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The role of cytoplasmic protrusions in the intercellular adhesion of rat leukemia cells (line irc 741) Yit, Dominic Kwok-Wo 1972

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THE ROLE OF CYTOPLASMIC PROTRUSIONS IN THE INTERCELLULAR ADHESION OF RAT LEUKEMIA CELLS (LINE IRC 741) by DOMINIC KWOK-WO YIP B. Sc., University of Br i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1972 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i c a t i on of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada - i i -ABSTRACT I. The function, structure and response to environmental factors of a cytoplasmic protrusion found in rat leukemia cells IRC 741 were investigated. A greater r i g i d i t y and adhesiveness of the protrusions, as compared to the main c e l l body, was demonstrated by time-lapse cinematography, and this functional difference was correlated with localized ultrastructural differences in the cytoplasm and on the c e l l surface, and with higher negative surface-charge density, as shown by c e l l electrophoresis. The formation or maintenance of the cytoplasmic protrusions depended on adequate nutritional conditions, and was interfered with by diminished intercellular .contact,, by environmental temperatures below 37°C, by alkaline pH and by calcium-ion depletion. The protrusion appears to represent a type of adhesive organelle not previously described in either cancer cells or normal c e l l s . II. In the course of the above work, a method was developed whereby the differential staining of viable and nonviable unfixed c e l l s , as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining i s due, at least in part, to structural differences that are retained following aldehyde-fixation. - i i i -TABLE OF CONTENTS Page INTRODUCTION 1 PART I The role of Cytoplasmic Protrusions in the Intercellular Adhesion of Rat Leukemia Cells (Line IRC 741) . 2 MATERIALS AND METHODS 8 1. General 8 2. Electron Microscopy 8 3. Histochemical Methods - Light Microscopy 9 4. Electron-microscopic Cytochemistry 10 5. Cell Electrophoresis ....11 6. Time-lapse Cinematography ..11 7. The Induction and Maintenance of Protrusions .12 RESULTS 16 1. Histochemistry 16 2. Electron Microscopic Cytochemistry .16 3. Enzymatic Digestion of Ultrathin Sections 19 4. Cell Electrophoresis ....19 5. Cinematography • 24 6. The Induction and Maintenance of the Protrusion 27 DISCUSSION 37 Cytochemistry and Electrocytochemistry 38 Cell Electrophoresis 40 Cinematography ......43 The Induction and Maintenance of Protrusion 49 - iv -Page PART II The Dye-Exclusion Test for Cell V i a b i l i t y : Persistance of Differential Staining Following Fixation 56 INTRODUCTION 57 MATERIALS AND METHODS .. ..58 Dyes 58 Cell Cultures .60 Fixation and Counting 60 RESULTS 62 DISCUSSION ...69 SUMMARY 75 BIBLIOGRAPHY 77 APPENDICES .' 84 1. Preparation of Cells for Microscopy ..................................85 2. The Chemistry of EDTA . ...86 - v -LIST OF TABLES Table Page I. Dyes Used for V i a b i l i t y Determinations, Arranged in Ascending Order of Molecular weights 59 II. The Number of Stained Cells (Per Thousand) .„ in Four Overgrown Cultures of IRC Cells, Prior to, and Following Glutaraldehyde Fixation 63 III. Effect of Storage Following Fixation on the Subsequent Staining of Cells with Alcian Blue 64 - vi -LIST OF FIGURES Figure Page 1 Living IRC cells in culture 4 2 Electron micrograph of IRC c e l l clump 5 3 The change of the proportion of pear-shaped c e l l s with c e l l density and culturing .time .13 4 Reaction of KCN/10% HCl with sections of IRC cel l s stained with colloidal iron 17 5 Electron micrographs of IRC c e l l s stained with colloidal iron 18 6 Trypsinized IRC cells stained with colloidal iron 20 7 Electron micrographs of epon sections immersed in phosphate buffer and in protease/phosphate buffer 21 8 Electron micrographs of epon sections immersed for 1 hr in 0.1 N HCl and in pepsin/0.1 N HCl 22 9 The change in orientation of the protrusion of IRC ce l l s with the change of the e l e c t r i c a l polarity in c e l l electrophoresis 23 10 The interaction of a single pear-shaped c e l l with an aggregate of three c e l l s 26 11 . The change of proportion of viable pear-shaped cel l s with c e l l density and culturing time ...28 12 Changes in the proportion of pear-shaped cells with changes in the degree of c e l l contact ...30 - v i i -Figure Page 13, Change in the proportion of pear-shaped ce l l s 14 15 ' with changes in environmental temperature .31, 32 16, 17 Proportion of pear-shaped ce l l s in c e l l samples suspended in Hanks' BSS, Moscona's BSS and various concentrations of EDTA at pH 6.5, 7.4 and 8. 34, 35 18 "': Effect of calcium-depletion on the proportion of pear-shaped ce l l s over 3 hours 36 19 Interaction of electron-dense particles .47 20 Rates of diffusion into fixed c e l l s of seven dyes, over one hour 66 21 I n i t i a l s t a b i l i t y of the proportion of alcian-blue stained c e l l s with continued exposure to the dye 67 22 Changes in the proportion of alcian-blue stained cells over 48 hours of exposure to the dye 67 23 The molecular structures and molecular weights of the dyes used in the present study 73 24 The formation of metal-ion-EDTA complex 87 25 The structure of EDTA in aqueous solution 87 26 Titration of EDTA with KOH 87 27 Moles of EDTA required to bind one mole of calcium ions at different pH's 87 - v i i i -ACKNOWLEDGEMENT I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o : Dr. R. L. Noble, D i r e c t o r o f the Cancer Research C e n t r e , f o r t h e o p p o r t u n i t y and f a c i l i t i e s to c a r r y o u t t h i s r e s e a r c h . Dr. N. A u e r s p e r g , Department o f Zoology and Cancer R e s e a r c h C e n t r e , f o r h e r a d v i c e , g u i d a n c e and encouragement t h r o u g h o u t t h i s i n v e s t i g a t i o n . D r s . A. B. A c t o n and P. F o r d , Department o f Zoology, f o r t h e i r h e l p , a d v i c e , i n s t r u c t i v e comments and r e v i e w o f the m a n u s c r i p t . Dr. G. E. Dower and Mr. W. Z i e g l e r , Department o f Pharmacology, f o r t h e i r p e r m i s s i o n and i n s t r u c t i o n s i n the use o f t h e cinema-t o g r a p h i c equipment. Dr. P. S. V a s s a r , Department o f P a t h o l o g y , f o r h i s p e r m i s s i o n and i n s t r u c t i o n s i n the use o f the a p p a r a t u s f o r - c e l l e l e c t r o p h o r e s i s . . D u r i n g the t e n u r e o f t h i s i n v e s t i g a t i o n , t h e a u t h o r was t h e r e c i p i e n t o f a s t u d e n t s h i p from the N a t i o n a l Cancer I n s t i t u t e o f Canada. The r e s e a r c h was s u p p o r t e d by a g r a n t from t h e N a t i o n a l Cancer I n s t i t u t e o f Canada. - 1 -INTRODUCTION The role of the c e l l surface i n development and i n cellular interactions has been receiving increasing attention (Weiss, 1967; Carr et a l . , 1970; Chedd, 1972). Among the surface properties of normal and malignant cells that have been studied extensively are the structural factors that underlie cellular adhesiveness., the maintenance of the ri g i d i t y of the plasma membrane and the basis for c e l l asymmetry. The malignant behaviour of tumour ce l l s , such as invasiveness, lack of contact inhibition (Abercrombie, 1966), or decrease of adhesiveness (Coman, 1944), have been thought to be due to modifications of surface properties. Although research has failed to demonstrate any consistent change i n the c e l l surface that i s unique to cancer (Weiss, 1967), there are many quantitative differences in surface properties between tumour ce l l s and normal c e l l s . Among others, lack of communication between tumour cells (Loewenstein and Kanno, 1966), a decreased requirement of calcium ions i n metabolism (Gilbert, 1972), and often a relative increase of electrophoretic mobility between tumour ce l l s and their normal counterparts (Ambrose, 1966), have been observed. Some demonstrable surface features, although not unique to cancer ce l l s , have been found to accompany malignant transformation. Examples of these are an increase i n the rate of c e l l coat synthesis and c e l l coat thickness (Majlucci et a l . , 1972) and in cellular bubbling (Price, 1967). Structural changes of the surface, specific to certain types of transformed c e l l s , resulting i n the emergence of tumour-specific antigens (Klein and Cochran, 1971) and in the agglutinatability - 2 -by Concanavalin A (Inbar et a l . , 1972), have also been reported. In studying these problems, i t i s often d i f f i c u l t to correlate the behaviour of cells with the characteristics of the c e l l surface. Cells from solid tissues, or from monolayer culture, which may have structurally and functionally distinct regions on the c e l l surface, usually have to be enzymatically dissociated before the study (Moscona, 1962) and this alters their surface properties. On the other hand, cells which grow in suspension cultures are characteristically symmetrical in shape and surface properties, and here one of the main problems in trying to correlate different functions with specific regions has been the disperse nature of structural and functional heterogeneities on the c e l l surface (Weiss, 1967). Topologic differences on the c e l l surface in chemical composition, electric charges or conformations of the plasma membrane, and the related distribution of sites of adhesive, locomotor, or metabolic a c t i v i t i e s , frequently are d i f f i c u l t to identify. This is true also for a l l leukemic and leukocytic suspension cultures studied to date (de Harven, 1967; Chandra et a l . , 1968). It is the aim of the present project to analyze the relationship between the shape, surface structure and adhesive functions of leukemic cells IRC 741 (Dunning and Curtis, 1957), as these cells have been found to have unusual properties which might make i t possible to correlate such surface characteristics more easily than with other existing models (Yip, 1970). When grown in stationary suspension culture, a proportion of these cells was pear-shaped, with a cytoplasmic - 3 -protrusion by which they preferentially adhere to each other (Figure 1). Ultrastrueturally, the asymmetric c e l l s showed an accumulation of organelles near the protrusion. The c e l l surface over the protrusion o had no m i c r o v i l l i , but was covered with 200-400 A electron-dense particles embedded in a thick c e l l coat. The c e l l surface over the main c e l l body had m i c r o v i l l i but no particles and a thin c e l l coat (Figure 2). Thus, i n contrast to other c e l l s , which in suspension culture show no consistent asymmetry of c e l l shape or surface, two structurally and functionally distinct parts could be identified on the IRC cells even at the light-microscopic level, and these provide an opportunity to relate specific adhesive and locomotor characteristics of the cells to specific areas on their surface and to characterize these areas chemically and morphologically, without resorting to enzymatic digestion. Much of the quantitation in this study involved c e l l counts and determinations of c e l l shapes and c e l l v i a b i l i t y . The existing dye-exclusion test for c e l l - v i a b i l i t y (Hanks and Wallace, 1958) i s performed on l i v i n g c e l l populations which change continuously, and the test i s therefore only valid i f done immediately. To avoid the interruptions of the main experimental work, as well as the va r i a b i l i t y in timing, introduced by the counting of large numbers of samples by this technique, a new staining method was developed which permitted the dye-exclusion test as well as the determination of c e l l shapes to be performed on fixed c e l l samples subsequent to the main experimental work. Without this method, many of the observations on the properties of the leukemic c e l l s , described in Part I of this - A -Figure 1. Living IRC cells in culture, protrusions with accumulation of organelles (Pn). Anoptral phase microscopy, x 4,000. - 5 -Figure 2. Electron micrograph showing IRC c e l l clump. Adhesion by protrusions with extracellular particles (ECP), ECP separated from plasma membrane by electron-lucent area. Thick arrow, area of close adhesion; broken arrow, area of loose adhesion. T-microtubule; F-microfilaments; V-microvillus. x 13,500. - 6 -thesis, would not have been possible. The development of this new test i s presented as Part II of this thesis. - 7 -PART I THE ROLE OF CYTOPLASMIC PROTRUSIONS IN THE INTERCELLULAR ADHESION OF RAT LEUKEMIA CELLS (LINE IRC 741) - 8 -MATERIALS AND METHODS 1. General (a) The Cell Line IRC 741 is a spontaneous transplantable rat monocytic leukemia which originated in 1955 (Dunning and Curtis, 1957) and has been maintained at U.B.C. both as an ascites tumour in rats and as a tissue culture c e l l line for about seven years. The present study dealt with the cultured c e l l s . (b) Tissue Culture The leukemic cells were maintained in stationary suspension culture at 37°C in Fischer's medium with 10% horse serum, 100 units/ml of p e n i c i l l i n and 100 jug/ml of streptomycin. Cells were transplanted every three days , when the c e l l density had reached about 6xl0^/ml, which is close to the maximum density without significant c e l l degeneration. 