Biochimica et Biophysica Acta 458 (1976) 1-72 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The Netherlands BBA 87023

TRANS-MEMBRANE T U M O R CELLS

C O N T R O L OF THE RECEPTORS O N N O R M A L A N D

II. S U R F A C E C H A N G E S ASSOCIATED W I T H T R A N S F O R M A T I O N MALIGNANCY

AND

G A R T H L. N I C O L S O N

Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, Calif. 92112, and Department of Developmental and Cell Biology, University of California, lrvine, Irvine, Calif. 92664 (U.S.A.) (Received J a n u a r y 22nd, 1976)

CONTENTS I.

II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

A. Cell surface organization . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

B. Cytoplasmic control of the cell surface . . . . . . . . . . . . . . . . . . . . .

3

Cell surface modifications after transmission . . . . . . . . . . . . . . . . . . . .

4

A. Surface composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

B. Surface enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9

C. Cellular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14

D. Other changes

14

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11I. Dynamics of surface receptors o n normal and transformed cells . . . . . . . . . . . A. Lectin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

1. Differences in cell agglutinability . . . . . . . . . . . . . . . . . . . . . .

19

2. Distribution and mobility o f lectin receptors . . . . . . . . . . . . . . . . .

23

3. Factors affecting cell agglutination . . . . . . . . . . . . . . . . . . . . .

25

B. Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34

l. T u m o r associated antigens . . . . . . . . . . . . . . . . . . . . . . . . .

34

2. Dynamics of antigens . . . . . . . . . . . . . . . . . . . . . . . . . . .

36

C. Surface modifications during the cell cycle and at cell contact . . . . . . . . . . . IV. Possible relevancy to cancer . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 43

A. Mechanisms of t u m o r immunity . . . . . . . . . . . . . . . . . . . . . . . .

43

1. H u m o r a l immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43

2. Cell-mediated immunity . . . . . . . . . . . . . . . . . . . . . . . . . .

44

B. T u m o r escape mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . .

45

I. Surface antigen modification and antigen loss 2. Antigen shedding and blocking factors 3. T u m o r enhancement V.

18

. . . . . . . . . . . . . . . .

45

. . . . . . . . . . . . . . . . . . .

50

. . . . . . . . . . . . . . . . . . . . . . . . . . .

52

Final c o m m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

References

55

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION The cell surface or periphery (plasma membrane and attached intra- [1] and extracellular [2] membrane-associated components) remains one of the most potentially useful avenues for attacking neoplastic diseases. This cellular structure is involved in a variety of physiological properties which directly relate to neoplastic transformation such as cell growth, division, communication, movement, differentiation, escape from immune destruction, and other characteristics that define states of tumor progression and sometimes invasion and metastasis. It would be convenient if these properties were definable by unique cell surface characteristics which were readily identifiable so that highly specific molecular approaches to fighting cancer could be developed. Unfortunately, such a simplified approach has not yielded much in the way of clinically beneficial results, but the potential to eventually disect and ultimately understand many of the basic cellular characteristics of tumor cells and use them to selectively control or kill wayward cells is excellent. This review will analyze many of the altered properties and components of the surfaces of "tumor" cells (transformed in vivo and for the most part grown in vivo) and "transformed" cells (transformed in vitro or in vivo and for the most part grown in vitro). The distinction between these two tools of cancer biology will be made throughout the text. This was not done because one system represents the "correct" system of study; instead, it was done because alterations in cell surface properties or components must be compared to the properties of closely related (counterpart) normal cell surfaces. In the case of tumor cells isolated during in vivo growth, it is very difficult to find suitable normal cells for direct comparison because tumors most often arise in vivo from unknown precursor cells. On the other hand, "normal" tissue culture cells (defined by their tumorigenicities [3-7], growth properties [4-6,8,9], adhesive specificities [2,10-14], migratory abilities [2,11,15], surface morphologies [ 16,17], agglutinabilities [18,19], serum requirements [8,9,21-23], anchorage dependencies [8,24, 25] and other properties [1,2,8,9,15,19,26-33]) can be transformed in vitro resulting in tumorigenic cell lines [3,5,6]. Changes in the above properties after neoplastic transformation correlates well with ability to form tumors in vivo, although some of these properties have recently been separated from the tumorigenic properties of certain virus-transformed cells [25]. Before turning to some of the surface alterations of neoplastic cells, it is necessary to introduce some current theories and models which seek to explain the organization and dynamic behavior of cell surfaces. A much more comprehensive review

Abbrevations: B cells, bone marrow-derived cells; BCG, Bacillus Calmette-Gu∈ CLL, chronic lymphocytic leukemia; CEA, carcinoembryonic antigen; CSA, cell surface antigens; CSP, cell surface protein; DEAE, diethylaminoethyl ether; Ig, immunoglobulin; surface-Ig, cell surfacebound immunoglobulin; IgA, immunoglobulin A; lgG, immunoglobulin G; IgM, immunoglobulin M; LETS, large-external, transformation-sensitive protein; MLV, murine leukemia virus; SA, soluble antigen; SDS, sodium dodecyl sulfate; SF, fibroblast surface antigen; T cells, thymusderived cells; TATA, tumor-associated transplantation antigen: VEA, virus-associated antigen.

of dynamic cell membrane organization can be found in the companion review to this article [1] and in other recent reviews [2,18,19,30,33--48].

IA. Cell surface organization The most important structure at the cell surface is the cell or plasma membrane which is composed of amphipathic lipids and proteins, some of which contain covalently attached oligosaccharides. It is now well documented that cell membrane lipids are arranged in a bilayer configuration [49-51], allowing their hydrophobic tails to associate with the exclusion of bulk water [52]. Membrane lipid molecules are capable of rapid lateral motion in the membrane [1,18,28,30,38,40,41,47] when in the fluid state, but they can also undergo phase separation into gel-like islands where their mobility is low [1]. These molecules are probably maintained in an asymmetric composition from one side of the bilayer compared to the other [40,53]. Integral membrane proteins and glycoproteins [38,46] associate with the lipid bilayer and appear to be intercalated to various depths into the fluid lipid matrix. Integral glycoproteins and proteins are capable of lateral movement, although the rates of mobility and inherent distributions of these integral membrane components can vary [1,30,44,47,48]. In the fluid mosaic model of membrane structure [38,46] most integral membrane components are free to diffuse laterally; however, some recent evidence suggests that complex restraining mechanisms exist which prevent some components from free movement and completely random distribution [1,18,30,45]. lB. Cytoplasmic control of the cell surface Non-random movements of cell surface receptors can occur under the influence of a membrane-associated cytoplasmic system containing cytoskeletal elements [1,18,30]. In particular, multivalent ligand binding to receptors on cell surfaces can cause ligand-induced redistribution of the receptor-ligand complexes into "clusters", larger "patches" and on some cells, eventually polar "caps" [I,18,30,47,54-57]. For example, the binding of antibodies to cell surface immunoglobulin (surface-Ig) molecules on lymphoid cells results in cap formation with eventual shedding or endocytosis of some of the surface-Ig-antibody complexes [1,56]. Capping requires metabolic energy [1,55,60-63] and can be blocked [64--67] or reversed [64,66] by drugs that act on microfilaments (cytochalasin B) in combination with drugs that disrupt microtubules (colchicine or vinblastine). These results and others (reviewed in ref. 1) suggest that the cell cytoskeletal system (microtubules and microfilaments) plays an active role in ligand-induced receptor capping and trans-membrane regulation of cell surface receptor expression. Interplay exists between the two cytoskeletal elements (microtubules and microfilaments) which probably determines the dynamics of at least certain receptors [1,67-70]. Capping of lymphoid cell receptors is unaffected or actually enhanced by microtubule-disruption with colchicine [65-70], but can be at least partially blocked by cytochalasin B [1,65,67]. Cap formation can also be inhibited by the binding of concanavalin A, but this inhibition in turn can be blocked by colchicine or vinblastine

4

[62,68-70] suggesting a trans-membrane "anchoring" role for microtubules in stabilizing surface receptor mobility [62,65-70]. Microfilaments containing actinomyosin activities [71,72] appear to play a more active role in the capping process and are probably responsible for trans-membrane energy-dependent translocation of cell surface receptors (reviewed in ref. 1). The association of the cytoskeletal elements to each other or to similar cell membrane attachment or nucleation points (Fig. l) and their respective opposing roles as skeletal or anchorage components (microtubules) and contractile or movement components (microfilaments) probably offers cells the necessary fine control over cell surface receptor dynamics. II. CELL SURFACE MODIFICATIONS AFTER TRANSFORMATION A variety of cell surface properties have been found in a modified state on tumor or transformed cells (Fig. 2), although few of these seem to be universal for the neoplastic state (reviewed in refs. 2,27-30,33,73). Most of the experimental studies on neoplastic cells have utilized cloned tissue culture cell lines which can be transformed by oncogenic viruses, chemical carcinogens, or radiation to provide ample numbers of similar cells. Alternatively, spontaneous transformants have been selected from "normal" cell culture populations. Tissue culture cells and their neoplastic transformants can be grown under identical, or at least reproducible, conditions, albeit in absence of host immune surveillance, enzymes, hormones, etc. The classification of these tissue culture models for a cancer as "normal" and "transformed" may be misleading due to possible changes in cell properties occurring after long periods of cultivation in vitro and the fact that some transformations are very inefficient, and transformation-selected cells are grown out of the untransformed population. It is likely that these transformants arise from variants within the "normal" cell population. Most established untransformed cell lines are aneuploid and do not "age" in tissue culture with reasonable times, and some of these established cell lines have additionally been found to be tumorigenic under certain conditions. For example, the mouse 3T3 (clone A.31) cell line that is in use in many laboratories throughout the world is classified as a "normal" cell model, because it is rarely tumorigenic when cell suspensions (up to l0 s cells) are injected into mice [3]. However, Boone [74] attached 3T3 cells to small glass beads before implantation into BALB/c mice and found that these "normal" 3T3 cells eventually formed hemangioendotheliomas. One could interpret Boone's [74] experiments on the grounds that the glass-attached 3T3 endothelial cells simply "overgrew" on this non-physiological substrate and spontaneous transformants were selected in vivo. The overgrowth procedure is well known to be one of the easiest methods for selecting spontaneous transformants in vitro, but these experiments are instructive, since they remind us of how difficult it is to find suitable model systems for normal/transformed cell pairs. In the following section some of the variety of cell surface changes which have been reported after transformation of tissue culture cells are listed, discussed and occasionally compared to the surface characteristics of actual tumor cells of in vivo origin.

11.4. Surface composition The important surface components of all cell types are proteins, glycoproteins, lipids, glycolipids and glycosaminoglycans. Some of these components have been implicated as antigens or other important surface recognition structures, and there have been many reports on the modifications, for example, of complex carbohydrates in tumor cells (reviewed in refs. 30, 75). Many inconsistencies exist in the literature from one transformed or tumor system to another in the amounts or types of surface components which are modified, but in some cases there are distinct, reliable differences. Some tumors have elevated sialic acid content, especially human tumors of the colon, stomach, breast [76] and other tissues [77,78]. In cultured transformed cell systems the most frequent change in sialic acid content is usually the reverse; lowered membrane sialic acid contents are found after transformation [79-81]. Thus, specific cell surface sialic acid changes do not appear to be a general property of the neoplastic state [82]. It should be mentioned that sialic acid is a common terminal saccharide residue on many surface glycoproteins and glycolipids, and only a few of these may be important in cancer-related properties of the cell surface. The most prominent complex cell surface carbohydrates are the glycosaminoglycans (complex polysaccharides containing hexosamines and hexuronic acids) which make up the "glycocalix" outside the integral membrane zone*. There are many reports on transformation-dependent changes in these molecules [83-93]. For example, Hamerman et al. [94] found that SV40- and polyoma-transformed mouse fibroblasts synthesize less hyaluronate than their untransformed counterparts, but Ishimoto et al. [95] reported that avian sarcoma virus-transformed chick fibroblasts have greater amounts of hyaluronic acid after transformation. Increased quantities of hyaluronic acid and chondroitin sulfate have been found in human hepatic carcinoma compared to normal liver tissue [96] and in transplantable tumor cell lines [97]. Virus-transformed cell lines show enhanced rates of synthesis and cell surface amounts of hyaluronic acid compared to untransformed parental cell lines [83,84]. Interestingly, the amounts of sulfated glycosaminoglycans synthesized by transformed cells are generally lower [84-87]. Few modifications of this type seem to be general; however, this may be an oversimplification due to the large number of different classes of glycosaminoglycans at the cell surface and the fact that few systems have been fully investigated [75]. General changes in the overall membrane composition of lipids and glycolipids have been found in a variety of tumors and transformed cells. In many malignant human tumors the contents of cholesterol and phospholipid are higher than the corresponding normal tissue or benign lesions [98,99]; however, mouse and rat * The glycocalix is a morphological term which is used to describe extracellular complex polysaccharides, proteoglycans and glycoproteins whether they are integral to the plasma membrane and anchored there by a hydrophobic "tail" embedded in the lipid bilayer, or peripheral membrane components which are bound to integral components by hydrogen bonding, salt bridges, and other forces (see Section IIA and B of ref. 1}.

