Easty, D.M., Easty, G.C. , Carter, R.L. , Monaghan, P., and Butler, L .J . (1981). Ten human carcinoma cel l l ines derived from squamous carcinomas of the head and neck. Brit ish J . Cancer 43:772. Ehr l ich, J . , Coffey, G .L . , Fisher, M.W., Hi 11igas, A .B . , Kohberger, D.L. , Machamer, H.E. , Rightsel, W.A., and Roegner, F.R. (1956). 6-diazo-5-oxo-L-norleucine. A new tumor-inhibitory substance. Anti . and Chemo. 6^:487-497. Ekblom, P., Lash, J.W., Lehtonen, E . , Nordling, S . , and Saxen, L. (1979). Inhibition of morphologic cel l interactions by 6-diazo-5-oxo-norleucine (DON). Expl. Cell Res. 121:121-126. Erl inger, S . , and Saier, M.H. (1982). Decrease in protein content and cel l volume of cultured dog kidney epithelial ce l ls during growth. Importance for transport measurements. In Vitro 18:196-202. F id ler , I.J. (1978). Tumor heterogeneity and the biology of cancer i n -vasion and metastasis. Cancer Res. 38:2651-2660. F id ler , I.J., and Hart, J .R. (1981). The origin of metastatic hetero-geniety in tumors. Europ. J . Cancer 17:487-494. Fischer, G. , and Schachner, M. (1982). In vitro reaggregation of d i s -sociated mouse cerebellar c e l l s . I. Demonstration of different aggregation mechanisms. Expl. Cell Res. 139:285-296. Funderburg, F.M., and Markwald, R.R. (1981). Decreased synthesis and secretion of matrix glycosaminoglycans (GAG) accompanies altered formation and migration of endocardial cushion tissue (ECT) cel ls in the embryonic chick. J . Cell B i o l , abstracts 91:160a. Ghosh, S . , Blumenthal, H.J . , Davidson, E. , and Roseman, S. (1960). Glucosamine metablolism. V. Enzymatic Synthesis of glucosamine-6-phosphate. J . B io l . Chem. 235:1265-1273. - 98 -Gorbsky, G. , and Steinberg, M.S. (1981). Isolation of the intercel lular glycoproteins of desmosomes. J . C e l l . B io l . 90:243-248. Greene, R.M., and Pratt, R.M. (1977). Inhibition by DON of rat palatal glycoprotein synthesis and epithel ial cel l adhesion in v i t ro . Expl. Cell Res. 105:27-37. Harris, A. (1973). Behavior of cultured cel ls on substrata of variable adhesiveness. Expl. Cell Res. 77;285-297. Hayashi, H., and Ishimaru, Y. (1981). Morphological and biochemical aspects of adhesiveness and dissociation of cancer c e l l s . Int. Rev. of Cytology 70:139-215. Heaysman, J . E . M . , and Pegrum, S.M. (1973). Early contacts between f ibroblasts . An ultrastructural study. Expl. Cell Res. 78:71-78. Hennings, H., and Holbrook, K.A. (1983). Calcium regulation of c e l l - c e l l contact and differentiation of epidermal cel ls in culture. An ultrastructural study. Expl. Cell Res. 143: 127-142. Heppner, G.H., Dexter, D.L. , DeNucci, T . , M i l le r , F.R., and Calabresi, P. (1978). Heterogeneity in drug sensit iv i ty among tumor cell sub-populations of a single mammary tumor. Cancer Res. 38:3758-3763. Hiremagalur, N. Jayaram, Cooney, D.A., Milman, H.A., Homan, E.R., and Rosenbluth, R.J. (1976). DON, CONV and DONV - I. Inhibition of L-asparagine synthetase in v i t ro. Bioc. Pharm. 25:1571-1582. Hurmerinta, K., Thesleff, I., and Saxen, L. (1979). Inhibition of tooth germ differentiation in vitro by diazo-oxo-norleucine (DON). J . Emb. Expl. Morpho. 