4 New c e l l cultures were set up at a density of 2x10 /ml. The doubling time of IRC cells i s about 12 hours. 2. Electron Microscopy (a) Fixation Unless stated otherwise, cells in culture medium were i n i t i a l l y fixed by adding 0.1% glutaraldehyde in phosphate buffer (Pease, 1964) to an equal volume of c e l l suspension in medium at pH 7.3 and at room temperature. After 5 minutes, the cells were centrifuged for four minutes. The recovered cells were then covered with 2.5% glutaraldehyde in buffer for another 30 minutes, washed - 9 -twice in the same buffer, fixed with buffered 1% OsO^ and again washed twice with buffer. (b) Embedding For electron microscopy, cells fixed as described above were embedded in 5% agar (Glauert, 1965), dehydrated through 70%, 90% and 100% alcohol and embedded in epon according to the routine procedure (Appendix 1). (c) Sectioning Sections, 0.4 microns thick, were cut with a MT-2 Porter-Blum ultramicrotome, stained with 1% toluidine blue/1% borax and observed with the light microscope. For electron microscopy, silver-gold sections were mounted on carbon-coated grids , stained with uranyl acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965) and examined with a Hitachi HS-7S electron microscope. 3. Histochemical Methods - Light Microscopy In order to find possible differences in protein composition or acid mucosubstance distribution between the c e l l surface outlining the protrusion and that outlining the rest of the c e l l body, several methods were used. Cell smears were prepared by fixation with absolute alcohol. Epon sections (0.5-10 /J) were cut from material prepared for electron microscopy as described above. (a) Acidic and Basic Proteins For the following, both smears and epon sections were used. Toluidine blue is a basic dye which in acid solution (pH 4.2 to 5.6) stains acidic residues blue (Chayen et a l . ,1969). Solochrome - 10 -cyanine in acid solution (pH 1.4 to 2.1) stains acidic proteins blue and basic proteins red (Pearse, 1961). Fast green i s another acidic dye which stains basic proteins green (Chayen et a l . , 1969). The preparation of solutions and the staining procedures were according to those outlined by the authors except for the solochrome cyanine method, where a slight modification of the original method was required for penetration of the stain into epon sections: very thin sections (about 0.4 ji) and hot solutions had to be used and hot water was required for subsequent differentiation, (b) Acid Mucosubstances IRC c e l l s , either untreated or pretreated with 0.12% trypsin/ BSS for 30 min. at pH 7.2 and 37°C, were fixed as for electron microscopy, stained overnight with colloidal iron (Rinehart et a l . , 1951) at pH 1.9, washed with 25% acetic acid at pH2 .2 and then processed and embedded as for electron microscopy (Wetzel et a l . , 1966). Epon sections, 0.5-1.0 ju thick, were cut and treated with 10% potassium ferrocyanide in 10% HCl for 60 minutes (Tanaka and Berschauer, 1969). 4. Electron-microscopic Cytochemistry (a) Acid Mucosubstances Ultrathin sections were cut from material prepared as described in 3(b), and were examined with an electron microscope without further staining. (b) Enzymatic Digestion of Ultrathin sections Uncoated gold grids (300 mesh) bearing silver epon sections were immersed in 3 to 4 ml of 5% H ^ at 37°C for 10 to 20 minutes (Anderson and Andre, 1968). After the oxidation, the grids were - 11 -thoroughly washed in d i s t i l l e d water and then were immersed in 1 to 2 ml of one of the following solutions at 37°C to 40°C for 1 to 4 hours: 1. Protease (Bovine pancreatic, partially purified, Nutritional Biochemicals Corp.) 0.03% solution in phosphate buffer at pH 6.8 (Douglas et a l . , 1970). Control : in same buffer at pH 6.8. 2. Pepsin (3X crystalline, N.B.C.), 0.2 to 0.5% i n 0.1 N HC1. Control : in 0.1 N HC1 (Anderson and Andre, 1968). 5. Cell Electrophoresis Since the electron cytochemical study showed that the surface of the cellular protrusion had a heavier layer of acid mucosubstances than the rest of the c e l l surface, experiments were carried out to establish whether these two parts of the cells also differ in their electrophoretic mobility. IRC ce l l s , washed with and suspended in medium without serum, were transferred to a cylindrical chamber equipped with Ag/AgCl electrodes (Vassar et a l . , 1969) at 25°C. The movements and the orientation of the protrusion and the c e l l body in an electric f i e l d were observed with the b u i l t - i n microscope. 6. Time-lapse Cinematography IRC cells were transferred to Sykes-Moore chambers (Sykes and Moore, 1959) which were placed on the stage of an inverted Wild microscope built inside a Prior incubator at 37°C. Pictures were taken with Kodak double-X negative black and white 16 mm film in - 12 -a Bolex movie camera at speeds varying from 1 to 10 frames per minute. 7. The Induction and Maintenance,of Protrusions In a stationary culture the proportion of pear-shaped cells 4 increases steadily until the density reaches about 20x10 /ml, and then drops as the culture ages (Yip, 1970 , Figure 3). Since this increase in c e l l density is associated with an increase in the degree of c e l l contact, the exhaustion of nutrients supply, the accumulation of waste products and changes of pH, these factors were investigated separately to determine their roles in the i n i t i a t i o n or maintenance of protrusions of IRC c e l l s . (a) Effect of Renewal of the Growth Medium Cells from a single population , diluted to lxlO^/ml, were divided among two groups of tubes. Both groups were placed on a rol l e r drum (4 rpm) and incubated at 37°C. Every 24 hours, four tubes from each group were fixed with glutaraldehyde while the remaining tubes were centrifuged at low speed. The medium in one group of tubes was renewed "daily for 4 days while the cells i n the control group were resuspended in their original medium. In this series of experiments, as well as in a l l subsequent experiments , the proportion of viable cells with protrusions and of non-viable cells was determined from the fixed samples by the alcian blue method described in Part II of this thesis. About 1,000 cells were counted for each sample. (b) Effect of Cell Contact To study the relationship of the degree of c e l l contact to the formation of protrusions, cells were grown, in addition to the - 13 -Figure 3. The change of the proportion of pear-shaped cells with c e l l density and culturing time. Proportions were estimated by drawing samples daily from five independent 4 cultures, a l l started at a density of 1x10 /ml , medium unchanged. 5 1 1 1 1 1 1 " T 2 0 40 6 0 CELL DENSITY! X 104/ ml) 2 4 4 8 72 96 CULTURING TIME ( HOURS ) 3 - 14 -standard stationary cultures , in spinner cultures and in gyratory shaker cultures. At moderate speeds (70-80 rpm) in the gyratory shaker cultures, the c e l l contact was increased over that in stationary cultures, while i n spinner cultures i t was reduced (Moscona , 1961; Henkart and Humphreys, 1970). Samples were drawn daily, fixed with glutaraldehyde, and the proportion of pear_shaped and dead cells was determined. The cells cultured in the gyratory shaker showed a consistent increase i n the proportion of pear-shaped cells (maximum 50-60% compared with 30-50% in the stationary suspension culture) and therefore the shaker system was employed as the culturing method for the subsequent experiments described below. Cells , grown in standard culture, were 4 transferred at a density of 1x10 cells/ml , at 20 ml of c e l l suspension per 50-ml Erlenmeyer flask, incubated on a gyratory shaker (70-80 rpm) 4 and were used after 48 hours (approximately 12x10 cells/ml) when the proportion of pear-shaped cells reached i t s maximum (Figure 12). (c) Effect of Temperature Cells grown in gyratory shaker cultures were mixed thoroughly in a 37°C water bath and then were transferred to spinner culture flasks (40 mis of cells per flask) on magnetic stirrers at either 4°C, 25°C (room temperature) or 37°C. At various time intervals, samples were drawn and fixed immediately with 2.5% glutaraldehyde for the determination of the proportions of viable cells with protrusions and of non-viable c e l l s . (d) Effect of pH and of Divalent Ions Cells from gyratory shaker cultures were mixed thoroughly and transferred, in 8-ml aliquots, into small test tubes i n a 37°C water - 15 -bath. After centrifugation in an incubator at 37°C, the supernatant was removed and the cells resuspended in the following solutions for various periods of time and pH's (measured with a Beckman pH-meter). 1. Hanks' balanced salt solution, 2. Calcium and magnesium free Hanks' solution (Moscona's BSS), 3. Moscona's BSS containing Versene (etylene-diamine-tetraacetic tetrasodium dihydrate, (Nutritional Biochemicals Corp.) at various concentrations and pH's. - 16 -RESULTS L. Histochemistry No difference in staining for acidic and basic proteins was found between the surface outlining the protrusion and that outlining the rest of the c e l l s . Sections containing colloidal-iron stained c e l l s treated with ferrocyanide showed the presence of mucosubstances over the whole c e l l surface, but stained particularly heavily at the area surrounding the protrusions. Control cells unstained with iron showed no reaction with ferrocyanide (Figure 4). 2. Electrojn Microscopic: Cytochemistry In response to colloidal iron staining for acid mucosubstances a rather sparse layer of iron particles was observed in the c e l l coat overlaying the main c e l l body (Figure 5). It was quite similar in appearance and distribution to the iron staining of the c e l l coat of the normal macrophage (Carr et a l . , 1970). Within the c e l l coat overlaying the protrusion, however, there was a heavier deposition of iron particles in the matrix between the extracellular particles , but the particles themselves did not stain. On the surface of m i c r o v i l l i and the particles, there were filamentous structures which were not visible after staining with lead citrate and uranyl acetate alone. Treatment of l i v e cells with 0.12% crystalline trypsin, in balanced salt solution at pH 7.2 and 37°C for 30 minutes prior to the above treatment, removed the extracellular particles and matrix from the surface of the protrusion. However , the c e l l coat over - 17 -Figure 4. Reaction of 10% KCN/10% HCl w i t h epon s e c t i o n s of IRC c e l l s f i x e d w i t h glutaraldehyde, w i t h (A) and without (B) c o l l o i d a l i r o n s t a i n i n g p r i o r to embedding. In A, the areas surrounding p r o t r u s i o n s were s t a i n e d more h e a v i l y , x 1,200. - 18 -Figure 5. Electron micrographs of IRC cells stained with colloidal iron. A. The protrusion of a pear-shaped c e l l i s covered o with a c e l l coat, 3,000-4,000 A thick (J)* Unstained extracellular particles (P) are embedded in an iron-positive matrix. The surface of the remaining part of the c e l l is covered with a thin layer of iron particles (arrow), x 36, 000. B. Filamentous structures (arrows) are present on the surface of mi c r o v i l l i and the ECP. x 46,000. - 19 -the remaining c e l l surface was s t i l l stained with iron (Figure 6). 3. Enzymatic Digestion of Ultrathin Sections Ultrathin sections mounted on copper grids , after enzymatic treatments, were often covered with a coat of particles, possibly due to a reaction of the copper with the chemicals, because i t did not occur on sections mounted on gold grids. The following results were obtained with gold grids. Treatment with protease/phosphate buffer at pH 6.8 (Figure 7B), 0.1 N HC1 (Figure 8A) and pepsin/0.1 N HC1 at pH 1.2-1.5 (Figure 8B,C) at 37°C for one hour a l l removed the amorphous matrix from among the extracellular particles. This change was not observed after treatment with the buffer alone. The particles themselves became more irregularly shaped with definite electron-lucent round areas in the centre. After four hours, the mitochondrial matrix and some of the non-particulate cytoplasm and nucleoplasm were removed. However, the morphology of the extracellular particles remained unchanged, and the ribosomes , endoplasmic-reticulum, microtubules and the various membranes also remained intact. No differences between the results of the 3 treatments were observed. 