leukemic lymphomas have similar amounts of cholesterol compared to normal lymphocytes [100]. lnbar and Shinitzky [101,102] have proposed that cholesterol content in murine ascites lymphoma plasma membranes has an important growth regulatory role by adjusting the fluidity of the integral membrane zone (see Section II1). Although provocative, this proposal will have to be more conclusively tested. Membrane phospholipids do not vary significantly in many tumor systems compared to normal or untransformed cells [103,104], although minor shifts in fatty acid composition have been reported [105]. Glycolipid changes associated with the transformed state have been extensively documented (see reviews 30,106-108]. In one system (hamster NIL fibroblasts and several viral transformants) glycolipid changes after transformation can be generalized as: (a) decreased amounts of more complex glycolipids or deletion of terminal saccharide residues; (b) inability of glycolipid synthesis to respond to cell contact ("contact-extension" [109,110]) with the resulting addition of terminal saccharide sequences; and (c) enhanced glycolipid accessibility to antibodies, enzymes and lectins (reviewed in refs. 30, 33, 107, 108, 111). These generalized changes in transformed NIL cell glycolipids have been found in other tumor systems, such as the simplification of mouse, human, rat and baboon complex glycolipids after transformation [108-110,112-115]. In some of these tissue culture cell systems the activities of glycolipid: glycosyltransferases responsible for the synthesis of higher glycolipids are altered [116-119]. Although convincing, these changes may not be related to normal growth control, because spontaneous transformants of mouse and hamster cell lines do not show glycolipid changes such as glycolipid simplification [108,120], and transformed cell revertants which show growth characteristics more similar to untransformed cells only partially regain normal glycolipids patterns [108,117,120]. However, this latter finding can be rationalized if the revertants only partially returned to the "normal" phenotype. Some of the most interesting cell surface changes found after transformation are changes in the "exposure" of proteins and glycoproteins to external labeling procedures (reviewed in refs. 30, 73, 121). Two currently popular methods, lactoperoxidase catalyzed z2sI_iodination [122] and galactose oxidase-tritiated borohydride labeling [111,123,124], have been used to study transformation-induced alterations. Intact cells are surface labeled and then solubilized in detergents. Most often radiolabeled and unlabeled components are separated by sodium dodecylsulfate-polyacrylamide slab gel electrophoresis [125], and the labeled components identified by radioautography*. The most obvious changes seen in transformed * Lactoperoxidase and galactose oxidase do not penetrate into cells due to their size, and it has been shown that they will not react with cytoplasmic components or inner surface plasma membrane components of intact cells. These reagents react with accessible tyrosine residues in proteins or terminal D-galactosyl residues in polysaccharides, glycoproteins and glycolipids, respectively. Only some surface components probably serve as suitable substrates due to absence of reacting groups or steric unavailability at the cell surface [126]. Molecular weight determinations of glycoproteins in sodium dodecylsulfate polyacrylamide gels are generally relative and are sensitive to carbohydrate content [127].

compared to untransformed cells is the disappearance of a high molecular weight glycoprotein (it can be detected by either technique) of approximate molecular weight 220000-250000. This normal cell glycoprotein has been called the 250K surface protein [121,128], LETS protein [73], component I [129], component Z [130], galactoprotein a [ 13 I, 132] or CSP [133]. It is sensitive to proteolytic enzyme digestion and is more exposed in the G1 phase of the cell cycle and in densely populated cultures [73,114,121,134-136]. There is a marked reduction in iodination of the 250K protein on cells blocked at mitosis [134,136] suggesting that untransformed cells in mitosis are similar to transformed cells as to expression of this surface component. Since it cannot be metabolically labeled in transformed cells, it is probably missing and not simply "masked" or "cryptic" on transformed cell surfaces [137]. Its absence could be due to blocked synthesis or to constant sublethal surface proteolysis [121,138,139]. The large molecular weight glycoprotein has been identified on untransformed (but not transformed) hamster [ 128,134,137, 140], mouse [ 121,141 ], rat [126] and chicken [121,126,129,142-148] cell lines. Yamada and Weston [133] have recently isolated and partially purified the high molecular weight cell surface (glyco)protein that they have called CSP from chick embryo and chick heart fibroblasts. It appears to be the surface-labeled approx. 220000 molecular weight glycoprotein component mentioned above which is missing after transformation. CSP was extracted from ceils by agitation in serum-free media containing 0.2 M urea and purified by slab gel electrophoresis. The isolation procedure removes only about 5 - 1 0 ~ of total amount of cell associated CSP, but its main advantage is that the treated cells are left intact and recover from the urea treatment. Yamada and Weston [133] were able to demonstrate that CSP is not collagen. Urea treatment also removes a factor from chick heart fibroblasts which restores contact-inhibition of movement to urea-treated cells [149], but Yamada and Weston [133] failed to demonstrate this activity in the CSP preparations. Graham et al. [150] used a different approach to purify the high molecular weight iodinatable glycoprotein they have termed LETS (large, external, transformation-sensitive). After iodination and disruption of hamster NIL8 cells, their membranes were fractionated. All of the iodinated proteins except LETS purified with the plasma membrane fraction. LETS purified predominately in a high density fraction containing mainly carbohydrate but little lipid, and electron microscopic examination of this fraction revealed a "fuzzy" amorphous morphology together with a few membranous structures. It is reasonable to conclude from these results that LETS could be a peripheral proteoglycan which is bound by non-hydrophobic forces to the extracellular plasma membrane surface or a large glycoprotein with only a small hydrophobic tail intercalated into the cell membrane. Hynes [121] has proposed that components like LETS could control the mobility of outer surface components by multiple interactions (such as in Fig. 1). If this turns out to be true, similar components might be involved in controlling the mobility and topography of a variety of outer surface components (possibly by trans-membrane linkages discussed in ref. 1 and Section IB and diagramed in Fig. 1) and could also serve cell matrix functions as well. Interestingly, Mallucci et al. [151] have removed a

Mg

MT

Fig. 1. Trans-membrane control over the distribution and mobility of cell surface receptors by peripheral and membrane-associated cytoskeletal components. In this example the mobility of integral glycoprotein complexes GP3 and GP4 is controlled by outer surface peripheral components and also trans-membrane linkages to membrane-associated cytoskeletal elements. In addition, complexes GPz and GP4 could be sequestered into a specific lipid domain indicated by the shaded area, while complex GP2 exists in a free or "unanchored" state and is capable of lateral motion. MF, microfilaments; MT, microtubules (from Nieolson [1]).

peripheral surface component which may be related to LETS. This component appears to be similar to a ruthenium red-stainable substance at the cell surface which in turn is closely associated with dense subplasma membrane microfilament networks. Similar localization results have been obtained for a normal fibroblast surface antigen (SF) located on an iodinable high molecular weight ( ~ 210000) component [143,152-154]. Fibroblast surface (SF) antigen is a normal cell antigen which appears to be carried on the high molecular weight normal cell glycoprotein [143,152-154]. Proteolysis of chick [155] or human [156] fibroblasts releases cell type-specific SF antigen which is also secreted or shed from cells in vivo and can be detected in serum. SF antigen is a major surface component(s) of fibroblasts (up to 0.5 % of the total protein of normal cultured fibroblasts) and is now thought to be a complex of at least three polypeptides (molecular weights approximately 210000, 145 000 and 45 000) [157,158]. One of these components, SF 210 ( ~ 210000 molecular weight), appears to be the high molecular weight surface glycoprotein labeled by lactoperoxidase-catalyzed iodination [73,126,128,129,133,141] and galactose oxidase-catalyzed tritiation [111, 131,140,159]. Interestingly, the approx. 45000 molecular weight component (SF 45) electrophoretically co-migrates with purified fibroblast actin suggesting the possibility that SF antigen molecules may be related to membrane actin [152]. Immunodiffusion data indicate that SF 210 and SF 145 carry similar antigens; however, the biochemical characteristics, metabolism and turnover of SF 210 and SF 145 are distinctly different. SF 210 is glycosylated, accessible to surface labeling and trypsin-sensitive, whereas SF 145 is characterized by a rapid turnover rate during pulse-chase studies [143,153, 1601.

Glycopeptide fragments removed from the cell surfaces of tumor and cultured cells by proteolytic enzymes show transformation-dependent differences when chromatographed on Sephadex gel filtration columns [161,162]. Trypsin treatment of untransformed and transformed hamster [161-163], mouse [164,165], chicken [166, 167] and other tumor cells [168] releases surface glycopeptides which can be further degraded by pronase treatment to low molecular weight fragments (average molecular weight around 4000). When these pronase-digested fragments are chromatographed, populations of glycopeptides obtained originally from transformed or tumor cells migrate differently (they have an "apparent" higher average molecular weight) from the glycopeptides obtained from untransformed cells. Neuraminidase treatment of transformed cell glycopeptides restores their gel chromatography elution profiles to those of glycopeptides from untransformed ceils, indicating that neuraminidasesusceptible sialic acid on transformed cell glycopeptides may contribute to the changes in surface glycoproteins accompanying neoplastic transformation [168,169]. Glick et al. [170] have found a positive correlation between the appearance of these faster migrating glycopeptides and the tumorigenic properties of the cell lines from which they were derived. When several types of transformed hamster cells and their revertants were examined after growth in culture, the transformation-characteristic rapidly migrating peptides were absent. However, when inoculated into animals, these same cells formed tumors, and the isolated tumor cells contained the faster migrating peptides associated previously with transformation. Glick et al. [170] concluded that the altered glycopeptide profiles correlate with tumorigenesis, rather than expression of transformed cell properties. It should be mentioned that gel elution profiles have not assisted in further characterization of these components which probably constitute very heterogeneous populations of different molecules, the identity of which await further investigation. A variety of other compositional differences between untransformed and transformed cells have been found, but their role in determining or maintaining the neoplastic state is unclear or tenuous. Some transformed cells are known to release more glycoproteins into the surrounding media than normal cells [171], possibly as the result of enzymatic degradation (see Section liB.). Chiarugi and Urbano [172] reported that the glycoproteins released by transformed hamster cells are more glycosylated than those released by untransformed cells, and the remaining membrane proteins are less glycosylated after transformation. Earlier articles on increased calcium content of tumor cells [173] led to proposals that calcium was important to transformation and metastatic processes [174].

liB. Surface enzymology The most dramatic transformation-dependent changes in surface enzymology are in degradative enzymes. Unfortunately, in some of these studies little attempt was made to distinguish between total cellular enzymes in cell homogenates and surface localized and secreted enzymes of intact ceils. Oligosaccharide hydrolytic enzymes or glycosidases have been reported to change after transformation. As with other

10 secreted, stored or surface displayed enzymes, measurements of glycosidase activity have been noted with specific substrates which may or may not be representative of the usual endogenous substrates. Also, it is difficult to assess whether in vitro assay conditions properly mimic actual in vivo conditions, and finally the enzyme preparation methods for measuring total cell activity as opposed to secreted enzyme activity or surface displayed activity vary with practically every laboratory. Even taking these points into consideration, there are several observations which are probably pertinent. A variety of glycosidase and protease activities of human malignant tissue have been compared to activities of adjacent normal tissue by Bosmann and Hall [175]. They found higher levels of fl-galactosidase, a-mannosidase, neuraminidase and acid protease in malignant breast and colon tissue homogenates compared to normal tissue homogenates. When pathologically determined premalignant tumors were assayed, intermediate activities were found. Although unusual numbers of leukocytes were not found during the investigation, these cells obviously could have affected the results. When tumorous tissue is surgically removed and assayed, it is very difficult to rule out contamination by normal cell enzymes. Host cells of endothelial and fibroblast origin and blood cells such as macrophages commonly contaminate most tumor specimens, and it is hard to control for their presence in tumors. In tissue culture systems transformation is known to increase cellular glycosidase levels. Kijimoto and Hakomori [176] found higher ceramide trihexoside fl-galactosidase activity after transformation of hamster fibroblast cells. Bosmann [91,177] used p-nitrophenyl sugars as substrates to demonstrate that a variety of glycosidase activities are elevated after transformation of mouse [177] and chicken [91] cell lines. Apparently conflicting data have been collected occasionally, but this may reflect different experimental conditions, material, substrates, etc. For example, ganglioside neuraminidase levels were reported to be elevated in transformed hamster cells [178], but in mouse cell lines no differences were found between untransformed and transformed cells [179]. Higher levels of proteolytic enzymes seem to be an important property of transformed cells. Schnebli [180] found that intact transformed mouse fibroblasts degraded a 3H-labeled protein substrate at a much higher rate than untransformed cells; however, these differences were not apparent when a prior osmotic shock treatment was included in the procedure to release intracellular enzymes. This suggested that transformed cells secreted or had greater surface exposure of proteolytic enzymes compared to their untransformed counterparts. Plasma from the interstitial fluids of mouse tumors contains higher levels of proteases compared to the corresponding activities of normal plasma or intraperitoneal fluid [181]. Homegenates of human breast and colon tumors [175] and virus-transformed mouse [177] and chick [91] cells have elevated cathepsin-like (measured at pH 3.4) protease activities. Sylv6n [ 182] has examined the cathepsin B activities of several tumors and transformed cell lines and has found this proteolytic activity to be elevated after transformation. Using fluorescent antibodies against cathepsin B, more enzyme was found localized

11 at the surfaces of transformed cells compared to untransformed cells. Lipkin and Knecht [183] have reported that malignant hamster cells bind large amounts of anti-cathepsin B1 antibody compared to a non-malignant precursor cell line. Interestingly, untransformed cells in culture appear to have a greater turnover rate of at least some surface proteins and glycoproteins upon reaching confluency. Thus, the turnover rates of surface constituents may determine the extent to which they can be modified by degradative enzymes. Collagenase activities of transformed and tumor cells have attracted attention due to their possible role in normal tissue matrix destruction and tumor infiltration [184-187]. Robertson and Williams [186] extracted collagenase from rat epithelioma tumors, added purified collagen and determined collagenase activity by an examination of the collagen degradation products. More recently tumorous human tissue has been examined for coUagenases. Riley and Peacock [188] studied collagenase activities of normal and neoplastic human tissues. To prove that these activities were due to the tumor cells and not to contaminating lymphoid or granular cells, Taylor et al. [187] cultured tumor fragments on collagen-coated films and monitored collagen destruction. Dresden et al. [189] scanned a variety of human neoplasms for their ability to produce collagenase in culture. Epithelial tumors, particularly colon tumors, squamous and basal cell carcinomas, produced high levels of collagenase, while tumors of mesenchymal origin and normal tissues rarely produced the enzyme. Human collagenolytic activity in squamous cell carcinoma [190] and malignant melanoma [191] are dramatically higher than in surrounding normal tissue. Strauch [192] has examined a wide number of benign and malignant tumors for collagenases. Benign tumors generally showed higher levels of collagenase activity, but there appeared to be a wide spread of enzyme levels in benign tumors, and occasionally low levels similar to normal surrounding tissues were found. Malignant tumors possessed high levels of collagenases, and the enzyme activities found in tissue slices recovered from invasive regions of tumors were higher than from non-invasive regions and surrounding normal tissues [192]. An enzymatic property of transformed cells which has generated a considerable amount of attention recently is the release by transformed cells of a serine protease [193,194] which converts serum plasminogen to plasmin. The activated plasmin acts to hydrolyze fibrin [195,196]. Plasminogen activator secretion by transformed mouse, hamster, rat [195] and chick [197,198] cells as well as human tumor cells [199] is higher than corresponding untransformed or normal cells*. The plasminogen activator-plasmin system appears to be necessary for maintenance of some morphological changes associated with transformation [201], but not for the cell growth characteristics of transformed cells. Reduction of fibrinolytic activity by added protease inhibitors [202], serum containing endogenous fibrinolysis inhibitors [203], or plasminogen-depleted serum [204] has little effect on the growth of transformed * Plasminogen-independent fibrinolysis has been reported to occur after Rous sarcoma virus-transformation of chick embryo fibroblasts by Chen and Buchanan [200].