50:99-109. Hurmerinta, K., and Thesleff, I. (1982). Diazo-oxo-norleucine (D0N)-induced alterations in the extracellular matrix of the mouse tooth germ. Cell D i f f . n_:107-113. Lanks, K.W., and Kasambalides, E.J . (1980). Factors that regulate prol i feration of normal and transformed cel ls in culture. Pathobiol. Ann. _L0:35-50. Lee, H.C. (1978). The role of calcium and extracellular materials in the vitro growth and contract i l i ty of carcinoma c e l l s . M.Sc. thesis , UBC, pp. 101-102. Linsenmayer, R.F. , and Kochhar, D.M. (1979). In vitro cartilage forma-t ion : Effects of 6-diazo—5-oxo-L-norleucine (DON) on glycosamino-glycan and collagen synthesis. Dev. B io l . 69:517-528. L io t ta , L .A. , Tryggrason, K., Garbisa, S . , Hart, I., Fo l tz , C M . , and Shafie, S. (1980). Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67-68. - 99 -Livingstone, R.B., Venditt i , J . M . , Cooney, D.A., and Carter, S.K. (1970). Glutamine antagonists in chemotherapy. Adv. Pharm. and Chemotherapy 8:57-120. Magnani, J . , Thomas, W.A., and Steinberg, M.S. (1981). 2 dist inct adhesion mechanisms in embryonic neural retina c e l l s . I. A Kinetic analysis. Dev. B io l . 81:96-105. Maroudas, N.G. (1973). Growth of fibroblasts on linear and planar anchorages of l imiting dimensions. Expl. Cell Res. 81:104-110. Marx, J . L . (1982). Tumors: A mixed bag of c e l l s . Sc i . 215:275-277. Maslow, D.E. , Mayhew, E . , and Minowada, J . (1976). Differential inhibit ion of embryonic cel l aggregation by cultured human cel ls with "malignant" or "normal" characterist ics. Cancer Res. 36:2707-2709. Maxwell, R.E. , and Nickel , V.S. (1957). 6-diazo-5-oxo-L-norleucine, a new tumor-inhibitory substance. V. Microbiologic studies of mode of action. Anti . and Chemo. _7:81-89. McGuire, E.J. (1972). A possible role for carbohydrates in c e l l - c e l l adhesion. In: Membrane Research, pp. 347-368. Mikuni-Takagaki, Y. , and Toole, B.P. (1980). Cel1-substratum attach ment and cel l surface hyaluronate of rous sarcoma virus-transformed chondrocytes. J . Cell B io l . 85:481-488. Mosher, D.F. , and Furcht, L.T. (1981). Fibronectin: Review of i ts structure and possible functions. J . Invest. Derm. 77:175-180. Overjera, A .A . , Houchens, D.P. , Catane, R., Sheridon, M.A., and Muggia, F.M. (1979). Efficacy of 6-diazo-5-oxo-norleucine and N-(N-o-glutamyl-6-diazo-5-oxo-norleuci ne) -6-diazo-5-oxo-norleuci ne agai n-st experimental tumors in conventional and nude mice. Cancer Res. 39:3220-3224. Overton, J . (1977). Formation of junctions and cell sorting in aggrega-tion of chick and mouse c e l l s . Dev. B io l . 55:103-116. Pessac, B., and Defendi, V. (1972). Cell aggregation: role of acid mucopolysaccharides. Sc i . 175:898-900. Petkau, J . , and Crapeau, H. (1983). Department of Mathematics, UBC. Personal communication. P i t t i l l o , R.F. , and Hunt, D.E. (1967). Azaserine and 6-diazo-5-oxo-L-norleucine (DON). In: Antibiotics I, pp. 481-493, Springer-Verlag, New York. Pratt , R.M., Goggins, J . F . , Wilk, A . L . , and King, C.T.G. (1973). Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation and fusion. Dev. B i o l . 32:230^237. - 100 -Rando, R.R. (1975). On the mechanism of action of antibiotics which act as irreversible enzyme inhibitors . Bioc. Pharm. 24:1153-1160. Roblin, R. (1981). Contributions of secreted tumor cell products to metastasis. In: Cancer Biology Reviews. Vol. 2^:59-94. Marcel Dekker, New York. Rosenbluth, R . J . , Cooney, D.A., Hiermagalur, N. Jayaram, Milman, H.A., and Homan, E.R. (1976). DON, CONV and DONV. II. Inhibition of L-asparagine synthetase in vivo. Bioc. Pharm. 