4. Cell Electrophoresis The electrophoretic movement of the cells is illustrated in Figure 9. When there was no electric potential applied through the medium, the asymmetric cells settled down slowly in response to gravity with the protrusion on the top (Figure 9A). When a voltage gradient of about 50 to 60 V was applied, the cells were attracted to - 20 -Figure 6. T r y p s i n i z e d IRC c e l l s s t a i n e d w i t h c o l l o i d a l i r o n . Most c e l l s were round but c e l l s w i t h p r o t r u s i o n s were a l s o present. The amount of e x t r a c e l l u l a r p a r t i c l e s (ECP) l e f t on the p r o t r u s i o n was v a r i a b l e . A and B are two micrographs of two s e c t i o n s of the same p r o t r u s i o n . A small d e p o s i t i o n of ECP i s present i n the area denoted by an arrow i n A but not i n the s i m i l a r area i n B. A l a r g e r amount of ECP was s p l i t o f f the p r o t r u s i o n . A t h i n l a y e r of c o l l o i d a l i r o n i s evident on the surface of the p r o t r u s i o n , the c e l l body and the e x t r a c e l l u l a r p a r t i c l e s ( P ) . A, x 15,600. B, x 18,000. - 21 -Figure 7. Electron micrographs of epon sections immersed for 1 hour in phosphate buffer at pH 6.8 (A) and in 0.03% protease/phosphate buffer (B). Poststained with lead citrate and uranyl acetate, x 32,000. The amorphous matrix among the extracellular particles was removed in B. The electron-lucent round areas in the centre of the particles became more apparent after digestion (B) . x 32 ,000. - 22 -F i g u r e 8. E l e c t r o n m i c r o g r a p h s of epon s e c t i o n s immersed f o r 1 hour i n 0.1 N HCl (A) and i n pepsin/0.1 N HCl (B,C). R e s u l t s were s i m i l a r to those d e s c r i b e d i n F i g u r e 7(B). A, x 4,600. B, x 1 ,600. C, x 50 p00. - 23 -Figure 9. The change in orientation of the protrusion of IRC cells with the change of the electrical polarity in c e l l electrophoresis. A, no electrical current ; B,C and D ill u s t r a t e changes of polarity. Large broken arrow, gravitational force; large solid arrow, electrical attractive force; small arrow, resultant direction of c e l l movement. 9 - 24 -the anode in a direction which was the resultant of the gravitational and the electrical attractive forces. This i s the usual picture in c e l l electrophoresis (Weiss , 1967). However, in the present case, the interesting observation was that the protrusion of the IRC c e l l s , as the whole c e l l was moving, gradually turned further towards the anode than the c e l l body. Thus the protrusion would turn l e f t or right in response to changes in the polarity of the current (Figure 9B, C and D). This peculiar behaviour of the protrusion was recognized in over 90% (34/37) of the observed pear_shaped cells on two separate occasions. The degreee of turning, i.e., the angle of deflection from the v e r t i c a l , seemed to vary with the size and shape of the protrusion ; and the time for assuming the maximum angle, although also variable, was around one minute. 5. Cinemato graphy (a) Cell Movement Gliding movement (Bhisey and Freed, 1971) was the characteristic movement in IRC c e l l locomotion. This particular movement was associated with the formation of a ruffled membrane at the leading edge of the cytoplasmic mass while the remainder of the c e l l mass moved in a rather rigid fashion. The microspikes appeared and disappeared rapidly on every part of the leading side, as observed with the 40X phase objective. In contrast, the protrusion, which was usually at the back, remained stable at the same site although i t s s i - z e v a r i e d a l l the time. There were no microspikes on i t s surface. Occasionally , a single pear-shaped c e l l might round up and then reform i t s protrusion. The pear-shaped - 25 -c e l l s were al s o able to move sideways or w i t h the p r o t r u s i o n at the le a d i n g s i d e , e s p e c i a l l y when c e l l s were coming together. O c c a s i o n a l l y , ameboid movement, which in v o l v e d s u b s t a n t i a l cytoplasmic streaming i n t o pseudopods, r e s u l t i n g i n elongating and shortening of the c e l l body, was observed i n some IRC c e l l s . The c e l l s u s u a l l y moved very s l o w l y , at about 0.1 p/sec. However, i n c e r t a i n cases, such as when two daughter c e l l s separated, when c e l l s were going to j o i n together by t h e i r p r o t r u s i o n s o r separate from adhesion at t h e i r p r o t r u s i o n s , the c e l l s might suddenly increase t h e i r speeds to 1.7 p/sec, which could be r e a l i z e d without d i f f i c u l t y by d i r e c t microscopic o b s e r v a t i o n (Figure 1 0 ) . (b) C e l l Adhesion I n t e r c e l l u l a r c o n t a c t , r e s u l t i n g from c o l l i s i o n s during random c e l l movement, almost i n v a r i a b l y r e s u l t e d i n f a i r l y s t a b l e i n t e r c e l l u l a r a s s o c i a t i o n s . When pear-shaped c e l l s came together, they tended to a t t a c h to one another d i r e c t l y by t h e i r p r o t r u s i o n s i f t h e i r p r o t r u s i o n s were f a c i n g one aiother at the time of i n i t i a l c o n t a c t . I f the p r o t r u s i o n of one c e l l met the c e l l body of another pear-shaped c e l l , they might f i r s t adhere together by the p r o t r u s i o n -c e l l body c o n t a c t , then the c e l l w i t h the p r o t r u s i o n might search around the other c e l l u n t i l f i n a l l y both p r o t r u s i o n s j o i n e d together. Adhesion by the pr o t r u s i o n s was not always immediate. A c e l l j o i n i n g a c e l l clump might undergo forward and backward movements s i m i l a r to a parking c a r , before f i n a l l y a r e l a t i v e l y s t a b l e p r o t r u s i o n - p r o t r u s i o n adhesion was formed (Figure 1 0 ) . This type of adhesion was s t a b l e - 26 -Figure 10. The interaction of a single pear-shaped c e l l with an aggregate of three cells i s illustrated by these composite diagrams made by tracing the images on the screen of a film editor. The numbers indicate the positions of the approaching c e l l in different frames. The time interval between consecutive numbers is 6 seconds. In A, a single c e l l advances with i t s protrusion in front at various speeds (1 to 3, 0.5 ju/sec; 3 to 4 , 1.2u/sec ; 4 to 5 , 1.7 ^i/sec). Then the protrusion turns towards the adhering protrusions until i t joins them (B). But the adhesion i s not yet stable. The newcomer separates and rejoins the group several times (C to E) until f i n a l l y a stable association is accomplished which lasts about one minute (not shown). F depicts the same c e l l moving away from the group. The speed was approximately 0.1 ju/sec. These diagrams illustrate that a pear-shaped c e l l can move in a l l directions at speeds varying from 0.1 p/sec to 1.7 yu/sec. They also suggest that the IRC cells "prefer" to adhere by their protrusions. - 2 7 -in spite of the vigorous motion of the c e l l s . However, occasionally, one of the ce l l s in an aggregate might separate from the rest and move away. Cells in aggregates also could divide. The protrusion seemed to disappear and the whole c e l l became larger and round. After division, both daughter cells stayed together and one of them s t i l l attached to other c e l l s at the same site where the protrusion had been observed before i f disappeared during prophase. Daughter oells immediately after division did not have protrusions. (c) Cell Aggregation Single c e l l s moved around and aggregates of two or three c e l l s were formed as the ce l l s collided. These small aggregates were also able to move as a whole while adhering by the protrusions, and the ruffled membranes were conspicous on the leading side of the leading ce l l s of the group. Small aggregates in turn formed larger c e l l clumps. Single c e l l s joining and , occasionaly, leaving the c e l l colonies were also observed. 6. The Induction and Maintenance of the Protrusion (a) Effect of Renewal of the Growth Medium There seemed to be l i t t l e difference in the proportion of pear-shaped c e l l s grown with or without daily renewal of culture medium 4 unti l after 48 hours of culturing or at a c e l l density of about 5x10 /ml (Figure 11). The proportion of pear-shaped ce l l s in the cultures with 4 unchanged medium reached about 35% at 25x10 /ml (48-72 hours) cells and then dropped with the culturing time u n t i l , at 96 hours, most cells Figure 11: The change of proportion of viable pear-shaped cells with c e l l density (live and dead) and culturing time in groups of cultures with (A) and without (•) daily renewal of medium. Each symbol represents a separate culture. |,proportions of pear-shaped cells in the group without daily renewal of medium co after 96 hours of culturing. PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) — K ) U> *»* L/l • • - 29 -were dead and only few showed protrusions. In contrast, the proportion of pear-shaped ce l l s in the group grown with daily renewal of medium 4 increased steadily to about 50% when the c e l l density was about 60x10 /ml at 72 hours, before i t dropped slowly as the culture continued, suggesting that beyond a certain c e l l density, the formation or maintenance of protrusions might be limited by nutritional factors. (b) Effect of Intercellular Contact The growth patterns of ce l l s maintained in gyratory, stationary and spinner cultures, illustrated in Figure 12, indicate that the formation of protrusions was proportional to intercellular contact. At low c e l l densities, the maximum proportion of pear-shaped c e l l s was consistently highest in gyratory cultures and lowest i n spinner cultures. The proportion dropped most slowly in the stationary cultures as the cultures aged. (c) Effect of Temperature In response to the change of environmental temperature from 37°C to either 25°C (Figure 13) or 4°C (Figure 14), the proportion of pear-shaped ce l l s dropped over one hour, when i t levelled off and was maintained at a rather steady level as long as 4 hours. If at this point the bottles were placed back into a 37°C environment, the proportion of pear-shaped c e l l s slowly went up, sometimes approaching the original level within 1 hour. When samples from a single c e l l population were placed in either 4°C or 25°C, the reduction in the proportion of pear-shaped ce l l s was similar in both temperatures (Figure 15). Furthermore, the recovery of the original proportion of pear-shaped cells was s t i l l observed after 5 hours of temperature depression. - 30 -Figure 12: Change in the proportion of pear-shaped cells with changes in the degree of c e l l contact. - control stationary culture; - gyratory culture; - spinner culture. o o m r — r — m c/> —I X o o & o PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) I C O C n • Figure 13. Change in the proportion of pear-shaped cells with changes in environmental temperature. A, control at 37°C; at 256C, after 4 hours, returned to 37°C. Each curve represents a spinner culture. Figure 14. Change in the proportion of pear-shaped cells with changes in environmental temperature. • , control at 37°C; 9 , at 4°C, after 4 hours, returned to 37°C. Each curve represents a spinner culture. Figure 15. Change in the proportion of pear-shaped ce l l s with changes in environmental temperature. 