12 cells to high densities in culture. Also, some untransformed mouse cell lines in log phase growth show fibrinolysis levels as high as their transformed counterparts [202]. The role of plasminogen activator in determining certain characteristics of the transformed phenotype is not really questionable. What remains unanswered, however, is the actual role this enzyme plays in processes important to transformed cell survival in vivo. Contrary to one proposal, plasminogen activator does not seem to be of overriding importance to tumor spread and metastasis. When Nicolson et al. [205] examined the rates of plasminogen activator production for a series of B16 melanomas of low and high metastatic potential [206], no differences were found among the various lines in contrast to the production of certain other degradative enzymes [207]. Proteases may be important in release from growth regulation of quiescent normal cells. Untransformed cells, when treated with low non-toxic concentrations of proteases, display several properties normally associated with transformation: enhanced lectin agglutinability [reviewed in 18,19], phosphate and sugar transport [208,209], mobility of surface components [18,210,211], reactivity with certain antibodies [212,213], and so on. The enzyme-treated cells usually go through another cell cycle and double in number [214,215]. It has been proposed that increased release or display of proteolytic and other hydrolytic enzymes by transformed cells results in cell surface alterations by "sublethal autolysis" [138,139]*. Manipulation of untransformed cell surfaces by proteolytic enzymes or infection by certain non-oncogenic viruses results in many cell surface characteristics indicative of the transformed state [216] which has led Poste and Weiss [139] to propose that these modifications are secondary and not the primary determinants of transformation. The glycosyltransferases are another important class of cell surface enzymes. Glycosyltransferases catalyze the transfer of a sugar residue from a donor, generally a sugar nucleotide such as UDP-D-galactose, to an acceptor, such as the nonreducing terminal sugar of a glycolipid or glycoprotein. Interesting new experiments have demonstrated that polyisoprenoid lipids may serve as intermediate sugar donors in glycosyltransferase reactions [119,217,218]. As expected, the Golgi apparatus contains the bulk of cellular glycosyltransferase activity and is the location where most glycoproteins receive their oligosaccharide components [219-224]. Some activity * Proteolytic activities of tumor cells are thought to be a prominent determinant of their phenotypic properties, because transformed and tumor cells release, and have at their surfaces, higher proteolytie activities than untransformed cells. To demonstrate the involvement of proteases in growth control and phenotypic surface modifications of cells after transformation, protease-inhibitors have been added to cell cultures [195,204,643,757-761]. In several of these studies, transformed cell growth was preferentially reduced, but other studies found no selectivity, and the toxicity of some protease inhibitors brings the general results into question [762]. One transformed cell property that was not modified by protease inhibitors was hexose transport which remained at the usual transformed cell level, even though the growth rate of transformed cells was reduced in the presence of proteolytic enzyme inhibitors [761]. The idea that "sub-lethal" proteolytic autolysis [728] could be, in part, responsible for many of the cell surface alterations and other properties of transformed and tumor cells is intriguing [73,121,139], but it is an extremely remote possibility that all the modifications caused by transformation could be due to proteolytic enzyme action.

13 seems to be associated with cell surfaces, although surface glycosyltransferase molecules may simply reach the outer cell surface as a consequence of plasma membrane biogenesis [225-227]. Roseman [228] and Roth [229] have proposed a theory where intercellular adhesion and cell communication involve binding of cell surface glycosyltransferases to their substrates on the surfaces of other cells. The activities of glycosyltransferases have been measured in whole cells or in cell homogenates using glycolipids or glycoproteins as acceptors. As previously mentioned, viral transformation reduces the amount of complex glycolipids and decreases the activity of at least one transferase needed for their synthesis. Patt and Grimes [116] found that viral transformation of mouse 3T3 cells reduces the transferase activities responsible for transferring sialic acid, galactose, and N-acetylgalactosamine from added sugar nucleotides to endogenous lipid. This is not a general reduction in activity for all monosaccharide transferases, because mannosyltransferase activities are increased. Deppert et al. [230] feel that several cell lines do not have significant cell surface galactosyltransferase activities, and incorporation of labeled sugars from nucleotide donors into cell surface glycoproteins and glycolipids is due to rapid external hydrolysis of added UDPgalactose followed by transport and intracellular metabolism of the labeled sugar. Contrary to this proposal, however, are the results of Yogeeswaran et al. [119] where hamster NIL and BHK cells transfer sialic acid and galactose from added sugar nucleotides or endogenous sugar donors to ceramide dihexoside covalently attached to glass beads. Polyoma-transformed cells transferred less of these sugars to the insolubilized ceramide dihexoside; these experiments strongly support the presence of surface glycosyltransferases. A variety of observations on glycosyltransferase activities have been made on untransformed, transformed, normal and tumor cells. Human breast and colonic tumor tissue homogenates have higher sialyltransferase activity toward endogenous and added glycoproteins than do homogenates of the nearest nonmalignant tissue [175]. This contrasts to sialyltransferases in mouse and hamster cell lines which show reduced activities after transformation [79]. Virally transformed hamster, chick, and mouse cell lines, however, show elevated sialyltransferase activities, if in place of added glycoproteins, glycoprotein fragments trypsinized from cells (or their pronase digests) are used as substrates [167,231]. Transformation tends to increase the glycosylation of endogenous surface glycoproteins, particularly when compared to nongrowing confluent untransformed cells [90,116,177,232]. Roth and White [233] suggested that glycosyltransferases and acceptors on the same untransformed cell surface are not accessible to each other and can only interact between adjacent contacting cells. Transformation modifies the cell surface to allow transferase and acceptor molecules on the same cell to interact, possibly because of release of trans-membrane restraints that may limit the mobility and distribution of surface components (Section III). Patt and Grimes [116] were unable to confirm the model of Roth and White [233], but they used different cells and different experimental conditions. It is important to remember that surface transferase reactions require active enzymes, suitable donors, suitable acceptors, and the mutual accessibility of all three on the cell surface. Although

14 transformation modifies some glycosyltransferase activities, the significance of these changes is unclear. HC. Cellular transport Due to the difficulties in accurately measuring cellular transport, most studies have utilized cultured untransformed and transformed cells. Many transport systems operate at greater rates after transformation. Transport of glucose and its relatively non-metabolizable* analogs, 2-deoxyglucose and 3-0-methylglucose as well as mannose, galactose and glucosamine, increase with transformation (reviewed by Hatanaka [235]), while that of other sugars (sucrose, fructose, ribose, deoxyribose, fucoses, glucose 1-phosphate and glucose 6-phosphate) remains unchanged. Transformed cells also have higher transport activities for certain amino acids and amino acid analogs such as glutamine [236], arginine glutamic acid [237], a-amino-isobutyric acid and cycloleucine [236,237]. Cunningham and Pardee [238] found that phosphate transport is increased after transformation, a result which can be duplicated in untransformed cells by treatment with proteases [208], glycosidases [239], serum or hormones [240]. Importantly, serum which can release density-inhibited untransformed cells from growth control, rapidly stimulates transport of 2-deoxyglucose [208,209], phosphate and uridine [238,240]. Concurrent with these transport changes are rapid increases in cellular cyclic G M P [241,242]. The involvement of cyclic nucleotides with growth control is reviewed elsewhere [243-246] and will not be dealt with in detail here. Changes in cyclic nucleotide levels seem to be related to changes in some transport activities [247], but many systems do not depend on cyclic nucleotide changes for stimulating alterations in transport activities [209,235,240,248,249]. It has been suggested in one system that the transport of a critical amino acid required for cell growth is regulated by cyclic AMP levels [250]. Restriction in transport of a critical metabolite(s) in normal cells and the escape of malignant cells from this restriction has been proposed as an important fundamental difference between normal and neoplastic cells leading to loss of growth regulation [245,251-253]. Pardee et al. [245] have advanced the idea that transformation permanently modifies plasma membrane adenyl cyclase to maintain constant lower cellular levels of cyclic AMP. The lowered cyclic AMP levels, in turn, are proposed to stimulate a variety of transport systems and maintain nutrients at required intracellular levels. Although this scheme must be reconciled with the observations that some important transport systems are not regulated by cellular cyclic nucleotide levels [240,248,249] and are independent of the stage of cell cycle during which virus transformation occurs [253], it appears to be a hypothesis well worthy of careful consideration and further investigation. IID. Other changes A variety of additional cell surface modifications have been reported after * Transported 2-deoxyglucose and 3-O-methylglucose are not metabolized within 30 min, so measurements of transport with these analogs are not plagued by isotope reutilization and excretion [234].

15 neoplastic transformation. Modifications in tumor cell density and electrophoretic mobility were once thought to be important parameters of transformation [254-257], but several subsequent studies have failed to find consistent differences between normal and tumor cells [258-263]. Another surface modification of transformed and tumor cells which has elicited attention is the histochemical staining of cell surface mucopolysaccharides by colloidal iron hydroxide, ruthenium red and phosphotungstic acid [264]. Defendi and Gasic [265] observed that transformed cell lines have increased thicknesses of colloidal iron stainable material at their surfaces compared to untransformed cells. This was subsequently confirmed in other systems using ruthenium red and phosphotungstic acid staining [266-268], with notable exceptions [269,270]. Indeed, when Dermer et al. [270] carefully examined untransformed normal rat kidney cells and compared them to sarcoma-transformed normal rat kidney cells for differences in ruthenium red or phosphotungstic acid staining surface material, they found no difference in the surface coats of these cells in subconfluent, rapidly growing cultures. However, in confluent cultures where the untransformed normal rat kidney cells were quiescent but the transformed cells continued growing, the former cells had thicker surface coats of ruthenium red stain. In contrast, Mallucci et al. [271] and Poste [272] used ellipsometry to measure the thicknesses of cell surface coats (presumably extracellular complex saccharides) on several untransformed and transformed cell lines and noted thicker surface coats on the transformed cells. Cell junctional formation was reported diminished in tumors [266, 273] and in transformed cell cultures [270, 274-276]. However, no differences in cell junctional structure or formation have been seen using freeze-fracture techniques in chick [277] or mouse [278] cell cultures compared with their transformed counterparts. Pinto da Silva and Gilula [277] also failed to find differences in the freeze-fracture morphology of untransformed and RNA virus-transformed chick fibroblasts. Scott and his collaborators [279,280] recently presented results indicating that the freeze-fracture particles in the plasma membranes of untransformed 3T3 cells progressively cluster with the establishment of cell-contact in culture, while the particles of transformed 3T3 cells remain dispersed, but these latter results are probably artifactual. Particle clustering observed by Scott et al. [279] and Barnett et al. [280] may have resulted from the use of unfixed cells and glycerol impregnation to avoid damage during rapid freezing [278,281]. Other investigators have used fixation procedures which prevent glycerolinduced aggregation of membrane-intercalated particles and have failed to show differences in plasma membrane particle distribution between untransformed and transformed mouse 3T3 [278] and hamster BHK21 [282] cells. Torpier et al. [282] did determine, however, that the densities of particles observed in sarcoma- and polyoma-transformed hamster BHK21 and Rous sarcoma transformed chick embryo membranes were higher than in untransformed parental lines. The significance of these results is uncertain, but a possible explanation may reside in the different amounts of membrane-associated cytoskeletal components attached to the plasma membranes of these cells. Untransformed tissue culture cell lines have dense networks of plasma membrane-associated microfilaments and microtubules while their

16

ALTERED SURFACE ENZYMES INCREASED LECTIN AGGLUTINABILI

ALTERED

OF MOSlLITY

SURFACE CHARGE DENSITY --

(~)

~) SURFACE ION DENSITY

0 NEW ANTIGENS

-..~..':~.. ~ e . A T l ' n N ~

: ...-.