25:1851-1858. Rosenfeld, H., and Roberts, J . (1981). Enhancement of antitumor act iv i ty of glutamine antagonists 6-diazo-5-oxo-L-norleucine and Aciv ic in in cel l culture by glutaminase-asparaginase. Cancer Res. 41:1324-1328. Spooner, B.S. , and Conrad, G.W. (1975). The role of extracellular mater-ial in cel l movement. I. Inhibition of mucopolysaccharide syn-thesis does not stop ruff l ing membrane activity of cel l membrane. J . Cell B io l . 65:286-297. Skyvova, M., Kocent, A . , and Vermouseki, I. (1973). Host-tumor re lat ion-ship. XXXI. Acid glycosaminoglycans in the plasma and in the asc i -t i c f lu id of rats during experimental tumor growth. Neoplasma 20:181-188. Staehelin, L .A. , and Hul l , B.E. (1978). Junctions between l iv ing c e l l s . Sc. Amer. 238:141-152. Steinberg, M., Armstrong, P.B. , and Granger, R.E. (1973). On the re-covery of adhesiveness by trypsin-dissociated c e l l s . J . Membr. B io l . 21 :98-128. Stoker, M., O 'Ne i l l , C , Berryman, S . , and Waxman, V. (1968). Anchorage and growth regulation in normal and virus-transformed ce l l s . Int. J . Cancer 2-*683-693. Takeichi, M. (1977). Functional correlation between cell adhesive properties and some cel l surface proteins. J . Cell B i o l . 75: 464-474. Toole, B.P. , Biswas, C , and Gross, J . (1979). Hyaluronate and invasive-ness of the rabbit V2 carcinoma. Proc. Natl. Acad. Sc. 76:6299-6303. Turley, E. (1980). The control of cytodifferentiation by extracellular matrix. D i f f . _T7:93-103. Weisman, L .L . , and St r ick ler , J . (1981). Desmosome frequency: experimen-tal alteration may correlate with differential cell adhesion. J . Cell S c i . 49:217-223. Weiss, L. (1977a). Tumor necrosis and cell detachment. Int. J . Cancer 20:87-92. - 101 -Weiss, L. (1977b). A pathobiologic overview of metastasis. Seminars in Oncology 4-:5-17. Weiss, L. (1978). Some mechanisms involved in cancer cell detachment by necrotic material. Int. J . Cancer 22:196-203. Weiss, L. , and Maslow, D.E. (1980). In vitro studies on the interactions of tumor and non-tumor c e l l s . In: Tissue culture in medical  research (II), pp. 117-124. Pergamon, N.Y. Yamada, K.M., and Olden, K. (1978). Fibronectins - adhesive glyco-proteins of cell surface and blood. Nature 275:179-184. Yogeeswaran, G. , and Salk, P.L. (1981). Metastatic potential is posi -t ively correlated with cel l surface sialy lat ion of cultured murine tumor cel l l ines . S c i . 212:1514-1516. APPENDIX I CHANGE IN VARIANCE IN COLONY FORM (x IP" 3 ) . Treatment single dose single dose single dose double dose t r ip le dose Treatment single dose single dose single dose double dose t r ip le dose Time of DON addition  (days after plating) 2 1 0 1 + 2 1, 2 + Time of DON addition  (days after plating) 2 1 0 1 + 2 1, 2 + 3 C-4I  small colony  day 4 day 6-7 L10 0 s10*0 clO'O jVo sio;o S 9. 7 L 7 4 s4.o C-4I L1 7 L1 7 L l ' 7 [i;7 L 7 . 4 L 3 . 7 L2.9 S 9 . 7 S 9 . 7 S 9 . 7 S9. 7 S9. 7 large colony  day 4 day 6-7 S 2 2 L 2 ' 2 L 2 ' 2 L 2 ' 2  2 ! 2 C-4II  small colony  day 4 i day 6-7 S l 9 . 2 S l 9 . 2  S 19.2 L24 7 L24*7 L24*7 S 2 4 ' . 7 S 22 # 0 S 22.0 s22.o L22 0 L 2 2 ;o C-4II large colony  day 4 day 6-7 L 16.4 12 5 L16 4 . L12 5 i16*4 ! .12*5 * ' LX5 L 13 7 L13*7 |_ 13 * 7 No d i f f . Ll3.> Superscripts.. .variance of colony form of control cultures (x 10~ 3). S . . .variance is smaller than that of the corresponding control variance. L . . .variance is larger than that of corresponding control variance. No d i f f . . . .variance is the same as that of corresponding control variance. 

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