4 cultures from a single c e l l population, kept at 37°C, were placed separately in either 25°C or 4°C. After 5 hours, a l l were returned to the 37°C environment. Each curve represents a spinner culture. Thick arrow indicates the i n i t i a l proportion of pear-shaped c e l l s . PROPORTION OF PEAR-SHAPED CELLS ( X 0.1) - 33 -(d) Effect of pH and EDTA The effects of Hanks' BSS, Moscona's BSS and EDTA solution at various pH's are represented in Figure 16. The groups not significantly different from one another are enclosed by IECtangles in Figure 17. In a l l solutions except the 2% EDTA there was a significant drop i n the proportion of pear-shaped ce l l s at pH 8 from that at pH 7.4. There was also a slight increase in the proportion of pear-shaped c e l l s at the acid pH. With respect to the effect of the concentration of EDTA, the lowering of the proportion by 2 % EDTA was remarkable and nicely separated from the others. In contrast, the difference in the effects of other solutions at pH 5.6 and 7.4 was not so clear-cut. Although st a t i s t i c s separates them into groups (Figure 17), the results were only based on 4 samples per group which seems inadequate for results that are close. Moreover, in the acid pH, many cells of various Irregular shapes were observed, which might have diminished the accuracy of the counts of pear-shaped c e l l s . However, the important observation in this series of experiments was that the pH by i t s e l f has an effect upon the protrusion. The rate of change in the proportion of pear-shaped ce l l s was proportional to the concentration of BDTA as demonstrated in Figure 18. In 2% EDTA, the proportion dropped to below 10 after 30 minutes. After 40 to 45 minutes, the difference between the effects of 0.2 and 0.02% EDTA became clearly discernible. After 3 hours, the effects of Hanks' BSS, Moscona's BSS, 0.02% and 0.2% EDTA were very variable, probably due to nutritional factors. - 34 -Figure 16. Proportion of pear-shaped cells in c e l l samples suspended in Hanks' BBS, Moscona's BBS and various concentrations of EDTA at pH 6.5, 7.4 and 8. Arrow and asterick indicate the i n i t i a l proportion at pH 7.4. Time of treatment, 15 minutes. Each dot represents the mean of four samples. 1 6 Figure 17. Same as Figure 16. The means which are not significantly different from one another are enclosed by rectangles. Analysis of Variance (Sokal and Rohlf, 1969) and Duncan's New Multiple Range Test (Steel and Torrie, 1960). 17 Figure 18. Effect of cation-depletion on the proportion of pear-shaped cells over 3 hours. The points in each group of four represent samples treated separately. TIME (HOURS) 1 8 DISCUSSION Cytoplasmic protrusions are common structures in animal c e l l s . They may be transient, as, for example, in cells undergoing ameboid movement where the pseudopods are formed by multidirectional streaming of the cytoplasm. Cellular bubbling, or blebbing, i.e. the rapid projection and retraction of the cytoplasm, represents another type of transient protrusion which.is not related to locomotion (Price, 1967) However, the cytoplasmic protrusion found in IRC c e l l s appears to differ in many aspects from those found in other c e l l s . Although i t does not seem to be a permanently differentiated structure, i t seems more stable than either the pseudopods observed in ameboid movement or the cellular blebs. Moreover, i t is not a locomotor organelle nor a sign of degeneration, but rather seems primarily a structure used for intercellular adhesion. In addition, this structure i s associated with a localized, unusual and complex type of c e l l coat. Extracellular materials (ECM) have been of major interest in studies on growth, developmental interactions and differentiation in multicellular systems (Moscona, 1962). Grobstein (1961) demonstrated that intercellular factors were responsible for the transmission of morphogenetic information during induction. On the other hand, Moscona was interested in demonstrating whether or not extracellular materials are responsible for c e l l aggregation. The results seemed to be positive He (Mosconaj 1962a) found that enzymatically dissociated c e l l s reaggregated rapidly at 38°C, but failed to do so at 25°C. If rotated rapidly, c e l l s at 38°C liberated to the medium a material containing - 38 -mucoprotein which was isolated and shown to be able to promote c e l l aggregation at 25°C. This cell-binding material was further demonstrated to show a certain amount of tissue sp e c i f i c i t y (Moscona, 1962a) and to be dependent on RNA and protein synthesis (Moscona and Moscona, 1963). Experiments with malignant ce l l s also gave similar results. Mouse malignant c e l l s formed aggregates in gyratory cultures at 6°C after mechanical dissociation but not after trypsinization (Pessac and A l l i o t , 1970). A l l these observations suggest the importance of ECM i n c e l l adhesion. An adhesive organelle, associated with a modified c e l l coat, like the one observed in the IRG c e l l s , has not been reported in other normal and malignant c e l l s . In the following, different aspects of the surface properties and shape of IRC c e l l s are discussed under appropriate sub-headings. Cytochemistry and Electron-cytochemistry The collodial iron method Indicates the presence of acid mucosubstances over the whole surface of the IRC cells as well as i n the matrix embedding the extracellular particles. In comparison with related normal c e l l s , i.e. macrophages (Carr e_t a l . , 1970), no increase in cell-surface mucosubstances could be demonstrated in these leukemic cel l s by this method, except for the localized region over the protrusion. The c e l l coat over the main c e l l body was v i s i b l e only after iron staining, while on the other hand, the matrix was discernible after uranyl acetate and lead citrate staining alone. This might be due to either quantitative or qualitative differences in components of - 39 -the coat over the two parts of the c e l l surface. The filamentous structures revealed by colloidal iron on m i c r o v i l l i and the c e l l coat over the protrusion resemble those demonstrated in carcinoma c e l l s (Auersperg, 1969), and in human trophoblasts (Bradbury et a l . , 1970), but have apparently not been reported i n other cells of hemopoietic origin. No differences in the staining for either acidic or basic proteins were demonstrated between the surface of the protrusion and that of the c e l l body. However, i t is possible that existing differences were too small, either quantitatively or qualitatively, to be detected by the methods employed in the present study. The removal, by trypsin treatment, of the thick coat covering the protrusion of l i v e c e l l s and the digestion of the matrix from epon sections with protease indicate the presence of a proteinaceous moiety, in addition to acid residues, in the matrix embedding the electron-dense particles. However, as the cells after trypsin digestion were stained by the colloidal iron, there was s t i l l a layer of mucoproteins l e f t behind after the trypsin treatment. This finding i s consistent with those obtained by Gasic and Loebel (1966) and Carr eit a l . , (1970). The former authors observed that the staining for s i a l i c acids in sections of fixed mouse ascites tumour cells was actually enhanced after trypsin digestion. In contrast to the matrix, the electron-dense particles seemed unstained by the colloidal iron, and protease-, HC1- and pepsin-resistant, and the ring-shaped structures of the particles after enzymatic digestion suggest a protease- and pepsin-resistant framework. - 40 -Since the matrix i s susceptible to both trypsin and the bovine pancreatic protease, which contains 140 units/mg of trypsin, i t could be suggested that the matrix contains a proteinaceous moiety rich in arginine and lysine. However, since both 0.1 N HCl and pepsin/0.1 N HCl removed the matrix, i t i s d i f f i c u l t to evaluate the action of pepsin upon the matrix. The removal of matrix by HCl i s of interest because i t indicates that the proteins or other structures -in the matrix are susceptible to weak acid treatment, although a stronger acid solution at higher temperatures (e.g. excess 6N HCl at 100°C to 120°C for 10 to 24 hours) i s usually heeded to achieve the complete hydrolysis of proteins (Lehninger, 1970). Although the results show that electron-dense particles are protease-, HCl- and/or pepsin-resistant, the results do not prove that the particles are not proteinaceous in nature. It i s well known that different kinds of proteinaceous structures are susceptible to different types of proteolytic enzymes (Anderson and Andre, 1968; Douglas et a l . , 1970); and in the present study, only protease and pepsin were used. The finding that the mitochondrial matrix was removed after 4 hours treatment of pepsin while the the cytoplasmic microtubules and some other protein-containing structures remained intact is similar to that obtained by Anderson and Andre (1968). Of course, the framework of the electron-dense particles could also consist of nucleic acids or l i p i d s , for example. Cell Electrophoresis The observation that the protrusion turned further towards the - 41 -anode than did the c e l l body indicates that i t has a higher electrophoretic mobility. This may indicate that the average surface-charge density i s higher in this particular area (Weiss, 1967). However, the electrophoretic mobility of a particle i s also influenced by i t s shape and size. To rule out the possibility that the protrusion was more strongly attracted because of Its smaller size, some theoretical considerations are necessary. For an e l e c t r i c a l l y charged sphere of radius a, the density of charge can be related to ^ , the viscosity of the suspending medium; u, the electrophoretic mobility in u/sec/volt/cm; and k, where 1/k is the Debye-Huckel parameter which indicates the "effective" thickness of the diffuse electrical double layer surrounding the c e l l surface, by the following equation (Tenforde, 1970): •<f = (tyu/a) (1+ka-).. (1) When ka is greater than 100, the above equation could be reduced to C = tyuk .. (2) The parameter 1/k depends on the ionic strength of the suspending medium. In a physiological situation, when the ionic strength is ° 7 - 1 about 0.145, 1/k equals 8 to 10 A and k approximates 10 cm (Weiss, 1967; Tenforde, 1970). From equation (2) i t is obvious that i f the size of the particle (i.e. a) i s large enough to make ka greater than 100, the parameter of size and shape of the particle can be eliminated from the calculation. In other words, i f the ionic strength and the viscosity of the suspending medium are kept constant, the charge density w i l l be - 42 -directly proportional to the electrophoretic mobility of the particle, without considering its size and shape. In the present case, the r a d i i of the c e l l body and the protrusion -4 -4 of IRC c e l l s are about 5 u (5x10 cm) and 1 u (1x10 cm) respectively. Accordingly the products ka for them are. 7 - 1 -4 3 ka = 10 cm x5xl0 cm = 5x10 and 7 - 1 - 4 3 ka = .1.0 .cm xlO cm =.1x10...; i.e., both are greater than 100. Therefore equation (2) i s valid and the difference in electrophoretic mobility between these two entities, as demonstrated by the present experiments, i s attributable to the difference in charge density over their surface. Thus the results indicate that the protrusion of IRC c e l l s not only has a thicker layer of acid mucoproteins on i t s surface (3,000 o to 7,000 A) as revealed by earlier ultrastructural studies, but also that the density of the negative charges of the c e l l coat on the protrusion i s higher than that of the c e l l body. It should be noted here, that electrophoretic mobility is not necessarily influenced by c e l l coat thickness, since i t i s determined only by charges within o the superficial layer of the c e l l coat (8-10 A). Heterogeneity in surface charges is believed to be present on the c e l l surface (Weiss, 1969), but i t is usually not demonstrable by c e l l electrophoresis, because either the sites of different charge density are more evenly distributed over the c e l l surface (Weiss et^ a l . , 1972; Weiss et a l . , 1972a) than in the IRC c e l l s , or the morphology of the suspended cells i s symmetrical. A localized surface charge difference that can be demonstrated by c e l l electrophoresis as i n the - 43 -case of the IRC c e l l s has not been reported in any other c e l l s . Moreover, this localized increase of charge density becomes more intriguing when i t is associated with the current concept that increased charge i s a recognized characteristic of rapidly growing and/or malignant ce l l s (Maddy, 1966). An increase of c e l l coat thickness has been demonstrated to accompany malignant transformation by virus (Mallucci ejt al_., 1971) and has been linked to the a b i l i t y of cancer c e l l s to overcome the immune rejection by the host (Hause et a l . , 1970). It may also account for the lack of communication between tumour cells observed by Loewenstein and Kanno (1966). The thickness of the c e l l coat varies with the c e l l type and i s apparently related to the a b i l i t y to form m i c r o v i l l i and of c e l l s to fuse (Poste, 1970, 1971). Similarly, the cinematographic study of IRC cells-showed that -the ruffled membrane appears usually on the surface of the c e l l body of the pear-shaped cel l s but rarely at the protrusion. Increased electrophoretic mobility has been