~J

SECRETION OR SHEDDING

LOST ANTIGENS

LOST OR MODIFIED GLYCOLIPIDS IMPAIRED INTERCELLULAR COMPONENTS COMMUNICATION AND DENSITY INHIBITION OF GROWTH ALTERED PERMEABILITY

ENDOCYTOSISLOST OR MODIFIED GLYCOPROTEINS ALTERED PHAGOCYTOSIS OR ALTERED TRANSPORT

MODIFIED ADHESION AND CONTACT INHIBITION OF MOVEMENT

Fig. 2. Cell surface alterations found after neoplastic transformation (modified from Robbins and Nicolson [30]).

transformed counterparts possess few membrane-associated cytoskeletal elements [270,274-276,283,284]. Glycerol impregnation could affect the membrane-associated cytoskeleton causing it to contract or aggregate. One of the most dramatic properties of normal and untransformed cells which is absent or modified after transformation, is contact inhibition of cell movement (reviewed by Abercrombie [285] and Harris [286]). This process was first described by Abercrombie and Heaysman [287,288] as the tendency of normal cells to inhibit the locomotion and overlapping (more properly underlapping) of adjacent normal cells when contacted. Closer analyses revealed that the characteristic blebing and ruffling movements of the leading edge of the cell were paralyzed at cell contact, but cell surface movement in other non-contacting areas continued [289]. Mouse sarcoma lines did not show contact inhibition of movement (or growth) and formed multilayers in culture [290]. The unrestricted movement of sarcoma cells compared to normal fibroblasts seemed analogous to the invasiveness of the tumor cells in vivo. Harris [286] has reviewed the various theories which have been proposed to explain contact inhibition of cell movement: differential adhesion, junctional formation, electrical coupling, diffusing low molecular weight inhibitors and peristaltic surface

17 motion. Unfortunately, dissenting evidence for all of these theories has been presented [291-297]. Contact inhibition of cell movement was once thought to be directly related to growth regulation ("contact inhibition of growth"); however, it is now generally accepted that these phenomena are separable [12,21,251,252,298-300], and the latter term has been replaced with "density-dependent inhibition of growth". Growth regulation of animal cells in culture involves complex interactions with many factors present in serum [8,9,21,22,251,299-301]. As mentioned previously serum is a complex mixture of proteins, vitamins, hormones, minerals and other factors that are usually essential for cell viability and growth. The density to which cells grow in culture under depletion conditions is dependent on the initial concentration of serum in the medium [8,21,22,302,303]. When the medium is depleted of essential components required for growth at a particular cell density, the cells stop growing and become "density inhibited" for growth. Addition of fresh complete medium or serum to the density inhibited culture results in a new round of cell division. The growth of normal cells is probably regulated by hormone or hormonelike growth factors present in serum [301], and one of the dramatic changes occurring after transformation is a diminished requirement of serum for survival or growth [8,21,251,252,301,303], Holley [251] has proposed a general theory for neoplastic growth in which modification of the plasma membrane resulting from genetic alterations by virus, radiation or carcinogens is thought to be responsible for the important phenotypic properties of tumor cells such as increased rates of transport. Another surface property of tumor cells which is relevant to their location and distribution in vivo is cell adhesiveness. Little is known concerning the actual cell surface structures involved in cell adhesion, but evidence suggests that they probably contain carbohydrate. Oppenheimer [304,305] has documented the fact that cell aggregation in vitro requires the cellular utilization of saccharide precursors which are synthesized into cell surface complex oligosaccharides. In other experiments Chipowsky et al. [306] found that transformed mouse fibroblasts adhered in vitro to D-galactose but not D-glucose or N-acetyl-D-glucosamine-derivatized Sephadex beads. Interestingly, Chipowsky and coworkers [306] determined that the binding of SV3T3 cells to the galactose beads promoted cell-to-cell adhesion (perhaps due to trans-membrane cooperative processes?) to the bead-bound cells leading to the formation of large aggregates of beads and cells. Untransformed 3T3 cells bound less efficiently to the galactose-beads. The role of adhesion in determining the metastatic spread of malignant cells was studied by Coman [307], who proposed on the basis of his experiments on the forces required to detach or separate cells, that malignant cells are less self (homotypic) adhesive than their normal counterparts. It seems reasonable that malignant cancer cells should have reduced homotypic adhesive properties to aid in release of cells from the primary tumor. Dorsey and Roth [308] attempted to test this hypothesis using untransformed and transformed mouse fibroblasts and monitored adhesive properties using the aggregate-cell capture assay [309] which measured the rate of single cell attachment (as opposed to Coman's [309] measurements on cell detachment) to cell

18 aggregates in suspension. This assay [309], along with a monolayer attachment assay developed by Walther et al. [310], showed the malignant SV3T3 cells to have higher homotypic and heterotypic adhesive rates compared to untransformed 3T3 cells. However, a spontaneously transformed line, 3T12, has even lower rates of adhesion in this assay than 3T3 cells, while a nonmalignant revertant line of SV3T3 resembled 3T3 cells. Using another assay, the quantitative loss of single cells from suspension to form cell aggregates (developed by Curtis and Greaves [311 ]), Dorsey and Roth [308] could not find differences in homotypic adhesive properties of these three cell lines. These authors concluded that adhesive properties do not correlate with transformation, but their experiments could be criticized on the grounds that trypsin was used to harvest cells, a questionable procedure due to cell surface proteolytic modification [312]. Cell adhesive properties are important in tumor cell implantation and metastasis [2,33]. Increased net attachment of blood-borne tumor cells to each other or to host blood cells could promote interaction of circulating tumor emboli to endothelial cells leading to enhanced tumor cell arrest [13,313-315]. Using mouse B 16 melanoma variants selected in vivo for enhanced metastatic properties and ability to specifically spread to the lungs [206,206a], Nicolson and co-workers [205,13,315] noticed that the highly metastatic cells adhered more readily to lung cells in vitro or to cultured mouse endothelial cell lines using the monolayer attachment assay [14,316]. The different rates of adhesion seen with the high and low metastatic cell variants suggested that adhesive properties may be more important in determining malignancy (metastatic properties), rather than normal versus neoplastic [316a]. The appearance of "new" antigens on the surfaces of tumor cells and modifications in cell interactions with lectins will be discussed in the next section. Ill. DYNAMICS OF SURFACE RECEPTORS ON NORMAL AND TRANSFORMED CELLS The involvement of trans-membrane processes in controlling cell surface events was established in ref. 1 and the preceding sections. Here the dynamics of surface receptors on normal cells and their transformed counterparts will be compared and contrasted. In most, but not all, of these studies established tissue culture cell lines (untransformed cells) were used for comparison before and after viral-, chemical- or radiation-mediated transformation, although some characteristics of these established cell lines are not "normal". For example, established cell lines usually do not become senescent in culture as do primary lines, and under certain conditions such as continued overgrowth, these untransformed lines can be spontaneously transformed to tumorigenic lines. Nonetheless, they still remain among the best model systems for studying the basic cellular changes associated with transformation to the neoplastic state. Many of the dynamic changes in cell surface receptors have been found both in tissue cultures and in vivo grown cells. It should be mentioned that none of these observations are truly universal; that is, they are not found in every transformed or tumor cell system which has been examined, and exceptions abound in some cases.

19

IliA. Lectin receptors (1) Differences in cell agglutinability. A well known property of most tumor or transformed cells is that they generally agglutinate at a much lower concentration of lectins compared to their untransformed counterparts (see reviews by Lis and Sharon [20]; Nicolson [18,317]; Rapin and Burger [19]; Burger [318]) (Table I). This was first noted by Aub and his collaborators [319,320] for a lipase preparation which contained wheat germ agglutinin. Burger and Goldberg [321] purified wheat germ agglutinin and observed that a variety of tumor cell lines were highly agglutinable, but untransformed cell lines were not agglutinable or showed lower agglutinability [322-324]. The degree of agglutination was subsequently correlated with loss of growth regulation in vitro [324] and with expression of genetic material specifying transformation [325-329]. Cell infection with non-transforming mutants of transforming viruses [325], or abortive transformation with certain oncogenic viruses [328], did not result in the enhanced lectin agglutinability. Salzberg and Green [330] have recently separated certain transformation events from helper virus infection and agglutinability in murine sarcoma virus (MSV)-transformed NIH/3T3 cells. These MSV-infected non-producer 3T3 lines were as unagglutinable as parental control cells unless superinfected with murine leukemia virus (MLV). MLV infection was required to activate expression of sarcoma genetic material. Changes in lectinmediated agglutinability have also been seen with normal cells obtained near malignant and "pre-malignant" tissues. Chaudhuri et al. [331] examined the agglutination of fibroblasts underlying human uterine cervical dysplasia, carcinoma in situ and invasive carcinoma, and recorded maximal concanavalin A agglutinability of normal fibroblasts that were obtained near invasive carcinoma. Fibroblasts in close proximity to cervical dysplasia gave minimal agglutinability, and those underlying carcinoma in situ were intermediate. Concanavalin A failed to agglutinate normal cervical fibroblasts. These exciting results indicate that malignant cells can also influence the surface properties of normal surrounding ceils. There are some interesting exclusions from the generalization that transformed cell lines agglutinate with lectins more readily than their untransformed counterparts. Sivak and Wolman [332] found that many "normal" adult rat and monkey cells were highly agglutinable. Gantt et al. [333,334], Glimelius et al. [335,336] and Van Nest and Grimes [337] noted numerous exceptions where untransformed cells manifested lectin agglutination properties similar to tumor cells or highly tumorigenic transformed cells, but one of these groups [336] still noticed that "a tendency exists for the tumor cells as a group to agglutinate more readily than normal cells". In certain cases where agglutination properties are not markedly divergent, enzymatic removal of cell surface hyaluronic acid was necessary to obtain distinctions in lectin agglutinability between untransformed and transformed cells [338]. Cell variants have been successfully used to study changes in lectin agglutinability associated with transformation. Selection of transformed cells which grow to low densities in cultures ("fiat revertants") has proven that at least some of the phenotypic properties of neoplastic cells can be abrogated. Rabinowitz and Sachs

20 TABLE I AGGLUTINATION OF SOME UNTRANSFORMED VERSUS TRANSFORMED CELLS BY LECTINS Species

Mouse

Lectin

Concanavalin A

Cells

3T3/SV3T3

3T3/SVT2 H6ts-SV3T3(38°)/ H6ts-SV3T3(32°) 3T3/3T12 3T3,Py3T3 3T3/MSV3T3 3T3/ST3T3 MEF/MSVMEF Wheat germ Aggtutinin

3T3/SV3T3 3T3/Py3T3 3T3/3T12 3T3/SV101 CHI/RSCHI MEC/MECT lymphocytes/HED lymphocytes/L # 2

322, 337 358,369, 370, 400 337 337 764 322, 323, 329 763, 765, 766 766 322 322 765 333 319 319

3T3/SV3T3 3T3/PySV3T3

317, 365, 763, 767 767

Soy bean agglutinin

3T3/SV3T3 3T3/Py3T3

766, 768 766, 768

Cortcanavalin A

hepatocytes/hepatoma RF/MSVRF RF/MLVRF RLB/RLT LWF/RSVLWF RLB/RLT RLB/hepatoma RLB/RLT RLB/hepatoma REC/SVREC

758, 769 764 764 758 770 758 758 758 758 768

LWF/RSVLWF

770

HEC/PyHEC HEC/SVHEC HEC/EMNA-HEC HEC/RSVHEC BHK/PyBHK

328, 360 360, 766 771 350 357, 358, 359

Wheat germ agglutinin Ricinus communis

agglutinin Soy bean agglutinin Pisum sativum

agglutinin Hamster

concanavalin A

No difference in agglutinability

317, 328, 329, 332, 344, 350, 355, 357, 358, 360, 369, 370, 400, 422, 609, 763 337 369,371

agglutinin

Ricinus communis

Rat

References Transformed cells more agglutinable

21

Species

Lectin

Wheat germ agglutinin Ricinus communis

agglutinin Soy bean agglutinin Chicken

Concanavalin A Wheat germ agglutinin

Human

Concanavalin A

Wheat germ agglutinin Ricinus communis

aggluti nin

Cells

BHK/BHKR BHK/BHKT ts3-PyBHK(39°)/ ts3-PyBHK(32 °) BHK/PyBHK NIL-Z/NIL-2T BHK/BHKR BHK/BHKT BHK/PyBHK ts3-PyBHK (39°)/ ts3-PyBHK(31 °) HEC/SVHEC HEC/RSVHEC HEC/DMNA-HEC

References Transformed cells more agglutinable

No difference in agglutinability 334 334

357, 368, 772 323 773 334 334 368, 767 368 768 768 768

CEF/RSVCEF CEF/RAVCEF CEF/RSVCEF CEF/RAVCEF

329, 338, 774

CEF/B77CEF CEF/RAV5OCEF

764

lymphocytes/CLL lymphocytes/Burkitt's lymphocytes/ lymphoma lymphocytes/ myeloma lymphocytes/ lymphoblastoid glia/gliaoma fibroblast/sarcoma leukocytes/leukemic

320, 336, 771 336

329, 338, 774

764 764 764 764 774

336 336 336, 775 335 335 319

lymphocytes/lymphoma lymphocytes/CLL lymphocytes/ lymphoblastoid glia/gliaoma

336 336 336 335

[339,340] grew polyoma-transformed hamster cells on aldehyde-fixed monolayers of normal cells and were able to select phenotypic revertants. The selected cells which had reverted to near normal growth saturation densities, cloning efficiencies and tumorigenicities possessed concanavalin A agglutination properties analogous to untransformed cells, although they continued to express a virus-specific nuclear (T) antigen [341]. Several laboratories have used selection by 5-fluoro-2'-deoxyuridine to obtain revertant lines which show diminished agglutination by lectins [322,342,343], and lectins themselves have proven useful as selection agents [344-347].

22 High saturation densities during in vitro cell growth are generally indicative of elevated tumorigenicity [3,348], and for many cells their lectin agglutination properties directly follow their ability to grow to high densities in vitro and form tumors in vivo [322,324,334,349,350]. When Inbar et al. [350] studied the relationship of concanavalin A agglutinability to tumorigenicity using chemically and virally transformed hamster fibroblasts, they found that cells plated in vitro were less agglutinable soon after subculturing but regained lectin agglutinability within four days. Harvesting the transformed cells at day 2 and day 4, they analyzed for the ability of these cells to form tumors in vivo and determined that the highly concanavalin A-agglutinable cells (4 days subcultured) were more tumorigenic. However, agglutination by soy bean and wheat germ agglutinins failed to correlate with tumorigenicity. Van Nest and Grimes [337] observed that the tumorigenicity of several transformed 3T3 lines generally correlated with concanavalin A agglutinability, but subsequent tumor regression in some of their transformed lines in vivo did not. Similarly, De Micco and Berebbi [351] found a close relationship between concanavalin A agglutinability and tumorigenicity using Chinese hamster embryo lines. Interestingly, after fusion of high and low tumorigenic lines to form somatic cell hybrids, intermediate tumorigenicities and lectin agglutinabilities were observed. In contrast to these results, Hozumi et al, [352] and Smets and Broekhuysen-Davies [353] observed an opposite correlation of tumorigenic and agglutination properties, while others could not find differences in lectin agglutinability among lines exhibiting differences in tumorigenicity [334,336,354]. Although most transformed cells are more agglutinable than their untransformed counterparts, they generally possess similar numbers of lectin surface receptors. The evidence seems quite overwhelming in this respect [344,350,355-368, and others], although in one laboratory contrary results have been obtained [369-371 ]. These latter authors [369,370] claim several-fold increases in the number of concanavalin A receptors on transformed or protease-treated cells, and they proposed [370] that other investigators failed to see these differences because they used procedures which led to high levels of lectin-induced endocytosis [372-374]. However, many of the studies which Noonan and Burger [370] criticized, in fact, used conditions of low temperature (0-4 °C) during quantitative labeling to drastically reduce or prevent endocytosis, and yet they still found little difference in lectin binding between untransformed and transformed cells [350,355,361,364,365,367,368]. Phillips et al. [363] isolated plasma membranes from 3T3 and SV3T3 cells previously labeled with 12SI-concanavalin A at 0-4 °C and demonstrated that the lectin bound to the same extent to untransformed or transformed membranes; it remained associated with the plasma membrane fraction after labeling and was not found in other membrane fractions, nor was it released to an appreciable extent during the isolation procedures. In one tissue culture system utilizing untransformed BHK, wild-type PyBHK and temperature-sensitive polyoma-transformed BHK cells (ts3-PyBHK), it was determined that the PyBHK and ts3-PyBHK (grown under permissive conditions) bound less x251-RCAI than did untransformed BHK cells during ten minute incubations