noted in dividing cells (Gerner et a l . , 1970) as well as in malignant liver c e l l s (Fuhrmann, 1965). This increased electrophoretic mobility has been considered to be due to a temporary increase of surface charge in the case of regenerating cel l s , and a permanent increase in malignant cells (Anderson, 1966). Cinematography The present study suggests that the main structure involved in IRC c e l l locomotion i s the ruffled membrane and not the protrusion. - 44 -Ruffled membranes developed in moving round or pear-shaped c e l l s , in the c e l l body of the leading cells when a group of four pear-shaped ce l l s , attached by their protrusions, moved together, and in the daughter cells after c e l l division and before separation. If IRC c e l l s are examined under the microscope, pairs or small groups of pear-shaped c e l l s , attached by their protrusions are observed which strongly suggests that the protrusion may be the preferential site of intercellular adhesion. The cinamatographic study provided direct proof for this view, as single pear-shaped c e l l s , upon contact,, always adhered by their protrusions. Thus the question as to the possible underlying mechanism is of interest. Since a l l mammalian cells so far examined carry a net negative charge at their surface (Weiss, 1967), i t has been suggested that contact between cells can be considered as similar to the contact of charged particles with e l e c t r i c a l double-layers, and consequently the DLVO theory, developed for the interaction of lyophobic colloid particles; may be applied (Poste, 1970). According to the DLVO theory, electrostatic forces tend to keep cells apart while attractive forces, largely London-Van der Waals forces, tend to favour close contact between c e l l surfaces. However, before two surfaces can come together, the repulsive electrostatic forces must be overcome usually o by locomotor energy, unt i l they are only few A apart, when the physical and chemical bonds between the surfaces are formed and the attractive forces created therefrom become effective. The repulsive forces encountered by adhering cells may be represented by the following formula (Poste, 1970): 7 -kd V = 1/2 Da/0 log e (1+e ) -45 -where: V R = potential repulsive energy barrier to close contact; D = dielectric constant of the medium separating the c e l l surfaces; a = the radius of curvature of the interacting c e l l surface or projections; 0 = the c e l l surface or zeta potential; 1/k = Debye-Huckel parameter; and d = distance separating the approaching surfaces of the two c e l l s . From the above formula, i t i s obvious that V D i s proportional to two of the important factors in considering the c e l l surface, i.e., the radius of curvature and the surface charge density. In other words, surfaces with smaller r a d i i of curvature and lower charge densities w i l l adhere more easily than surfaces with larger r a d i i of curvature and higher charge densities. As a matter of fact, Poste (1970) has demonstrated this by showing that m i c r o v i l l i (radius of curvature 0.1 p) are important in the primary adhesion leading to c e l l fusion. Thus i f the above argument is applied to the IRC c e l l s , i t should be expected that i t would be the c e l l body, with m i c r o v i l l i and a lower charge density, instead of the protrusion, with no mi c r o v i l l i and a higher surface charge density, that is the preferred site of intercellular adhesion. However, as shown by the present experiments, this i s not the case. There are at least two possible explanations for this discrepancy. F i r s t , the high dynamic energy, resulting from cellular locomotion (Weiss, 1967), may overcome the - 46 -strong repulsive forces on the protrusion. This notion i s supported by the sudden acceleration of motion observed when a c e l l was approaching the protrusion of other cells with i t s own protrusion. Another explanation i s the possi b i l i t y that the r a d i i of curvature of the approaching surfaces that should be considered when two protrusions come together are not the whole surface of the protrusions, but those of the electron-dense particles. According to the calculation of Poste (1970), two approaching cells (of 12 p radius with surface potential - 15 mV) w i l l not experience any repulsive forces o u n t i l they are about 50 A apart. As the c e l l s come closer, the repulsive forces become stronger. Therefore, the protrusions can come o as close as 100 A apart without experiencing any repulsive forces. When the protrusions come s t i l l closer, within the interaction o distance of about 50-60 A, the -surfaces contributing to-the repulsive forces are those of the electron-dense-particles because the diameter of the particles i s about 250 A (Figure 19). Since the radius of curvature of the particle (0.01 p) is much smaller than that of a microvillus (0.1 p), the repulsive forces generated when two protrusions come together must be very small irrespective of the relatively higher average charge density at this area, and consequently the adhesion by the protrusion i s much easier than that by the mi c r o v i l l i . Another reason for the c e l l s not to adhere by the c e l l body could also be the great activity of the ruffled membrane (Carter, 1968). Since the microspikes appeared and disappeared rapidly in every part of the c e l l body, close contact in this area might be d i f f i c u l t and stable -47 -Figure 19 A. Diagram showing the loose-adhesion area from Figure 2. B. Interaction of two electron-dense particles,. The diagram shows that two protrusions may approach freely to a distance of 60 A and at this distance • only the surfaces of the particles w i l l contribute to the electrostatic repulsive forces (see text). CELL A •'•.•'.••A^-i CELL B 60A I J tOO A B H ELECTRON-DENSE PARTICLES MATRIX CONTAINING PROTEINS AND MUCOSUBSTANCES SURFACE CONTRIBUTING TO THE FLECTROSTATIC REPULSIVE FORCES 1 9 - 48 -adhesion by the tips of microspikes was not formed. Light-microscopically and functionally, the protrusion of IRC cells resembles the "uropod" of the pear-shaped activated normal lymphocytes (McFarland et a l . , 1966). In response i n vitro to specific and non-specific antigens, as observed by cinematography, these lymphocytes produced an anatomically distinct cytoplasmic process with which they attached to other c e l l s , debris and glass surface. Adhesion between uropods of different lymphocytes was also observed. The pseudopods tended to appear and disappear at the anterior end of the c e l l but the uropod remained prominent and relatively constant at the rear. After c e l l division, the daughter ce l l s remained immobile in contact, and asymmetrical for a variable period of 15 to 90 minutes. The uropod developed in the area of the last point of contact between the daughter c e l l s . Then the pseudopods appeared and the cells rapidly elongated into the characteristic pear-shape (McFarland et a l . , 1966). In contrast, in the IRC c e l l s , the daughter ce l l s seemed to remain immobile for about 3 hours, then the ruffled membrane developed and they separated rapidly. But the protrusion was not noticed at that stage. Another difference between the protrusion of IRC cells and the uropod of the activated lymphocytes is that in the latter there were no electron-dense particles similar to those outlining the surface of protrusion of IRC c e l l s . Instead, there were numerous long m i c r o v i l l i (McFarland, 1969). Adhesion between the m i c r o v i l l i and the surface of other cells was also observed (Smith et a l . , 1971). Thus i t i s apparent that although both the protrusion of IRC ce l l s and the uropod of lymphocytes are "tools" - 49 -for adhesion, the underlying mechanism of adhesion is probably different in these two systems. While the electron-^dense particles on the surface of IRC ce l l s could play an important role, the uropods may adhere by m i c r o v i l l i , as many other mammalian cells do. There are s t i l l several important questions not answered by the present cinematographic study. Due to the rapid movement of the IRC c e l l s , i t has been impossible to follow one single c e l l through the whole division cycle. Thus it was not possible to determine whether the protrusion appears i n response to c e l l contact or also i n single cells i n certain stages in their l i f e cycle. Nor was i t possible to determine whether the protrusion ever separated from the c e l l . The Induction and Maintenance of Protrusions The experiments on the effect of renewal of medium suggest that beyond a certain c e l l density, the formation or maintenance of protrusions is limited by nutritional factors. These could be the deficiency in nutrients, the accumulation of waste products or the drop i n pH. However, the acid pH in the exhausted culture medium is unlikely to play a part as shown in experiments designed specially to test pH effects. The observation that at low c e l l density the proportion of pear-shaped cells was independent of nutritional factors was important, because these factors could therefore be ignored when cells at low density were used to study other environmental influences. Although the results obtained from the present study are - 50 -hot sufficient to ju s t i f y the conclusion that the protrusion i s induced by c e l l contact, they nevertheless indicate that the proportion of pear-shaped ce l l s i s proportional to the degree of intercellular contact which may therefore play a part in the induction and/or maintenance of this structure. While the proportion of pear-shaped cells increased and dropped slowly in the control suspension culture, i t increased and dropped rapidly i n the gyratory culture. The rapid drop in the proportion of pear-shaped ce l l s in the latter case may have been due to nutritional factors. The considerable increase 4 of pear-shaped cells at the density of about 10x10 /ml might have consumed so much nutrients that only a small portion was l e f t for the later stages. If this was the case, then i t may have been possible to maintain the high proportion of pear-shaped cells in gyratory shaker cultures for longer periods of time i f 'the medium was renewed more frequently. The drop i n the proportion of pear-shaped ce l l s after the temperature was lowered to either 25.0°C or 4°C indicates that the maintenance of the protrusion is dependent on biochemical processes which require temperatures above 25°C. For example, the production of ATP and the integrity of microtubules, which may be important factors in maintaining the protrusion, are both temperature sensitive. The similarity in the degree of lowering of the pear-shaped c e l l proportion at 25°C and 4°C i s quite different from the usual picture depicting an enzymatic activity-temperature relationship. The most apparent explanations for the maintenance of the protrusions at temperatures above 25°C include the involvement of some enzymes - 51 -which are active only above 25°C, of some necessary products which are degraded below this temperature, or of some temperature-sensitive conformational changes induced in the structural proteins. The levelling off of the proportion of pear-shaped c e l l s after one hour at both temperatures suggests that there could be several stages'-in. the development of protrusions and that, in about 10% of the total population, they were not temperature-dependent for their maintenance.:* Another interesting point i s the reve r s i b i l i t y of the protrusion after the temperature depression. The underlying mechanism determining the reappearance of the protrusion i s unknown but i t obviously did not require intercellular contact. It i s l i k e l y that the thick c e l l coat covering the protrusion may be involved in this process. Thus, in ameba i t has been shown that the production of pseudopods can be induced by a local concentration on the c e l l surface of proteins, some substituted amines and long-chain aliphatic substituted amines (Brewer and Bell, 1970). In IRC c e l l s , although i t has not been demonstrated by cinematography, the results from the gyratory shaker experiments indicate that the induction of the protrusion i s related to intercellular contact. Then one could speculate that some "activators" similar to the chemicals mentioned above might be secreted when two round cells form contact by m i c r o v i l l i (Yip, 1970) as a c e l l may secrete proteinaceous substances when i t contacts a substratum (Weiss, 1972), or that the thick c e l l coat on the protrusion of a pear-shaped c e l l could induce the protrusion on a round one. EDTA decreased the proportion of pear-shaped cells and there was a direct correlation between the concentration of EDTA and the - 52 -rate of loss of protrusions. Although the ionization and hence the calcium ion binding capacity of EDTA increases with the increase of pH, this did not seem to be the basis for the increased effect of 0.2 and 0.02% EDTA at pH 8 because, f i r s t , a similar decrease in the proportion of pear-shaped ce l l s occurred in Hanks' solution alone and secondly, EDTA achieves almost i t s f u l l calcium-ion binding-capacity at about pH 7 (Chaberek and Martell, 1959; see Appendix 2). Therefore, the decrease in the percentage of the pear-shaped cells • + • at pH 8 in any of the solutions tested (Hanks, Moscona, - EDTA), must be due to a direct effect on the c e l l surface. Divalent ions, especially calcium ions, have long been related to the maintenance of plasma membrane structure and r i g i d i t y . For example, in treating cells with EDTA solution, the degree of c e l l deformation is proportional to the concentration of EDTA used (Weiss, 1967a). When rat intestine is injected in vivo with EDTA solution, loss of architectural details of the intermediate junctions with separation of the dense border in the epithelium of the intestinal mucosa was observed (Cassidy and Tidball, 1967). The mechanism of the maintenance of membrane structure by calcium ions may be related to i t s interaction with proteins in the c e l l membrane arid coat. It is well known that structural alteration and inactivation (in the case of enzyme) of proteins are induced by the removal (by EDTA) of divalent ions. Reaction and structural stabilization of the altered or inactivated proteins are restored by incubating them in solutions containing divalent ions including calcium and magnesium. This type of cation-protein interaction is also - 53 -^influenced bypH (Kingdon et a_l., 1968;" Shapiro and Ginsburg, 1968). The effect of EDTA upon the protrusion of IRC cells may also be due to i t s action in removing divalent ions from the cytoplasm. For example i t was noted that EDTA diminishes the viscosity of the peripheral cytoplasm in mouse ascites cells (Nishimura et a l . , 1955). EDTA could also remove the divalent ions which are necessary for various bio-chemical processes involved in the synthesis or function of either the microtubules or microfilaments (Wessels et a l . , 1971), or the substances in or overlaying the c e l l membrane at the protrusion. It has been demonstrated that electrophoretic mobility is lowered by incubating cells in calcium solution (Collins, 1966; Kiremidjian and Kopac, 1972) and i s increased by treating cells with EDTA (Weiss, 1967). These observations indicate that calcium ions bind to the negative charges at a depth less that 10 A from the c e l l surface. The calcium binding sites could be the phosphate groups of the phospholipids, or the polyphosphoinositides with sialate groups on the c e l l surface, as demonstrated in human red cells (Gent,et a l . , 1969). Since the surface negative charges are dependent on pH (Collins, 1966), an increase of pH would decrease the amount of calcium ions bound. As a matter of fact, the difference of electrophoretic mobility between the conditions with and without the presence of calcium ions i s highest at pH 5 (at lower pH's, where the sites become increasingly protonated, calcium reduces the net charges considerably less) and decreases as the pH goes higher. Since the lowering of electrophoretic mobility is due to the binding of calcium ions, the results mentioned above then indicate that fewer - 54 -calcium ions are bound a t p l l 8 (Collins, 1966) than at pH 7.4. This could account for the drop of the proportion of pear-shaped cells at the former pH. Changes of surface properties induced by changes in both pH and calcium ion concentration have been described in many systems. For example, Holtfreter in 1948 already noted that "isotonic saline solutions lacking calcium or having a pH above 9 were among the agents which"weaken or liquefy the c e l l membrane" and caused changes of surface properties in cells isolated from amphibian gastrulae. One of the important results obtained from the present study is perhaps the effect of pH upon the change in shape of the IRC c e l l s . Although EDTA has been extensively used in various studies including c e l l deformation, tissue dissociation etc., the pH of the solution used was hot uniform or sometimes not reported. The pH effect may not be that important i f only the dissociation of the tissue i s desired. However, in the studies of the effect of EDTA in c e l l deformation (Weiss, 1967) or other cellular interactions involving EDTA (Kay, 1971), different pH's of the solution used may give entirely different pictures i n the results. An electron-microscopic study on IRC cells treated with different concentrations of EDTA might show whether the thick c e l l coat over protrusions was removed by such treatment and would help to define the role of both ECM and calcium ions i n intercellular adhesion. The thick c e l l coat at the protrusion of IRC ce l l s with i t s stronger adhesiveness, may be an important factor for the expression of the malignancy of IRC c e l l s . For example, this stronger - 55 -adhesiveness might f a c i l i t a t e the attachment of the pear-shaped c e l l to the epithelial wall of a blood vessel or to other tissues and might also enable the cells to aggregate, thus preventing an attack by the normal leukocytes. If this was so, i t would be worthwhile to determine whether or not the round c e l l s , without the protrusion, are less malignant. If this was the case, then we would have an example of cancer cells that possess a transitional expression of malignancy which can be a characterized in functional, structural and chemical terms. - 56 -PART II THE DYE-EXCLUSION TEST FOR CELL VIABILITY: PERSISTENCE OF DIFFERENTIAL STAINING FOLLOWING FIXATION - 57 INTRODUCTION o The proportion of viable cells in a c e l l population can be estimated in various ways including methods based on the reduction of 2,3,5-triphenyltetrazolium chloride (Hoskins et a l . , 1956), v i t a l staining, and the uptake of labelled thymidine (Wojciech et _al_., 1967), but the simplest one and the one most widely used is the so-called 'dye-exclusion' method (Hanks and Wallace, 1958), which depends on the phenomenon that many stains are excluded by l i v i n g cells but not by dead c e l l s . While the staining procedure i s quite simple, i t is d i f f i c u l t to concurrently process large numbers of samples, particularly where the exact timing of progressive cytotoxic effects i s required. It therefore seemed desirable to determine whether the d i f f e r e n t i a l staining of dead and l i v e cells was based on structural differences that would persist following fixation, so that the dye-exclusion test could be performed on fixed cells at convenient times after the main experimental work. The present communication describes such a method. - 58 -•MATERIALS AND METHODS Dyes Seven dyes, l i s t e d in Table 1, were studied. Six of these, i.e., safranin (Paul, 1961), eosin (Phillips and Terryberry, 1957), Congo red (Geschickter, 1936), erythrocin (Phillips and Terryberry, 1957), trypan blue (Wojciech et a_l., 1967), and nigrosin (Kaltenbach et al_., 1958), had been used previously for dye-exclusion tests. Alcian blue, a water soluble stain with a relatively high molecular weight (M.W. 1341), was added because preliminary work showed that, over brief periods of time, trypan blue (M.W. 961) but not safranin (M.W. 351) or eosin (M.W. 692) stained only a small proportion of glutaraldehyde-fixed c e l l s , and this observation suggested a possible dependence of the differential staining on the molecular weight of the dye used. In order to compare the staining properties and the rate of diffusion into cells of these dyes, they were made to a uniform molal -4 concentration. The concentration that was selected (2.26 x 10 m) was the lowest one among those reported for v i a b i l i t y determinations (Table I) to minimize possible toxic effects of the dyes on li v i n g c e l l s . Stock solutions (x 10) were made i n Hanks' BSS except for alcian blue, which at this concentration precipitates in BSS or in phosphate buffer within a few hours. Therefore, a solution of 6.0% alcian blue/95% ethanol was prepared and diluted with d i s t i l l e d water 1:20, to obtain an aqueous stock solution (x 10) of 0.3% alcian blue and 4.5% ethanol. This solution was stable for several months. - 59 -TABLE I DYES USED FOR VIABILITY DETERMINATIONS,, ARRANGED IN ASCENDING ORDER OF MOLECULAR WEIGHTS Dye (Source) Molecular weight Concentration reported f o r v i a b i l i t y counts gm/1 equivalent . m o l a l i t y Concentration equivalent,to 2.26 x 10 m a gm/1 Safranin (B.D.H.) Eosin Y (B.D.H.) Congo red (Gurr) Trypan blue (B.D.H.) 351 692 697 Ery t h r o c i n B 880 (Fisher Sci.Co.) 961 Nigrosin b ( A l l i e d Chem.Co.) A l c i a n blue (Harleco) 1341 not given 0.2 2.17 x 10 not given -4 0.2 2.26 x 10 1.0 1.04 x T 0 ~ 3 0.5 0.080 0.156 0.157 0.200 0.217 r d 0.226 • 0.300 a - The m o l a l i t y of a l l stains was adjusted to the lowest one reported ( i . e . that of erythrocin ( P h i l l i p s and Terryberry, 1957)), to minimize possible toxic e f f e c t s of i n d i v i d u a l dyes on l i v i n g c e l l s . b - Nigrosin i s a mixture of complex compounds of unknown c o n s t i t u t i o n and has not been assigned a molecular weight ( L i l l i e , 1969). c - Not previously used. d - On the basis of an a r b i t r a r y M.W. of 1000. - 60 -Cell Culture The following c e l l lines were used: (a) IRC (Dunning and Curtis, 1957), a strain of rat leukemia, cultured as stationa suspension cultures i n Fischer's medium with 10% horse serum, 100 units/ml of p e n i c i l l i n and 100 ug/ml of streptomycin. (b) KB 14, a human carcinoma line, cultured as a monolayer i n Waymouth's medium MB 752/1 with 10% fetal calf serum, 100 units/ml of p e n i c i l l i n and 100 ug/ml of streptomycin. Cell death or damage that resulted in staining by the dye exclusion test was induced by heating, by repeated freezing and thawing, by treatment with 4-Nitroquinoline-l-oxide, or by overgrowth of the cells in exhausted culture medium. Fixation and Counting The effects of alcohol-, heat- and glutaraldehyde fixation were screened, but only the latter was used for detailed studies. Cells were suspended i n 2.0-5.0 ml of culture medium (following trypsinisation in the case of monolayer cultures) and were fixed with an equal volume of cold 2.5% glutaraldehyde In Millonig's phosphate buffer (Pease, 1964), at pH 7.4 for 60 minutes in an ice bath. The cells were then centrifuged and resuspended i n glucose - containing Millonig's buffer for staining and counting. Unfixed cells were stained and counted directly in the culture medium. For dye exclusion tests on either unfixed or fixed c e l l s , 0.1 ml of the 10 x stock solution of dye was added to 0.9 ml of c e l l suspension and the mixture kept at room temperature for 10 minutes unless stated - 6 1 -otherwise. Then two drops of suspension were transferred to the two sides of a hemocytometer and five hundreds cells were counted on each side. The number of stained cells out of a total of one thousand was recorded. Congo red and alcian blue stained some glutaraldehyde-fixed cells partially, i.e. a small portion of the membrane or cytoplasm was stained deeply while the rest was completely unstained. These cells were counted as unstained. RESULTS The proportion of cells stained by the seven dyes in non-fixed and fixed aliquots of the same c e l l suspension was compared in four different c e l l populations (Table II). The number of c e l l s stained with any one of the dyes including alcian blue, was consistently similar in the unfixed samples. However, after glutaraldehy.de fixation the proportion .of stained cells varied considerably, although consistently, depending on the dye used. With alcian blue, the numbers of fixed stained cells did not d i f f e r significantly from those obtained in the corresponding unfixed c e l l preparations. In contrast to these results with glutaraldehyde, fixation with either heat or alcohol resulted in the rapid staining of a l l cells by a l l dyes. On the basis of these preliminary studies, the staining of glutaraldehyde-fixed cells by alcian blue was examined in de t a i l . To determine the period of time over which the glutaraldehyde-fixed cells could be stored without a significant change in their staining properties, c e l l suspensions were fixed, and separate portions stored at 4°C i n the glutaraldehyde fixative or in buffer respectively. The proportion of cells stained with alcian blue was determined after 1 hour of fixation, and again after 24, 48 and 72 hours, after one week and after one month of storage. The results, summarized in Table III, indicate that there was a small but significant increase i n the number of stained cells already after 24 hours of storage i n buffer. On the other hand, cells stored in glutaraldehyde fixative and resuspended i n - 63 -TABLE II THE NUMBER OF STAINED CELLS (PER THOUSAND) IN FOUR OVERGROWN CULTURES OF IRC CELLS, PRIOR TO, AND FOLLOWING GLUTARALDEHYDE FIXATION. 