23 at 0-4 °C with saturating concentrations of lectin, although the transformed or ts3 cells displaying the transformed phenotype were more agglutinable at 22 °C [368]. In this last study endocytosis during quantitative labeling at 0-4 °C was not appreciable, and its extent was monitored ultrastructurally with ferritin-lectin conjugates [368]. Thus, there is no obvious relationship between the number of lectin receptors on a given cell and its agglutination characteristics. (2) Distribution and mobility of lectin receptors. Fluorescent microscopy and electron microscopy have been used to localize lectin receptors and determine their distribution and relative mobility on untransformed and transformed cell surfaces. Employing ferritin-concanavalin A Nicolson [375] reported that the distribution of concanavalin A receptors on mounted plasma membranes isolated from mouse SV3T3 cells were in a more clustered distribution at 20 °C compared to the receptors on 3T3 plasma membranes. Martinez-Palomo et al. [376] and Bretton et al. [377] used concanavalin A-peroxidase techniques to study the distribution of concanavalin A receptors on untransformed hamster cell lines and the same lines transformed with polyoma or SV40 viruses. They discovered that the electron-dense product of concanavalin A-peroxidase was generally more patchy on transformed than on untransformed ceils labeled at room temperature. Similar results have been published for many other untransformed/transformed cell pairs using fluorescent [317,378-385] or electron microscopic [210, 317,366-368,378,385-392] techniques on mouse [210, 317,355,375,378-383,387,393-395], hamster [376,377,379,384,388,391-393,396], rat [389,390,397] and chicken [366] cells. However, it is now clear that the discontinuous localizations of lectin-binding sites seen on most transformed cells is due to lectininduced redistribution of an inherently random display of receptors to clusters [210, 317,355,368,378,381,385,388,391-393,398,399,400-402]. Clustered surface distributions of lectin receptors presumably arise by lectin-receptor diffusion in the membrane plane which leads to crosslinking of additional lectin-binding sites by the polyvalent lectin molecules [63,210,317,381,383,402,403]. When cells are prefixed with formaldehyde or glutaraldehyde before labeling with lectin probes, the distributions of cell surface lectin receptors appear to be randomly dispersed [210,381,383,388,391,392, 400,401,404]. Therefore, it is more appropriate to interpret these results in terms of a greater relative mobility of lectin receptors on most transformed cells compared to their untransformed counterparts. Exceptions have been seen where the lectin receptors on some transformed cells did not appear to have greater relative mobilities than on comparable untransformed cells [388,392,400,401,405-408]. It does not seem likely that the discrepancies between these latter findings and the majority of the investigations on the relative differences in receptor mobility between untransformed and transformed cells will be easily resolved. Dissimilar results between one group and another could have been due to unlike cell systems (or even different clones of the same cell line), divergent labeling procedures and utilization of difficult, but usually reliable lectin receptor localization techniques [18]. Occasionally these techniques have come into question [409-411] concerning the large sizes of ultrastructural markers (for example, ferritin and hemocyanin are approximately 110/~ and 100 ×

24 350 A in size, respectively), possible preferences for certain receptor affinity classes [412,413] and differential release of receptors and/or labels during the manipulations required for each technique [396,404,407,414]. Alternatively, these exceptions point out the difficulties in trying to make "universal" observations on entirely diverse cellular systems. Untransformed cells can be rendered as agglutinable as transformed cells by brief proteolytic enzyme treatment [18,19,214,323]. This property of normal and untransformed cells seems to hold for a wide variety of different cell types and agglutinins [18-20,317,318] and led to a proposal that "cryptic" lectin receptors were unmasked by protease treatment [214,323]. However, several laboratories have discerned that protease treatment can increase lectin agglutinability of normal and untransformed cells without changing the total number of lectin-binding sites [210, 350,357,359,361,364,380,383,384,413]. Protease treatment may also free surface receptors from peripheral transmembrane restraints, because their relative mobility is enhanced in most cases [210,211,392,415,416]. Naturally, protease treatment causes many surface modifications as well as changes in surface zeta potential [417,418], charge distribution [419-421 ] and others [18,211 ]. Infection of normal cells by some non-oncogenic viruses results in enhanced lectin agglutination similar to transformation (reviewed in Poste [216] and Nicolson [18]). Zarling and Tevethia [422] observed that shortly after vaccinia virus infection ( ~ 2 h), concanavalin A agglutinability increased abruptly without the requirement of viral or host DNA synthesis. Similarly, infection of chick and hamster embryo cells by Newcastle disease, influenza, Flow plague, vesicular stomatitis, Simliki, Sindbis and SV5 viruses heightened their susceptibility to lectin agglutination [216, 423-425], The accession of lectin agglutination is not accompanied by an increase in the number of lectin-binding sites on the infected cells [216,426], but a correlation was found with increases in cell coat thickness measured by ellipsometry [423]. Poste [138] has stated that these alterations may be due to enzymatic modification of the cell surface caused by host lysosomal enzyme release, because they occur before the synthesis and appearance of virus-coded proteins. This proposal is consistent with the observation that virus infection enhances the relative mobility of lectin receptors commensurate with increased agglutinability and causes loss of some cell surface proteins [210,211,216,426,427]. In addition, viruses released by infected cells commonly contain glycoproteins which must be cleaved by proteases to achieve virulence [216,427]. In developing procedures to quantitate the mobility of lectin receptors on cell surfaces, Shinitzky et al. [428] adapted fluorescent polarization techniques to study the rotational mobility of surface-bound lectin molecules. Inbar et al. [429] found that the rotational mobility of fluorescent-concanavalin A molecules on transformed 3T3 cells measured by the fluorescent relaxation time was approximately one half that noted for the same probe on untransformed 3T3 cells. The very rapid rotational relaxation times found for fluorescent-concanavalin A on SV3T3 cells (73 ns) compared to its rotation in buffer alone (58 ns) suggests that the surface-bound concana-

25 valin A molecules are rotating very rapidly, indeed. This finding brings up an important point: In order for lectin-induced redistribution of receptors to occur, crosslinking must take place which should cause a decrease in rotational freedom. A possible explanation could be that Inbar et al. [429] used saturating concentrations of lectin which disfavored receptor crosslinking and allowed maximum-rotational freedom. Interestingly, high concentrations of Ricinus communis I agglutinin led to agglutination of both 3T3 and SV3T3 cells without significant clustering of surface receptors [317], and cells attached to fibers and treated with high concentrations of concanavalin A do not show observable clustering of surface receptors [62,430,431]. (3) Factors afJecting cell agglutination. An assortment of factors influence the agglutination properties of cells [18,19,317] (Fig. 3). Some of these factors enhance agglutinability while others are inhibitory. In this section several, but perhaps not all of these factors will be reviewed and an assessment of their respective roles in determining the agglutination properties of transformed cells discussed. Lectins and antibodies are polyvalent molecules, a necessary requirement for cell agglutination. Modification of these molecules by proteolytic enzyme treatment is known to reduce their capacity to cause cell agglutination. Noonan and Burger [432] used a trypsinized non-agglutinating concanavalin A preparation to inhibit the growth of transformed 3T3 cells, and a similar procedure was used by Steinberg and Gipner [433] to produce a non-agglutinating lectin with chymotrypsin. Unfortunately proteolytic digestion of lectins such as concanavalin A (in contrast to papain treatment of immunoglobulins [434]) yields a complex mixture of unmodified concanavalin A, totally inactive digestion products [403] and molecules of variable valencies [435]. Also, these procedures seem to work for some lectins but not for others [436]. A more promising procedure was developed by Gunther et al. [437] where tetravalent concanavalin A was succinylated to produce a divalent molecule which is only 1/500 as effective in agglutinating sheep erythrocytes and 1/10 as effective with mouse splenic lymphocytes. Conversely, cell agglutination properties can be augmented by coupling agglutinin molecules together. Lotan et al. [438] crosslinked soy bean agglutinin to make lectin dimers, trimers, etc. The lectin oligomers were orders of magnitude more effective as agglutinators. Some lectins are sensitive to low temperatures which affects their valencies. Concanavalin A will undergo a tetramer to dimer transition in the cold [402,439] which partially accounts for its diminished ability to agglutinate cells at low temperature [350,369,440]. Local concentrations of agglutinin molecules in the proper display between adjacent cells and receptor mobility seem to be very important for cell agglutination. The requirement for a critical number of agglutinin molecules to initiate the cell aggregation has been discussed by Hoyer and Trabold [441], but in many cases agglutination occurs via a minor specialized class of receptors [442-444]. The vertical location of the receptor is probably an important determinant, because receptors close to the lipid bilayer surface could be masked by long glycoprotein chains with their attached oligosaccharides radiating out into the surrounding media. Interestingly, transformed cells have been reported to possess thicker surface "coats"

oo

Z

O

==

(3"

m

0Q

o

O

c. Mobility of Sites

b. Membrane Location

o. Number of Sites

Z. NATURE OF THE LECTIN RECEPTORS

d. Size

c. Net Charge

b. Number of Binding Sites

o. Saccharide Binding Constant

~

~ -+-C:~ -- ÷

~ e < : ) p.-,,
n-t>n-n) [451]. Polarization fluorescence has been used to measure the rotational mobility of fluorescent-concanavalin A on untransformed and transformed cells, and the relaxation times for concanavalin A bound to its receptor on SV3T3 was approximately half that for concanavalin A bound to 3T3 cells indicating greater receptor rotational freedom on transformed cells [428,429]. Rutishauser and Sachs [430,431,453] carefully examined whether receptor mobility was an important determinant in cell agglutination by immobilizing one group of cells to nylon fibers [454] and then adding another assemblage of free cells to combine with the immobilized cells. Agglutination was scored by lectin-induced attachment of free cells to immobilized cells. By this elegant method the two groups of cells could be independently manipulated. These authors found that prior aldehyde fixation of both free and fiber-bound cells blocked lectin-mediated agglutination, while fixation of only one cell group allowed greater agglutinability. Using normal hamster embryo fibroblasts they observed that treatment of either one or both classes of cells with trypsin heightened lectin agglutination to levels similar to transformed cells [431]. Rutishauser and Sachs [431 ] postulated that agglutination of two cells requires receptor mobility on at least one of the cell surfaces and an alignment of complementary receptors. They also ascertained that

28 large scale lectin-induced lateral rearrangements of receptors inhibits agglutination, consistent with previous studies [378,384,455]. This last point is not at all unreasonable, since processes such as large scale patching and capping actually remove receptors from large areas of the cell surface, reducing the chance of a cell collision occurring in an area of displayed receptors. Previous electron microscopic studies with ferritin-concanavalin A in this laboratory have implicated lateral rearrangements as necessary, leading to the formation of multiple crossbridges or small clusters between adjacent cells [211,317]. In these experiments trypsinized-3T3 [211] or untreated SV3T3 [317] cells were agglutinated with low concns, of ferritinconcanavalin A. Ferritin-lectin binding induced a topographic rearrangement of receptors leading to higher local densities of surface bound lectin in distinct regions which were observed to be directly involved in SV3T3 and trypsinized-3T3 cell-to-cell attachment. These rearrangements were observed at low ferritin-lectin conchs, where binding to untransformed 3T3 cells occurred, but did not induce receptor rearrangement, nor cell agglutination. Using another lectin, Ricinus communis agglutinin [368], at higq conchs, where most cell surface receptors are occupied on 3T3, trypsinized-3T3 and SV3T3, all cells agglutinated and receptor redistribution did not occur to any significant extent. Thus receptor redistribution per se is not essential for agglutination, but it may enhance lectin-induced cell aggregation at low agglutinin concentrations where only a few binding sites are occupied, and these have relatively good lateral mobility. To determine more precisely the requirements of receptor mobility for cell-fiber attachment and its relationship to agglutination Rutishauser and Sachs [453] derivatized nylon fibers with various densities of lectins using serum albumin as non-agglutinating spacer molecules on the fiber surface, and then analyzed cell binding to the derivatized fibers. The number of cells specifically attached to the fibers could be directly modulated by changing the local densities of lectin molecules (ratio of lectin: albumin) on the fibers, the densities of lectin receptors on the cells (by enzyme treatment) or by modifying the mobility of lectin receptors (by chemical fixation) on the cells. They found that increased local densities of lectin molecules and enhanced lateral mobilities and numbers of lectin receptors favored agglutination. Fixation of cells by glutaraldehyde or formaldehyde drastically reduces cell agglutination commensurate with a loss in receptor mobility, but fixation does not modify the number of surface lectin-binding sites [210,317,370,379,381,383,384,400]. Rutishauser and Sachs [431] observed that cell attachment to lectin-derivatized fibers was also inhibited by metabolic inhibitors or low temperatures. However, the binding of cells to soy bean agglutinin-derivatized fibers was unaffected by low temperature, as was cell-cell agglutination in suspension [350]. Thus, rapid lateral receptor mobility promoted agglutination by lectins for most cells, particularly transformed fibroblastic cells. In mouse fibroblasts the receptor redistribution or alignment observed between adjacent agglutinating cells [317] may not be required on both cells [430,431], and on some cells the proper display of agglutination sites may exist de novo such that inhibition of receptor mobility is without effect on cell agglutination properties.