3 Treat-ment Experi-ment No. Safranin Eosin Congo red Erythro-cin Trypan blue Nigrosin Alcian blue 1 56 52 52 53 52 48 53 2 57 60 57 69 60 65 65 Lxed 3 55 56 50 58 52 55 61 Unf; 4 70 66 65 66 68 68 68 mean 59.5 58.5 56.0 61.5 58.3 58.8 6.1.8 1 992 900 150 925 341 149 52 2 976 956 163 952 380 143 63 Q> 3 988 860 180 844 368 125 50 •rl 4 . 976 920 160 905 357 130 66 mean 983.0 910.5 163.3 906.5 361.5 136.8 57.8 a - No significant difference at the p< 0.05 level among the seven non-fixed groups and including the fixed group stained with alcian blue ( s t a t i s t i c a l analysis by analysis of variance (Sokal and Rohlf, 1969) plus Duncan's new multiple range test (Steel and Torrie, 1960). - 64 -TABLE III EFFECT OF STORAGE FOLLOWING FIXATION ON THE SUBSEQUENT STAINING OF CELLS WITH ALCIAN BLUE 3 Storage after fixation Numb er of stained cells (per thousand) Time Solution " ' • .. ^  1 hour 24 hours 1 week 1 month Phosphate buffer - 64.3 - 3.21 80.8 - 10.00 149.3 - 24.60 c Glutaraldehyde fixative 45.8 - 3.95 49.09^ 5.47d 49.5 - 3.69d 80.6 - 8.20 a - Means - S.D., quadruplicate samples, overgrown IRC c e l l s , b - Millonig's buffer (Pease, 1964). c - Equal volumes of 2.5% glutaraldehyde/phosphate buffer and of culture medium, d - Not significantly different from 1 hour specimen. - 65 -buffer immediately before staining gave sim i l a r counts for one week, although there was an increase i n the proportion of stained c e l l s after one month. The rate of penetration by various dyes into glutaraldehyde-fixed c e l l s was examined to determine, i n p a r t i c u l a r , how stable the proportion of stained c e l l s was following the addition of dyes. Figure 20 i l l u s t r a t e s the results obtained with a c e l l suspension where pr i o r to f i x a t i o n , only a small proportion (.7%) of c e l l s was. nonviable by dye exclusion. Following f i x a t i o n , safranin, with a molecular weight of 351, stained almost a l l c e l l s within 5 minutes. In contrast, the number of c e l l s stained with a l c i a n blue (M.W. 1341) a f t e r 5 minutes was not s i g n i f i c a n t l y d i f f e r e n t from that of the unfixed controls. The other dyes diffused into the c e l l s at intermediate rates. A more detailed study of a l c i a n blue staining showed that the proportion of c e l l s stained at 5 minutes remained constant for another 15 minutes (Figure '21). Then there was a small increase i n the number of stained c e l l s and a f t e r a few hours, a steady l e v e l was reached which did not change substantially for 2 days (Figure 22), and, as shown by other s i m i l a r experiments, not even after 4 days. Renewal of the dye after 2 days did not change th i s pattern. On the basis of these re s u l t s , the following routine procedure was adopted: 1. A 2.0-5.0 ml sample of a c e l l suspension i s transferred to a centrifuge tube. 2. An equal amount of cold 2.5% glutaraldehyde-phosphate buffer (pH 7.4) i s added and f i x a t i o n continued for one hour i n an i c e bath. Subsequently the mixture can be stored at 4°C for up to one week. - 66 -Figure 20. Rates of d i f f u s i o n i n t o f i x e d c e l l s of seven dyes, over one -4 hour. Overgrown, g l u t a r a l d e h y d e - f i x e d IRC c e l l s i n 2.26 x 10 m of dye/phosphate b u f f e r . Arrow i n d i c a t e s number of trypan-blue s t a i n e d c e l l s / t h o u s a n d i n the u n f i x e d c o n t r o l sample. 2 0 4 0 6 0 STAINING TIME (MINUTES) 2 0 - 67 -Figure 21. I n i t i a l s t a b i l i t y of the proportion of alcian- b l u e stained c e l l s with continued exposure to the dye. 4 - N i t r o q u i n o l i n e - l -oxide-treated, glutaraldehyde-fixed IRC c e l l s i n 0.03% (2.26 x -4 10 m) a l c i a n blue/phosphate b u f f e r . Each point represents the mean of seven samples. Arrow in d i c a t e s number of trypan-blue stained cells/thousand i n the unfixed c o n t r o l sample. Figure 22. Changes i n the proportion of al c i a n - b l u e stained c e l l s over 48 hours of exposure to the dye. Overgrown, glutaraldehyde-fixed IRC c e l l s i n 0.03% a l c i a n blue/phosphate b u f f e r . Each point represents the mean of four samples. Arrow i n d i c a t e s number of trypan-blue stained cells/thousand i n the unfixed control sample. 2 5 0 - , <c ..... I— • I O L U 1 0 0 -— I — I 1 1 I I 2 6 1 0 2 0 3 0 4 0 5 0 STAINING TIME (MINUTES) 6 0 21 STAINING TIME (HOURS) 2 2 - 68 -3. Immediately before counting, the c e l l s are recovered by centrifugation and resuspended i n a measured volume of phosphate buffer. (This step can be omitted i f the: suspension medium at the time of fixation did not contain serum. Glutaraldehyde-fixed serum seems to interfere with the staining of fixed c e l l s by alcian blue). Cells clumps are broken up by pipetting. 4. 0.1 ml of 0.3% alcian blue/R^O i s added to 0.9 ml of the c e l l suspension, and 5 minutes later the cells are-counted with a hemocytometer. Cells that are completely and deeply stained are scored as nonviable prior to fixation, and those that are unstained or partially stained at the periphery are considered as viable prior to fixation. Using the above procedure, results comparable to those obtained by the dye exclusion test on unfixed cells were obtained with IRC cells and with KB 14 c e l l s , in preparations where c e l l death was caused either by heating, by freezing, by treatment with 4-Nitroquinoline-l-oxide, or by overgrowth i n exhausted culture medium. DISCUSSION The reproducibility, by alcian-blue staining of glutaraldehyde-fixed ce l l s , of the results obtained by the standard dye-exclusion test on unfixed preparations, suggests that cells with a similar degree of damage or loss of v i a b i l i t y are stained by both methods. It suggests further, that the differential staining of viable and non-viable unfixed c e l l s i s ,based, at least i n part, on structural differences i n the region of the c e l l membrane and that these differences are preserved by glutaraldehyde fixation but not by fixation methods involving extensive protein denaturation. A l l dyes penetrated into non-viable c e l l s equally rapidly before and after fixation, and were excluded by unfixed viable c e l l s . However, after viable cells were fixed, their rate of staining by different dyes varied considerably. This variation was, to a degree, inversely proportional to the molecular weight of the dyes and was presumably due to a limited increase in membrane permeability of the viable cells following fixation. The increase of membrane permeability after fixation could be due to various factors. The f i r s t i s the loss of material during fixation. It has been demonstrated, for instance, that glutaraldehyde fixes mainly the proteins, while the l i p i d s are retained but not fixed (Sabatini et a l . , 1963; Korn and Weisman, 1966; Hayat,1970). Korn and Weisman1s results also indicate that a l l the neutral l i p i d s and almost a l l the phospholipids are extracted by ethanol during- the subsequent dehydration, although the demonstrated amount of l i p i d s extracted during glutaraldehyde fixation is only 1%. With respect to the proteins, although they are generally well fixed by glutaraldehyde, a loss of about 8% from tissue thus - 70 -fixed has been demonstrated (Luft and Wood, 1963). Loss of l i p i d s and proteins was also observed by these authors when other fixatives are used. Moreover, other chemical groups may probably be lost during chemical fixation. As a matter of fact, before these experiments were done, the phenomenon of the loss of material during fixation had been considered by Wolman (1955), who once remarked that "fixation i s always attended by diffusion and loss of some chemical constituents" and that "not a l l the proteins are insolubilized by fixatives, not even by those which are known as protein precipitants. Many low molecular weight proteins might remain soluble in water when the tissue is treated by the usual fixatives." From the above evidence and discussion, a loss of material from the cells , especially from the plasma membrane, is obvious. Another possible change induced by fixation upon the plasma membrane is in it s chemical organization. For example, cross-linking of the protein molecules by aldehydes i s demonstrated (Sabatini et a l . , 1963). This conformational change which may increase the size of the inter-molecular spaces and the loss of materials from the plasma membrane as discussed above apparantly has significant effects on the permeability of the plasma membrane regardless of i t s organization in the l i v i n g state. There i s , moreover, another possible mechanism to be considered. There could be some active systems involved in the exclusion of these dyes in l i v e c e l l s . Although, in the present study, there i s a definitive increase of membrane permeability after fixation, i t is not possible to judge whether this increase is due to the loss of an active mechanism - 71 -or to the overall increase in leakiness of the membrane resulting from the loss of material and from conformational changes. The difference in staining between viable and non-viable c e l l s was preserved for longer periods after fixation i f the cells were stored in the glutaraldehyde mixture than i f they were stored in buffer. Presumably, loss of cellular constituents continued upon storage in either solution, but more rapidly in the lat t e r . A loss of cellular materials similar to that observed in glutaraldehyde, has been demonstrated to occur during the washing of fixed tissues in buffer (Korn and Weisman, 1966; Trump and Ericsson, 1965). It has been shown also that nonelectrolytes augment the extraction of cellular materials during fixation and washing (Hayat, 1970), and therefore , one factor contributing to the faster increase in the number of stained cells in buffer may have been the presence of glucose, an ingredient of Millonig's Buffer (Millonig, 1962) as used for storage but not when used as a vehicle for glutaraldehyde (Pease, 1964). It i s important to note that the staining kinetics observed upon the addition of alcian blue to glutaraldehyde-fixed cells suggested the differential staining of c e l l groups with varying properties, rather than the gradual diffusion of the dye into a uniform c e l l population. Thus, a proportion of fixed c e l l s , similar to that of nonviable cells in the unfixed controls, was always stained within 5 minutes or less. No further increase in staining occurred for about 15 minutes, when an additional limited and gradual increase in the number of stained cells was observed. This increase levelled off after a few hours, and the rest of the cells remained unstained even - 72 -after several days of exposure to the dye. It Is l i k e l y that those cells that stained most rapidly correspond to the nonviable cells as detected by the dye exclusion test i n unfixed preparations, while the group that stains more slowly, after 20 minutes, might represent either cells with lesser membrane damage that were not detected prior to fixation, or c e l l s whose membrane-permeability was excessively increased during the processing. Finally, i t would appear that the a b i l i t y to exclude alcian blue for long periods was preserved In a considerable proportion of the c e l l s following fixation. While this staining pattern of fixed c e l l s by alcian blue confines the time for obtaining reliable v i a b i l i t y counts to about 15 minutes following the addition of the dye, this limitation rarely poses a practical problem. It should be noted also that a similar gradual increase i n the number of stained c e l l s , with exposure to some of the dyes used for v i a b i l i t y counts, has been observed i n unfixed c e l l preparations (Hanks and Wallace, 1958; Philips and Terryberry, 1957). The staining of biological tissues i s controlled by several factors including the molecular size and net charge of the dye molecules, the temperature and the pH of the dye solution (Singer, 1952). The present results i l l u s t r a t e that, in