29 Cell surface structures play a role in cell agglutination. Most cells have specialized surface protrusions such as pseudopodia and microvilli, and these structures could aid in cell associations by presenting specialized low radius-of-curvature surfaces [456458]. Weiss and Subjeck [459] examined the density of receptors for positively charged particles on mouse Ehrlich ascites tumor cells and found higher densities on microvilli than surrounding membrane regions. Similarly Grinnell et al. [460] labeled baby hamster kidney cells with polycationic ferritin and noticed higher densities of anionic sites on the microvilli. These membrane components may be concentrated into microfilament-ladened villi by trans-membrane restraints (as envisaged in Fig. 13 of ref. 1). Microvilli could serve to "trap" other cells which make close approach, so that direct cell surface contact is avoided. Most cells have a large net negative surface charge which aids in cell repulsion [419]; lowering this repulsion (zeta potential) should allow greater cell association [418]. Willingham and Pastan [461,462] have proposed that agglutination of transformed cells is due entirely to the abundance on their surfaces of microvilli which are, in turn, controlled by the cellular concentration of cyclic AMP. It was known for some time that addition of dibutyryl cyclic AMP to Chinese hamster embryo cells reduced their agglutinability (slightly) with wheat germ agglutinin and converted them to a more "fibroblastic" form [463,464,465]. Similar experiments have been reported by Sheppard [466] using transformed fibroblasts. The cyclic nucleotide-induced morphologic conversion was blocked with colcemid and vinblastine sulfate suggesting microtubule involvement [464,467,468]. Willingham and Pastan [462] used dark field microscopy to observe what they claim are microvilli trapping cells in a lectin-dependent process which is also strictly regulated by cyclic AMP. Unfortunately, not all transformed cells are blessed with the large number of microvilli seen on mouse L cells. In fact, other studies show exactly the opposite: untransformed cells have numerous microvilli but their more agglutinable transformed counterparts do not [469,470,470a]. Loor [471] has observed that metabolic inhibitors which block agglutination actually stimulate the formation of microvilli [472]. In addition, examination of contact points between agglutinated transformed fibroblasts does not point to a universal microvilli-microvilli theory for agglutination [21 !,317,400]; however, these structures may be important in certain systems. Cell rigidity may also be a determining factor in cell agglutination. The deformability of a cell could affect the amount of surface area on adjacent cells which can be brought into close contact [18,19,400]. Homotypic cell adhesion effects can also modify lectin-mediated agglutination. Cells such as fibroblasts tend to associate homotypically or adhere within several minutes at 37 °C [14,205,308,310], and O'Neill and Burnett [473] have noted that this specific adhesion process in some cell systems is initially inhibited by lectins at low concentrations. Modification of cell surface oligosaccharides by growth in 2-deoxy-D-glucose also alters agglutinability [474]. One of the more interesting ways in which cell agglutination may be controlled is through outer surface peripheral and trans-membrane peripheral and membraneassociated restraints. Hynes [121] has proposed that outer surface peripheral com-

30 portents such as the LETS glycoprotein may control the mobility of surface glycoproteins and therefore regulate cell agglutination properties. The possible mechanisms and interactions involved in this type of restraint system have been discussed previously [1,18]. Inner surface peripheral membrane control over the mobility and distribution of erythrocyte glycoproteins by a spectrin-actin network has been proposed [475-478], and recent evidence suggests that this system influences the agglutination properties of the erythrocyte ghost [479]. Evidence for control over cell agglutination by membrane-associated cytoskeletal systems has come mainly from drug studies. Colchicine, colcemid and vinblastine sulfate, which disrupt microtubules [479-481], reduce cell agglutination of polymorphonuclear leukocytes [482] and transformed fibroblasts [455]*. These pioneering experiments first suggested trans-membrane linkage of surface receptors to cytoskeletal systems. Kaneko et al. [484] later discovered that metabolic inhibitors and microfilament-disrupting cytochalasin B inhibited lectin agglutination of lymphoma cells, but did not affect lectin binding to the cell surface. In an analogous study Loor [471] determined that a metabolically active cell was important to the agglutination process, as sodium azide and cytochalasin B reduced lymphocyte lectin-mediated agglutination. Somewhat different results were found with untransformed and transformed fibroblasts by Vlodavsky et al. [485]. Low cellular concentrations of ATP characteristic of transformed cells correlated with concanavalin A agglutinability, but cells grown with a high cellular concentration of ATP were relatively unagglutinable. The involvement of microfilament and microtubule systems in controlling the distribution and mobility of surface receptors on lymphoid cells was discussed in ref. 1 and briefly presented in Section lB. In light of so many determinants and effectors of cell agglutination, it is important to ask what are the factors that direct the agglutination properties of transformed cells? The answer to this question has eluded many an investigator seeking to find the answer. We must face the fact that there may not be one simple answer (such as microvilli [462]) which can explain the differential agglutinability of transformed ceils in every system, just as there are no two tumor systems that have exactly the same cell surface properties. One surface characteristic which has been found generally to be a significant factor in cell agglutination is enhancement of receptor mobility. Assuming that this difference is due to a general and important modification in cell surface control after transformation, the altered mobility of components could be due to alterations in plasma membrane fluidity, a change in the structure * In a more recent study [483] the effects of colchicine were more extensively examined. Brief times (5-10 min) after colchicine addition at 22 °C resulted in enhanced cell agglutination while at later times (30-60 min) agglutination was suppressed. At 37 °C only suppression of agglutination occurred. The explanation for this effect is that colchicine-induced uncoupling of surface receptors allows greater receptor lateral mobility and initially enhances lectin-mediated agglutinability, but in time more of the receptors are freed from anchoring components, and lectin binding and receptor redistribution induces capping [378] probably due to microfilament contraction. This latter event reduces agglutinability by sequestration of receptors into caps making random cell collisions which lead to agglutination less probable as proposed by Ukena et al. [378].

31 of the receptor itself or a variation in one or more of the cell surface restraint systems. An analysis of the available evidence suggests the latter as the most probable answer. The intrinsic fluidities of the plasma membranes of transformed cells has been measured by electron spin resonance and fluorescent-polarization techniques. Barnett et al. [280] probed the membranes of 3T3 and SV3T3 cells with the spin label sodium 6-(4',4'-dimethyloxazolidinyl-N-oxyl)heptadecanoate by incubating cells in situ with the probe for 30 min at 37 °C. Cells were washed and then removed from substrate by scraping and paramagnetic spectras were taken at 33 °C. Barnett et al. [280] found that transformed 3T3 cells had order parameters S ~ 0.52-0.55 while confluent 3T3 cells had S values near 0.6. From this minor change in order parameter they concluded that the membranes of transformed cells are slightly more fluid. In a later correction Barnett et al. [486] conceded that the probe used could partition into various membranes within the cells they examined, unfortunately, bringing their results into question. Gaffney [487] has directly challenged the data of Barnett et al. [280]. She carefully examined the fluidity of untransformed and transformed mouse chick cell lines using fatty acid spin-labels and found no significant differences in membrane fluidity in these cells [487,488]. Additionally, Yau et al. [489] probed exponentially growing normal and Rous sarcoma virus-transformed chick embryo fibroblasts with stearic acid nitroxide analogs and observed that the transformed cell membranes had slightly less fluidity! The lipid composition of Rous sarcomatransformed chick cells also does not support the thesis that fibroblast transformation results in a more fluid plasma membrane [490]. Yau et al. [489] analyzed the fatty acid compositions of normal and transformed chick fibroblasts and determined that there was a pronounced decrease in arachidonate (20 : 4) concomitant with an equivalent rise in oleate (18 : 1). These results, along with measurements of surfacebound particle movement* [491,492], argue against a significant increase in fluidity after transformation of fibroblastic cells. However, the opposite situation may prevail in lymphoma cells compared to normal lymphocytes. Although one can argue that comparisons of a heterogeneous mixture of normal lymphoid cells with a cultured lymphoma cell line have dubious validity, the findings of differing plasma membrane fluidity between lymphoma cells and lymphocytes are nonetheless interesting. Shinitzky and lnbar [493] studied microviscosity determined by fluorescence polarization of the probe 1,6-diphenyl-l,3,5-hexatriene, and found that lymphoma plasma membranes had distinctly lower microviscosities. By "loading" the membranes with lecithin-cholesterol (1 : 1) liposomes, the fluidity of the lymphoma membranes could be lowered to the normal level [101], and upon returning the

* Albrecht-Buhler [491,492] measured the movements of gold particles attached to the surfaces of 3T3 and SV3T3 fibroblasts and observed greater rates of mobility on the untransformed cell upper surfaces. The movement of these attached particles may be under trans-membrane control, so conclusions on cell membrane fluidity may be meaningless in this case.

32 cholesterol-"rich" lymphoma cells to animals, Inbar and Shinitzky [101] were able to inhibit the rate of killing~compared to cholesterol-"poor"~ l y m p h o m a ceils*. More direct evidence for the involvement of membrane fluidity in cell agglutination has been obtained by modifying plasma membrane lipid composition [496-499]. Horwitz et al. [500] used lipid-depleted serum supplemented with different fatty acids to modify 3T3 and SVI01 cell membranes. Transformed SVI01 cells normally show marked temperature-dependent concanavalin A agglutinability with a transition from a relatively low to a highly agglutinable state at 14-18 °C. With oleate or elaidate supplemented cells this transition temperature changes to 6-8 °C or 26-28 °C, respectively. Although 3T3 cells are much less susceptible to agglutination, they possess similar transition temperatures for concanavalin A agglutination after fatty acid incorporation. Wheat germ agglutinin-mediated cell agglutination is usually independent of temperature from 0-37 °C, but cells supplemented with elaidate are markedly temperature-dependent, with reduced agglutinability below 33-35 °C. Alteration of the fatty acid composition of mouse LM fibroblasts also modified the temperature dependency of concanavalin A agglutination. Rittenhouse et al. [440] lowered the normal transition temperature of agglutination (15-19 °C) to 7-11 °C by supplementing cells with a higher proportion of polyunsaturated fatty acids and raised the transition to 22-28 °C by incorporating a greater percentage of saturated fatty acids. Lectin receptors may be modified after transformation. Jansons and Burger [501] isolated and purified a lectin receptor for wheat germ agglutinin from mouse LI210 lymphoma cells. Antisera against the purified receptor reacted with L1210, P y B H K and Py3T3 cells, but did not react to an appreciable extent with normal mouse lymphocytes. Unfortunately, Jansons and her colleagues did not check the reactivity with untransformed 3T3 and B H K fibroblasts. It is possible that the antisera which Jansons and Burger [501] used reacts against an alloantigenic determinant such as Thy-1 (0), known to be present on fibroblasts and T-lymphomas, but present on only a subpopulation of splenic lymphocytes. As discussed in Section IIIA(2), untransformed and transformed cells appear to have similar numbers of wheat germ agglutinin receptors [357,364,368]. Dievard and Bourrillon [502] have isolated lectin

* lnbar and Shinitzky [102] have proposed that cholesterol is a bioregulator in lymphoma and leukemic cell proliferation. The lowered cholesterol content of the tumor cells reduces their microviscosity compared to the average of a normal lymphocyte population, and this somehow regulates cell proliferation. In support of this hypothesis. Inbar and Shinitzky [494] observed that a decrease in microviscosity accompanies normal lymphocyte mitogenic stimulation induced by concanavalin A. The determining factor, they claim, in leukemia is blood cholesterol level which is usually (but not always!) reported to be low in reticular endothelial proliferative disorders. According to their scheme (Fig. 2 of ref. 102) leukemia can be inhibited by cholesterol, an enormous boost to dietary claims for cancer cures! It seems more likely that the cholesterol-"loaded" cells were not viable when injected or expired rapidly in the hostile in vivo environment, perhaps, because of a more rigid (hence, more fragile) surface membrane. Alternatively, the more rigid high cholesterol plasma membranes may have facilitated cytotoxic reactivity against the lymphoma cells simdar to the observations of Humphries and McConnell [495].

33 receptor fragments from normal rat hepatic cells and hepatoma and judged them to be unique. Evidence exists indicating that the membrane-associated cytoskeletal systems are drastically altered by transformation. McNutt et al. [274,276] observed that untransformed 3T3 and flat revertants of SV3T3 cells develop an extensive microfilament-microtubule system at confluency, while transformed 3T3 cells failed to elaborate an intense subplasma membrane cytoskeletal system after cell contact in culture. Incidentally, the agglutination properties ofuntransformed 3T3 cells parallel membrane-associated cytoskeletal development, and the concentration of cyclic AMP also rises at confluence [244,503-505]. Sparse 3T3 cells were found to be more agglutinable with concanavalin A [317,355] and Ricinus communis agglutinin [317,365] compared to contacting- or confluent-3T3 cells. A decrease in plasma membrane-associated actin occurring after transformation has now been documented in mouse [270,274,276,283,506-508], chicken [275,284], rat [283] and human [508] cell lines. Cytoplasmic myosin also decreases after transformation [509]. Microfilaments contain appreciable amounts of actin [506, 507,510-513] and are commonly present in two cellular configurations: sheath or a-microfilaments (immediately adjacent to and running parallel with the plasma membrane and bundles penetrating into the cell cytoplasm) and lattice or network microfilament. The subplasma membrane sheath microfilaments have been observed to be present in areas where the cell surface is noticeably refractile and immobile, and have been proposed to offer the cell structural rigidity [514]. The mobility of surface (SF) antigens and concanavalin A receptors correlates with the suggestion of Wessells et al. [514] that microfilament sheaths immobilize the plasma membrane in certain regions. Cell surface SF antigens were found to be associated with fibrillar structures such as microfilament bundles, surface ridges and other appendages on untransformed chick fibroblasts [154]. Nicolson [355] found that concanavalin A-induced redistribution was not detectable on confluent 3T3 cells (labeled in situ) in regions immediately over membrane-associated microfilaments, but lectin-induced redistribution was observed in regions of the 3T3 cell surface not associated with cytoskeletal elements. When transformed cells were examined under identical conditions, only one class of receptor mobility (highly mobile) was detected [355]. The similarities between the distribution of concanavalin A receptors [355] and surface (SF) antigens [154] on untransformed fibroblasts and their connection with cytoskeletal systems is probably not fortuitous, since LETS or SF 210 possesses concanavalin A-binding sites [73]. These results strongly implicate the involvement of a cyclic nucleotide-regulated membrane-associated cytoskeletal system in modulating the mobility and topographic distribution of lectin, antigen and probably other receptors on untransformed fibroblasts [1,65,66]. Transformation results in partial disorganization of the cytoskeletal system and an uncoupling of surface receptors from cytoplasmic trans-membrane control. Elevated levels of ATP and cyclic AMP are required for and appear to stimulate cytoskeletal organization and correspondingly, suppress lectin-mediated cell agglutinability. The fluctuations in cellular cyclic nucleotides with the cell cycle