addition, the penetration of dyes into the fixed viable c e l l s is inversely proportional to their molecular weights. It is obvious that molecular weight is not the only factor determining the molecular size, as, for example, molecular structure is also an important factor. Thus, although eosin and erythrocin, two dyes of very similar structure (Figure 23), dif f e r by almost 200 i n molecular weight, they have very similar penetration patterns (Figure 20). Figure 23. The molecular structures and molecular weights of the dyes used in the present study. Alcian blue from Scott et a l . , 1964; others from Gurr, 1960. i CO Erythrocin B (880) Trypan blue (961) Alcian blue (1341) 2 3 - 74 -Furthermore, dyes may aggregate (congo red) with a resulting reduction in penetration rate. The fact that congo red and alcian blue stained some ce l l s "partially" suggests that in these cells there may be a localized leakage on the plasma membrane and, second, that both congo red and alcian blue diffuse very slowly through the fixed cytoplasm. Since the method described here i s presumably dependent on the preservation of membrane structure, the quality of fixation i s of utmost importance. Various factors such as excessively high speed centrifugation of the cells prior to fixation or variations in pH and temperature during the processing may cause some v a r i a b i l i t y in the results obtained. On the other hand, the i n i t i a l staining properties of the cells could be more stable following fixation with a glutar-aldehyde-osmium tetroxide -mixture (.Trump and Bulger., 1966) because both proteins and l i p i d s , and hence the structure of the plasma membrane, might be preserved more completely. It i s possible that the membrane damage caused by preparative and fixing conditions may not be detected by electron microscopy, but could increase substantially the number of stained c e l l s . Therefore, the present method could be a useful tool to detect the membrane damage caused by various physical and chemical agents, and to compare the performance of various fixating and fixing vehicles employed in electron microscopy. Furthermore, i t could also be used to detect the location of necrotic areas in fixed monolayer tissue cultures. - 75 -SUMMARY 1. About 20 to 40% of IRC 741 rat leukemia cells grown i n stationary suspension culture have single cytoplasmic protrusions which are vis i b l e by light microscopy and by which the cells preferentially adhere to each other. Ultrastructurally, these pear-shaped cells show an accumulation of organelles near the protrusion. The c e l l surface over the protrusion has no m i c r o v i l l i but i s covered with 200-400 A electron-dense particles embedded i n a matrix, while the c e l l surface over the remaining c e l l body has m i c r o v i l l i but no particles. It was the aim of the present investigation to analyse the relationship between the shape, surface structure and adhesive function of these two parts of the IRC c e l l s . 2. Cinematographic results suggest that the cells move mainly by ruffled membranes and adhere to each other by their protrusions, which are therefore adhesive rather than locomotor structures. 3. By light and electron microscopic cytochemistry, i t was shown that the electron-dense particles overlaying the protrusion are protease-, HCl- and/or pepsin resistant while the surrounding matrix contains proteins and acidic moieties. 4. The c e l l coat over the protrusion i s not only thicker but also has a charge density higher than that on the remaining c e l l body, as shown by c e l l electrophoresis. The paradoxical observation that the region of highest charge density is also one of increased adhesiveness might be explained by the reduced radius of curvature - 76 -of the protrusions and of the overlaying extracellular particles. 5. The induction and/or maintenance of the protrusions i s sensitive to environmental factors. The proportion of pear-shaped cells increases i n relation to the degree of intercellular contact. Acid pH does not affect this proportion but alkaline pH lowers i t , as does exposure to temperatures below 37°C or suspension i n 0.2-2.0% EDTA (calcium depletion). The proportion of pear-shaped cel l s also decreases in aging c e l l cultures, probably due to deficiency in nutrients and/or the accumulation of waste products in the medium. 6. The protrusion appears to represent a type of adhesive organelle not previously described in either cancer c e l l s or normal c e l l s . Its specialized function can be related to i t s special ,ultrastructure and surface properties. The localized stronger adhesiveness over the protrusion may be important for the expression of the malignancy of IRC ce l l s by f a c i l i t a t i n g their attachment to normal tissues, or by protection from host immune mechanisms through enhancement of the aggregation of the IRC c e l l s . 7. In the course of the above work, a new dye-exclusion test was developed which permits v i a b i l i t y counts as well as the determination of c e l l shapes to be performed on fixed c e l l samples subsequent to the main experimental work. This method employs alcian blue which has a molecular weight larger than that of dyes used in the standard dye-exclusion test and i s excluded by the viable cells even after they are fixed in glutaraldehyde. - 77 -BIBLIOGRAPHY Abercrombie, M. (1966). Contact inhibition: The phenomenon and Its biological implications. In: Decennial Review Conference  on Cell, Tissue and Organ Culture, Nat. Cancer Inst. Monogr. 26: 249-277. Ambrose, E.J. (1966). The surface properties of tumour c e l l s . In: The Biology of Cancer, D. \an Nostrand Co. Ltd. (London). Anderson, M.R. (1966). Cell Surfaces. Brit. J. Cancer, 20: 299-306. Anderson, W.A. and J. Andre. (1968). The extraction of some c e l l components with pronase-and pepsin from thin sections of tissue embedded in an epon-araldite mixture. J. Microscopie, 7: - 343-354. Auersperg, N. (1969). Histogenetic behaviour of tumors. II. Roles of cellular and environmental factors in the jLn vitro growth of carcinoma c e l l s . J. Nat. Cancer Inst., 43: 175-190. Bhisey, A.N. and J.J. Freed. (1971). Ameboid movement induced in cultured macrophages by colchicine or vinblastine. Expt. Cell Res., 64: 419-429. Bradbury, S., W.D. Billington, D.R.S. Kirby and E.A. William. (1970). Histochemical characterization of the surface mucoprotein of normal arid abnormal human trophbblast. Histochem. J. 2: 263-274. Brewer, J.E. and L.G.E. Bell. (1970). Specificity of pseudopodium induction by the action of cations on Amoeba proteus. J. Cell Sci., 7: 549-555. Carr, I., G. Everson, A. Rankin, and J.Rutherford. (1970). 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Human leukemic c e l l s i n tissue culture: an electron microscopic survey. Cancer Res., 27: 2447-2464. Hause, L.L., R.A. P a t t i l l o , A. Sances, Jr. and R. F. Mattingly. (1970). Cell surface coatings and membrane potentials of malignant and nonmalignant c e l l s . Science, 1969: 601-603. Hayat, M.A. (1970). Principles and Techniques of Electron Microscopy. Volume 1, Van Nostrand Reinhold Co. (New York). Henkart, P. and T. Humphreys. (1970). Cell aggregation in small volumes on a gyratory shaker. Expt. Cell Res., 63: 224-227. Holtfreter, J. (1948). Significance of the c e l l membrane i n embryonic processes. Ann. New York Acad. Sci., 49: 709-768. Hoskins, J.M., G.G. Meynell and F.K. Sanders. (1956). A comparison of methods for estimating the viable count of a suspension of tumour c e l l s . Expt. Cell Res., 11: 297-305. Inbar, M., H. Ben-Bassat and L. Sachs. (1972). Membrane changes associated with malignancy. Nature New Biology, 236: 3-4, 16. Kaltenbach, J.P., M.H. 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Thesis, Biology,Programme, Faculty of Science, University of Bri t i s h Columbia. - 84 -APPENDICES - 85 -APPENDIX I Preparation of Cells for Microscopy , (a) Dehydration and Epon Embedding Procedure 1. 70% EtOH, 5 to 10 minutes. 2. 90% " 2 I I I I I I I I 4. 100% " i. H 5. . . . . . . . . 6. " " /Propylene oxide (1:1), 10 minutes. 7. Propylene oxide, 10 minutes, g .. it I I it 9. " " /Epon (1:1), 1 hour. 10. " " " (1:3), " 11. Epon, 1 hour. 12. Fresh Epon, 24 hours at 37°C then overnight at 56°C. (b) Epon Solution Stock solution A: Epon 812 Resin 62 ml DDSA (Dodecenyl Succinyl Anhydride) 100 ml Stock solution B: Epon 812 Resin 100 ml NMA (Nadic Methyl Anhydride) 89 ml Working solution: Mix solution A and B (1:1) then add 1.5 to 2.0% by volume, the accelerator DMP-30-Tris (Dimethylaminomethyl) phenol. - 86 -APPENDIX 2 The Chemistry of EDTA The aminocarboxylic acid ethylene-diamine-tetraacetic acid, or EDTA in short, belongs to the group of chemical compounds, commonly known as chelators, chelons or chelating agents (from greek "cheele", O^ ity'O the crab's claw) for their a b i l i t y in forming complexes with many divalent metal ions. The formation of metal-ion-EDTA complex is illustrated in Figure 24 (Chenoweth, 1956). Many divalent metal ions have binding a f f i n i t y for EDTA. The following are important ones, arranged in the order of decreasing chelate s t a b i l i t y (Meites, 1963, pp 1-45); 4H" ,^.4*4" „ 4*4" _ f I _ ++ _ "f• f" "H~ _ ~\"I Cu , Ni , Pb i Zn , Co , Fe , Mn , Ca , Mg EDTA as a free acid is only slightly soluble in water. Therefore i t i s usually employed as a solution of sodium (or ammonium) salts and the term EDTA usually implies an anionic form (Meites, 1963). The following table summarizes the four important sodium salts of EDTA. a) Monosodium EDTA, NaH^EDTA b) Disodium EDTA (Sequestrene NA^) Na^EDTA c) Trisodium EDTA (Versene-9) Na^HEDTA d) Tetrasodium EDTA (Versene) Na^EDTA It should be noted that in the above table, i . The free acid form of EDTA i s represented by H^EDTA; i i . The names in parenthesis are commnon trade names (Stecher, 1968); i i i . A l l except monosodium EDTA are chelating agents. - 87 -Figure 24. The formation of metal-ion-EDTA complex (Chenoweth, 1956). Figure 25. The structure of EDTA in aqueous solution (Chaberek, 1959). Figure 26. Titration of EDTA with KOH. "a" denotes moles of base added per mole of EDTA. Twice-ionic and tripl e - i o n i c forms of EDTA occur at pH 4 and pH 8 respectively. (Modified from Chaberek and Martell, 1959, p. 312). Figure 27. Moles of EDTA required to bind one mole of calcium ions at different pH's. (Modified from Chaberek and Martell, 1959, p. 63). - 88 -In aqueous solution, the sodium salts of EDTA dissociate, e.g., Na. EDTA 4Na+ + EDTA4" k and assume the free acid form, 4-EDTA + 4H„0 H. EDTA + 40H . 2 4 Since excessive OH are produced, the aqueous solution of sodium EDTA is alkaline. The generally accepted structure of EDTA in aqueous solution is that, shown i n Figure 25, in which two of the carboxylic acid groups have interacted with amino nitrogen to form a double dipolar ion (Chaberek and Mertell, 1959). The pK values for the successive dissociation of the four hydrogen of EDTA are as follows (Chaberek and Martell, 1959, p. 128): = 2.00 : (H^EDTA H^EDTA + H ) PK2 = 2.67 (H3EDTA~ H2EDTA2~ +.'H+) PK3 = 6.13 (H2EDTA2~ — — HEDTA3" + H +) P K 4 = 10.26 (HEDTA3" EDTA4" + H+) The tit r a t i o n of EDTA with KOH is represented in Figure 26. In the graph, the appearance of the f i r s t inflection after two moles of base have been added per mole of amino acid corresponds to the complete neutralization of the two relatively strongly acidic car-boxylic hydrogen (pK 2.00 and 2.67). According to the Henderson-Hasselbalch equation (pH=pK + log [A~] - log [HA]), half of the EDTA 3- 4-molecules present are in the HA form at pH=6.13 and in the HA form at pH=10.26. At somewhere between pH 6.13 and 10.26, a l l the 3-EDTA molecules are converted to HA . This point is the pH«8.1, as - 89 -indicated in the ti t r a t i o n curve in Figure 26. The capacity of EDTA for calcium ions have been calculated from titration data and the results are shown in Figure 27. The ordinate represents the number of moles of EDTA required to complex one mode of calcium ion. EDTA exhibits i t s f u l l chelating capacity for calcium 3-ipns at around pH=7. At this pH, most EDTA molecules are in HA form (Figure 26) as discussed earlier. REFERENCES 1. Chenoweth, M.B. (1956). Chelation as a mechanism of pharmacological action. Pharmac. Rev. , 8: 57-82. 2. Meites, L. (ed.) (1963). Handbook of Analytical Chemistry. McGraw-H i l l (New York). 3. Chaberek, S. and Martell, A.E. (1959). Organic Sequestering Agents. John Wiley & Sons, Inc. (New York). 4. Stecher, P.G. (ed.) (1968). The Merck Index 8th Edition. MercK & Co., Inc. (Rahway, N.J.). 

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