34 and with transformation (reviewed by Pastan and Johnson [246], Goldberg et al. [515] and Sheppard and Bannai [516]) and the pleiotropic control which these classes of molecules exert over a variety of cellular functions [517] suggest that trans-membrane control of the cell surface is only one of many physiological processes under strict control in normal cells. In conclusion, many physical and biochemical factors regulate and determine cell agglutination (Fig. 3), and the proper combination or balance of these factors will determine agglutinability in any cell system. As stated previously [18], agglutination will occur when the sum of the factors favoring agglutination outweighs opposing factors. IIIB. Antigens Tumor cells possess a variety of surface antigens which may have different structures, antigenic specificities, location, mobility and roles in tumor escape or host rejection. These antigens, particularly viral antigens, embryonic antigens, etc. have been strongly implicated in host surveillance against neoplasia [518-524]. (1) Tumor-associated an tigens. Tumor-associated antigens are antigen s which are found on tumor cells, but they may not be tumor-specific and could be present on normal cells during specific embryonic stages or during oncogenic virus infection that does not lead to successful transformation. Embryonic antigens or fetal antigens have been found on a number of tumor cells and may universally appear after transformation; this topic has been thorougly reviewed by Coggin and Anderson [524,525]. Fetal antigens seem in at least some cases to be phase-specific during embryonic development and are usually not found in appreciable amounts on normal adult cells, although they can be detected in small quantities in normal adult tissue [524,526,527]. An example of the latter is the carcinoembryonic antigen (CEA) which is high in serum of patients with gastrointestinal neoplasms and is expressed on the surfaces of colon tumor cells [528-531 ]. Although CEA was once thought to be absent in normal adults [532], it is now known to appear in small amounts in blood and on normal colonic tissue cells [533-535]. In human neoplastic disorders CEA is found in high levels in the serum (as soluble antigen [SA]) in the majority of carcinomas of the gastrointestinal tract [536,537]. Unfortunately for diagnosticians, CEA blood levels also rise during some non-neoplastic diseases [535]. The structure of CEA has been quite elusive, probably due to the number of different molecular components carrying CEA specificities and to heterogeneity of the antigen itself. Krupey et al. [538] characterized CEA as a single homogeneous glycoprotein, but subsequent studies demonstrated that CEA is not homogeneous, and its antigenic specificities can be resolved into a number of different components [537,539-541]. Another circulating human antigen once thought to be exclusively embryonic, a-fetoprotein (see review by Abelev [542]), has been found at low levels in most normal people [543,544]. Tumor-specific transplantation antigens (or more accurately they have been called tumor-associated transplantation antigens or TATAs)are antigens which induce transplantation rejection immunity (delayed-type hypersensitivity) in autoch-

35 thonous or syngeneic hosts (reviewed by Herberman [521], Baldwin [545] and Butel et al. [546]). In rodent systems tumors induced by a given tumor virus carry the same distinct TATAs, and within the same host strain different oncogenic viruses elicit different TATAs. Moreover, the immunological cross-reactivity occurring between dissimilar tumor cells transformed by the same virus is not duplicated by transformations of different cells with the same chemical carcinogen. Transformation by chemical carcinogens results in non-crossreactive transplantation immunity. However, in certain virus-transformed mouse mammary tumors, individually unique TATAs have been found in addition to the common virus-related antigens produced, for example, after MTV-transformation [547]. It is assumed that TATAs are present at the cell surface, and they seem to be distinct from other tumor-associated cell surface antigens (CSAs) which do not produce transplantation immunity [548]. Not all TATAS appear to be virus antigens, because some have been found on nonproducer cell lines which do not express viral components [549,550]. Virus antigens induced by oncogenic RNA tumor viruses have been found on the surfaces of tumor cells and also on normal cells lytically infected by these viruses [551]. Khera et al. [552] found that immunity to virus particles can occur independently from immunity to TATAs, again strongly suggesting that at least some of these antigens are distinct and different. The external antigens carried by tumor viruses are on proteins (probably all glycoproteins) which are biochemically and immunologically unique. Antigenic cross-reactivity exists within the envelope glycoproteins of a given virus strain, and these virus surface components seem to be of similar size and are cross-antigenic for virus strains of the same or related species. Several classes of virally associated envelope antigen (VEA) specificities exist in RNA tumor viruses: (a) interspecies-specific VEA, found in many viruses from different mammalian species; (b) group-specific VEA, found in all viruses within a species; (c) subgroup-specific VEA, found in some strains of viruses within a species and different species; (d) type-specific VEA, found only in a given virus within a species; and (e) subtype-specific VEA, found in mixed form on individual viruses of some populations and demonstrable by different hyperimmune sera [553,554] (Fig. 4). The relationships of these VEAs have been reviewed recently by Bauer [555] and Aoki and Sibal [553]. In addition to intact virions, VEAs appear on the surfaces of virusinfected (plus exogenously infected [556-558] and more recently uninfected [559-561 ]) cells as cell surface antigens (CSAs) and free from cells or viruses as circulating or soluble antigens (SAs) (Fig. 4). Additionally, certain chemically-transformed tumors express VEAs at their surface [557,562,563] implying the activation of endogenous viruses. The function of VEA expression on normal cells is unknown; in a current editorial by Kurth [564] it was even suggested that VEAs might have a cellular role as surface receptors in the induction and maintenance of established morphogenetic patterns. Normal antigens are present on tumor cells and occasionally are derepressed or repressed by transformation [565]. An example of this is the inverse display of H-2 antigens with the appearance of VEA [566] or thymus-leukemia (TL) [567]

36

(

(GROUP-SPECIFIC

ENVELOPE ~ SUBGROUP-SPECIFIC (YEA)

VIRUS-ASSOCIATED (VEA) ANTIGENS

] TYPE-SPECIFIC

(SUBTYPE-SPECIFIC

(gs-COMPONENTS (TYPE C VIRUS) k.INTRAVIRAL ~s-COMPONENTS (TYPE B VIRUS) /.REVERSE TRANSCRIPTASE

: .:,:

TUMOR-ASSOCIATED TRANSPLANTATION (TATA) VIRUS-ASSOCIATED ENVELOPE (VEA) EMBRYONIC OR FETAL OTHER SEROLOGICAL CLASSES

(VIRUS

SOLUBLE (SA) (CELL SURFACE ANTIGENS /`INTRACELLULAR

Fig. 4. Classification of antigens associated with RNA tumor virus transformation (modified from Aoki [776]). antigens. Although some of these antigens are associated with oncogenic virus infection (for example, mouse antigens: G~x [568], TL [569], PC-l [570], among others), these are considered to be "normal" alloantigens and appear on specific normal cells of different animal strains. The listing of these alloantigens as normal is somewhat curious. In mouse lymphoid cells the expression of the G~x alloantigen has been invariably correlated with the presence of the group-specific VEA (gp 69/71) from a murine leukemia virus. Obata et al. [561] discovered that anti-gp 69/71 was cytotoxic for G~x+-thymocytes and partially blocked the cytotoxic activity of anti-G=x. A protein was then isolated from thymocytes ( ~ 7 0 0 0 0 molecular weight) which had identical biochemical properties to the gp 69/71 VEA of murine leukemia virus [571]. (2) Dynamics of antigens. There have been several reports on the localization of antigens on cells, but in most cases the dynamics of these cell surface receptors on normal and neoplastic cells were not investigated. The most well studied CSAs are the normal alloantigens of mouse cells. Aoki et al. [572] used indirect immunoelectron microscopy to examine the localization of H-2, Thy-1 (0) and TL alloantigens on mouse reticular cells, lymphocytes, plasma cells, eosinophils, etc. Although Aoki et al. [572] found CSAs distributed in clustered and patchy formations, it is now accepted that alloantigens are present in an inherently random dispersed display on these and other cells [56,573-576]. After binding antibody the surface receptors undergo ligand-induced redistribution as described previously (ref. 1 and Sections IB and IliA (2)). Differences have been found in the relative mobilities of antigens on untransformed compared to transformed fibroblastic cells. Edidin and Weiss [575] examined antibody-induced capping of H-2 and HL-A antigens on cultured mouse and human fibroblast and endothelial cells. Labeling C1 ld (a transformed mouse L cell deriv-

37 ative) with fluorescent-anti-H-2 resulted in rapid cap formation in perinuclear areas without the requirement for an added sandwich of antiimmunoglobulin. H-2 capping was inhibited by dinitrophenol, colcemid, sodium azide and temperatures below 15 °C. Human untransformed epithelial VA-2 cells labeled with anti-HL-A did not cap, but instead formed multiple clusters. Since the CI ld fibroblasts which Edidin and Weiss [575] used were transformed, these results could indicate that histocompatibility antigens are more mobile on transformed fibroblast cell surfaces. Comparing untransformed and transformed mouse fibroblasts for anti-H-2-induced capping, Edidin and Weiss [451] noted that the antibody alone failed to cause capping on untransformed cells, but rapidly capped on transformed cell lines. In a more elaborate study using the degree of antigen intermixing after the formation of fusion heterokaryons from two unlike cells as a criterion for antigen mobility [452,577], Edidin and Weiss [451] also demonstrated that surface antigens on transformedtransformed (t-t) hybrids intermixed more rapidly than on normal-normal (n-n) heterokaryons, with normal-transformed cells showing intermediate rates of intermixing ( t - t > n - t > n - n ) . These results [451] are consistent with most reports on the enhanced mobility of lectin receptors on fibroblastic ceils after transformation. On lymphoid cells a more confusing situation exists as to the relative mobility of antigens and other surface receptors. Many authors have used capping as a criterion for antigen mobility, and in most cases experiments have been performed with indirect antibody sandwich techniques. In addition to antigen mobility and redistribution (most likely into clusters and patches), capping involves a transmembrane cytoskeletal system for gathering the antigen-antibody aggregates into caps and maintaining the caps once they are formed (ref. 1 and Section lB.). Thus, capping experiments are more a reflection of transmembrane cytoskeletal control than receptor mobility, although the latter is undoubtedly required before the former can take place. Stackpole et al. [578] studied both patch and cap formation of TL alloantigens on normal mouse TL÷-thymocytes and on T-derived RADA1 leukemia cells using fluorescent-antibody methods. Patch and cap formation took place on both cell types, but it required at least twice as much time to observe patches and later caps on the normal cells. Somewhat different results have been obtained by Yefenof and Klein [579] using indirect labeling techniques. Studying the redistribution of H-2 antigens into caps on normal mouse thymocytes and various T-derived ascites tumors, they found that the normal thymocytes capped better and faster than three comparable tumor lines. However, the tumor cells which did cap were observably different from normal thymocytes in that their caps consisted of conglomerates of aggregated patches and clusters appearing over approximately one half of the cell surface. Since these workers did not follow cluster and patch formation and used indirect antibody sandwich labeling techniques, it could be argued that their experiments bear little relationship to antigen mobility, but reflect a modification (defect?) in the trans-membrane capping apparatus of their ascites tumor cells. However, these experiments are similar to the capping phenomenon produced by lectins on mouse lymphocytes and malignant lymphoma cell lines [379,380,580]. Lengerovfi

38 et al. [576] examined the events leading to patch and cap formation on leukemic EL4 cells and noted rapid capping and eventually internalization of antibody-receptor complexes. Other surface components such as tumor-associated antigens and virus receptors cap on normal and tumor cells, with some exceptions. Yefenof and Klein [579] examined cap formation by antibodies against MLV-induced CSA (MCSA) in several lymphoma lines and noticed that anti-MCSA incubation resulted in only ~15~o capped cells, but other tumor-associated antigens capped well. Increasing MCSA antigen surface concentration correlated with decreased efficiency of anti-H-2induced capping on YAC cells, and it was suggested that the poorly-capped MCSA viral antigens may interfere with the free movement of H-2 receptors. Tumor-associated antigens on many types of neoplastic cells are capable of redistribution. Leonard [581 ] followed the distribution of tumor-associated hepatoma CSAs with indirect fluorescent-antibody techniques and reported an initially dispersed display which slowly became irregular (clusters and patches) with time (2-4 h) at 37 °C. By 4-7 h less fluorescence was associated with the cells and shedding had occurred. Anti-tumor antibody alone was insufficient to cause redistribution and subsequent shedding. VEAs have been localized on a number of tumor cell surfaces at budding viruses or on other membrane regions [549,582-590]. Phillips and Perdue [583] investigated the distribution and mobility of VEAs using hybrid antibodyhemocyanin replica techniques on Schmidt-Ruppin sarcoma and leukosis virusinfected normal chick embryo fibroblasts during the course of productive infection. Within four days after infection several cells were positive for VEAs with a spectrum of antigen densities on individual cells. Fixed cells generally revealed a random diffuse distribution of VEAs, but with preferential labeling along cell edges, processes and microvilli. VEAs on unfixed cells labeled at room temperature either remained dispersed or underwent rapid antibody-induced partial redistribution at the cell edges and on processes, while the residual VEAs remained dispersed. Labeling cells at 37 °C peculiarily resulted in less peripheral antigen aggregation. After three or four subculturings of leukosis virus-infected cells, they displayed VEAs in an inherently clustered form superimposed on a random antigen display, but only some of these clusters appeared to be budding virions. VEAs on sarcoma virus-infected cells tended to cap when treated with antibody-hemocyanin. The binding of virions to their receptors at the surfaces of cells also results in cap formation [591,592], presumably because of the multiple binding sites contained on the envelope components in each virion. The most clinically interesting receptor redistributions which have been seen are the capping of surface-Ig. A high percentage of patients with chronic lymphocytic leukemia (CLL) have readily detectable surface-Ig on their lymphocytes [593]. In one study 28 ~o of the normal lymphocytes had detectable surface-Ig of lgG ( ~ 1 5 ~o), IgM (~8~o) and lgA (~6~o) types, whereas patients with CLL tended to have "monoclonal" lg expression of a given class [594,595]. Several groups have studied the dynamics of surface-lg on normal lymphocytes and on CLL cells and have found differences in anti-lg-induced capping. Normal cells cap rapidly after incubation

39 NORMAL LYMPHOCYTES

OoO0Ooo CLL LYMPHOCY/ES

¢

D

Fig. 5. Schematic representation of antibody-induced Ig redistribution (capping) on normal lymphocytes and lymphocytes from patients with chronic lymphocytic leukemia (CLL). The dark dots represent fluorescent anti-Ig. Most normal lymphocytescapped in the (A) configuration at 30 minutes with a few showing more extensive uropod cap formation (B). Most CLL cells were in the uncapped configuration (C) with a few showing some polar redistribution (D). (from Cohen and Gilbertsen [596]). with anti-lg (Fig. 5A, B), but CLL lymphocytes, on the other hand, did not cap in the same manner as normal cells and rarely demonstrated unidirectional surface movement of patches to form caps. On many cells the clustered surface-Ig receptors remained dispersed (Fig. 5C) or only partially capped (Fig. 5D) [596]. In another study the percentage of surface-Ig capping cells appeared to be within the normal range, but the structures of the caps formed were not reported [597]. Similar results have been reported for HL-A by Menne and Flad [598]. Although CLL cells did not show a diminished fluorescent-antibody labeling index, HL-A cap formation on CLL cells was almost completely lacking. Mintz and Sachs [580] have examined the concanavalin-A-induced cap forming ability of CLL lymphocytes and observed that the range of cap forming cells for normals was 2 5 - 3 4 ~ while 5-9 ~ of lymphocytes capped in all CLL patients and only 1-3 % capped in patients with Hodgkin's disease. They also found that CLL patients in remission had a normal percentage of cap forming ceils. Fine needle aspiration or operative biopsy was used by S~llstr6m et al. [599] to remove lymphoid cells for testing with concanavalin A. They observed that the proportion of cap forming cells in biopsies of normal tonsils and lymph nodes did not differ significantly but were lower than cells from thymus. Biopsies from the lymph nodes of patients with lymphosarcoma, reticulum cell sarcoma and Hodgkin's disease showed depressed percentages of cap forming cells compared to normal lymph node cells. IIIC. Surface modifications during the cell cycle and at cell contact The surfaces of normal cells in mitosis differ from non-mitotic cells [600]. It has been noted for many years that tissue culture cells tend to round up in mitosis,

40 and generally take on a surface morphology distinct from cells not in mitosis [601]. Porter and his colleagues [16,602,603] examined the surface morphology of mouse 3T3 fibroblasts and observed that these cells underwent morphological changes characteristic of each stage in the cell cycle. Specifically, confluent 3T3 cells had a smooth flattened morphology but during mitosis the cells developed dense networks of surface blebs and protrusions which tended to increase their surface areas [602-604]. Several quantitative studies have indicated that cells express different amounts of surface components during mitosis. Fox et al. [605] used fluorescent-wheat germ agglutinin to study cell cycle-dependent changes on untransformed fibroblasts and found that under the conditions of in situ labeling they used, only the mitotic cells bound appreciable fluorescent-lectin. Succeeding experiments established that fluorescent-concanavalin A acted similarly on untransformed mouse [600] and hamster [606,607] fibroblasts. The differences between mitotic and nonmitotic cells were observable only at very low lectin concentrations where preferential binding to mitotic cells may occur [606,607]; at high lectin concentrations all of the cells tended conspicuously to bind fluorescent-lectins [317,381,606,608]. Shoham and Sachs [606] discerned that untransformed hamster fibroblasts in interphase were not observably fluorescent when treated with fluorescein labeled-concanavalin A, unless concentrations greater than 5 #g/ml lectin were used. Mitotic-untransformed or interphase-chemically transformed hamster fibroblasts were fluorescent when labeled with 1 /zg/ml lectin, but the percentage of visibly fluorescent cells was intermediate between untransformed- and transformed-interphase cells. Interphase- and mitotic-untransformed and -transformed hamster cells bound equivalent amounts of 3H-concanavalin A when corrected for the increase in cell surface area during M [607] ruling out a net increase in receptors during mitosis. Brief trypsinization rendered the interphase-untransformed cells capable of visible fluorescence similar to nonmitotic-transformed cells suggesting that protease-treatment imitates some surface properties which are shared by the transformed state. Turner and Burger [600] obtained results diametrically opposite those for transformed mouse fibroblasts in mitosis [607], although differences in the techniques might explain these incompatible results. Labeling cells at all stages of the cell cycle with fluorescent-concanavalin A, they found that from prophase to early G1 transformed cells attached to coverslips were observably more fluorescent compared to interphase-transformed cells and quantitatively bound about three times more radiolabeled concanavalin A [369,609]. Somewhat intermediate results between the quantitative data of Shoham and Sachs [607] and Noonan and Burger [369,609] were found using mitotic and nonmitotic 3T3 cells. During mitosis the untransformed 3T3 cells quantitatively bound more concanavalin A and Ricinus cornmunis agglutinin, but when corrected for increases in cell surface area during mitosis these differences were not dramatic [610]. Agglutination changes have been detected with lectins during the cell cycle. Shoham and Sachs [607] observed an interesting relationship between the cell cycle and lectin agglutination of untransformed mouse and hamster fibroblasts and their

41 chemically and virally transformed counterparts. When untransformed cells were probed with lectins, they were agglutinable by concanavalin A and wheat germ agglutinin at mitosis, but not at interphase. Conversely, mitotic-transformed cells were relatively unagglutinable with concanavalin A and wheat germ agglutinin. Smets [611] and Collard et al. [469] have carefully determined concanavalin A agglutinability of 3T3 and SV3T3 cells at various phases of the cell cycle. In contrast to Shoham and Sachs [607], they found that agglutinability of SV3T3 cells using 25/~g/ml concanavalin A was high in mitosis and early G~, but decreased gradually into S and remained low through S. The transition from low agglutinability in G2 to high agglutinability in M occurred rather abruptly between late prophase and metaphase [611]. Untransformed 3T3 cells were also highly agglutinable in M, but were unagglutinable in all other phases. Garrido [450] has examined the distribution and mobility of concanavalin A and wheat germ agglutinin receptors on synchronized cultures of Chinese hamster embryo (CHO) cells and an SV40-transformed hamster cell line. Labeling cells in monolayer cultures using short incubation times at room temperature (to allow some lectin-induced redistribution), Garrido [450] detected a moderately discontinuous pattern of surface label during G1, S and G2. Cells identified as mitotic had strikingly aggregated or clustered lectin receptors indicating higher relative receptor mobilities during M. Cells labeled in suspension were not identifiably distinct in their lectin binding patterns, but this may have been due to the arbitrary labeling conditions employed. Using colcemid treatment to prevent passage through mitosis Garrido [450] was able to disassociate the surface modifications connected with mitosis from nuclear events. Garrido's [450] results are consistent with the proposal that mitotic cells have some surface properties more related to transformed cell surfaces than to untransformed-interphase cells. Specific cell surface antigens are displayed during certain stages of the cell cycle. Kuhns and Bramson [612] observed increased blood group H reactivity from M through early G1 in HeLa cells, and Thomas [613] found maximal H activity during mitosis in mouse mastocytomas. H-2 alloantigens are also displayed in a cell cycle-dependent manner and appear prominent during G~ and decreased in M on tumor cells [614-616] along with Moloney virus antigens [615]. Similarly, surface-lg on human lymphomas is decreased during cell division [617]. Burger [212,618] reported that Forssmann antigen is expressed on mouse fibroblasts predominately during mitosis and is also "permanently" displayed after virus transformation [456]. An assortment of other changes occurs at mitosis. Cells are more susceptible to virus-induced fusion [619], and when mitotic cells were assayed for release of extracellular mucopolysaccharides, Kraemer and Tobey [620] noted that certain cells release surprising amounts of surface heparin sulfate. Cell contact has been found to result in modified surface properties. Hakomori and Kijimoto [621] detected Forssmann antigen on hamster NIL cells with ~4Clabeled anti-Forssmann, and they noted intensified reactivity after cell-to-cell contact in culture. Contact-dependent changes in lectin agglutinability [317,355,365,622], membrane-intercalated particle distribution [279,280,623], cyclic nucleotide con-

42 centrations [244,503,504], cell motility [287,288], surface glycoproteins [134,624], transport [208,625,626] and cytoskeletal organization [274-276] have been discussed in previous sections. Modifications in plasma membrane glycolipids at cell contact ("contact-response" [110]) have been studied extensively [110,120,176,627,628]. However, in some systems such as mouse fibroblasts cell contact-dependent changes have not been seen [108,629], although Yogeeswaran and Hakomori [630] found increases in one ganglioside (GD1a) at contact in 3T3 cultures which did not occur in SV3T3 or SVPy3T3 cultures. These latter authors [630] also found that neuraminidase activity decreased at cell contact. Synthesis and exposure of glycolipids and glycoproteins vary through the cell cycle. Gahmberg and Hakomori [114,631] found that the display of particular glycolipids monitored by galactose oxidase-borotritide labeling on hamster NIL, but not PyNIL, fibroblasts was cell cycle-dependent. Ceramide tri-, tetra- and pentahexosides of NIL cells were maximally labeled during GI and minimally during S, although the chemical quantities remained invariant during the cell cycle [1 t4,631]. Bosmann and Winston [632] claim that most glycolipids are synthesized during G2 in L51781 lymphoma cells; however, their results have been criticized by Wolf and Robbins [633] who found synthesis of glycolipids occurred to some extent during all phases of the cell cycle in a hamster NIL fibroblast cell line as did Warmsley et al. [634]. Some glycolipids are made and exposed predominately in G~ and early S [633,635] in agreement with the studies of Glick et al. [636]. Critchley et al. [635] surveyed the cell cycle-dependency of several glycolipids in hamster NIL fibroblasts and found that ceramide trihexoside synthesis was stimulated in G1, whereas synthesis of ceramide tetra- and pentahexoside were not. Glycoprotein changes have been noticed during the cell cycle [636-638], but the most dramatic change appears to be the loss of exposure to lactoperoxidase catalyzed iodination of the high molecular weight transformation-sensitive (LETS) glycoprotein. Hynes and Bye [134] found that high levels of LETS exposure correlated with the G1 stage of the cell cycle when cells were arrested in this state by serum starvation or because of high cell density. Addition of serum to stimulate release from G~ resulted in a decrease of LETS exposure [134]. During mitosis LETS was almost undetectable, again mimicking the surface properties of transformed cells. As mentioned in Section IIA, brief trypsin treatment removes LETS glycoprotein very rapidly [128], and proteases can also (but not always [640]) stimulate growth of quiescent cells in culture [214,215]. On the basis of these and other experiments [641] it has been suggested [121,139,212,214,215,640,642 and others] that proteases may control cell growth and at least some of the surface properties attributed to the transformed state such as changes in cell morphology, agglutinability, receptor mobility, antigen, glycolipid and glycoprotein display, transport, and cyclic nucleotide levels. Although the evidence for this proposal is not decisive, available data indicate that a strong juncture exists between the properties of mitotic-normal cells and transformed cells.

43 iv. POSSIBLE RELEVANCY TO CANCER Several different immune defense mechanisms exist, and they probably play some role in the prevention of neoplastic disorders (reviewed in [518-520,522, 644-646]). The synergistic or anergistic effects of these systems in clinical situations are almost completely unknown and may vary with the individual. Because of genetic variability, inbred animal models have been used to study immunological surveillance mechanisms [645,647]. It is well documented that immunosuppression [648-652] increases the incidence of neoplasia, but some investigators doubt the reliability of immunological surveillance in coping with the variety of spontaneouslytransformed cells that show very low immunogenicity [653-656]. In fact, immune surveillance mechanisms should depend on thymus function, but the incidence of tumors in congenitally athymic nude [655,657] or neonatally thymectomized [658] mice is near-normal. However, thymus-independence of tumor incidence does not seem to be the case when strongly immunogenic tumors are induced by chemical carcinogens. Chemically induced tumors appear to elicit a strong immune response which can lead to rejection [659-661]. A neutral attitude toward these proposals will be taken by this author; it suffices to mention here that although immunological surveillance is generally accepted, it can fail, resulting in tumor progression. Immunological reactivity to tumors can be augmented by agents such as Bacillus Calmette-GuOrin (BCG), occasionally producing impressive remissions [662]. Some possible avenues for tumor escape from immune destruction will be discussed in IVB following a brief description of the immune mechanisms responding to neoplasia.

IVA. Mechanisms of tumor immunity The two basic arms of the immune system, humoral and cell-mediated, both appear to be involved in host response to tumors. As introduced cursorily in preceding sections, tumors are characterized by the appearance of "new" or increases in "old" antigens on their surfaces; these antigens may be embryonic or tumor virus envelope antigens or other TATAs. The two arms of host immunity recognizing and responding to these antigens are characterized by their antibody (humoral immunity)or cell (cell-mediated immunity)-dependency. (1) Humoral immunity. Efferent humoral immunity to tumors has been conjectured in several systems where immunoglobulins have been eluted from biopsy tissue, notably human sarcomas [663] and melanomas [664], or obtained from rodent hepatoma [665] and SV40-transformed [666] tumors. The presence of antibody bound to tumor cells has also been confirmed using anti-Ig immunofluorescence [667-669], but it is very difficult to determine the effectiveness of circulating antibody in a complement-mediated response against tumors in vivo. Witz et al. [670] have used 12SI-labeled anti-lg to examine quantitatively the amount of Ig present on the cell surfaces of various mouse ascites tumors in vivo. The amounts of surface-bound Ig generally increased in tumor bearing animals with time after tumor inoculation. Mice which had been irradiated prior to tumor inoculation have less Ig bound to tumor cell surfaces than unirradiated mice, suggesting a specific humoral immune

44 response was responsible for the increased antibody binding to the tumor cells in hosts capable of immunologically responding to the tumor [670]. Antibody-complement reactivity has also correlated with the absence of metastasis in one recent study. Bodurtha et al. [671] measured complement-dependent cytotoxic anti-CSA in the sera of twenty-one melanoma patients and found antibody reactivity in nine of ten patients with localized or regional melanoma, but in only one of eleven patients with disseminated metastases. Even normal sera from nontumor-bearing mice have been shown to be reactive to VEA expressed on some lymphoma [672,673] and myeloma [570] cell lines. Martin and Martin [674] detected normal serum IgM antibodies against a variety of tumors of many histological types which arose in liver lung and nervous tissue. They also found anti-tumor antibodies in the sera of nude mice, suggesting the thymus-independent appearance of these immunoglobulins [674,675]. However, Martin and Martin [674] noted that complement fixation to IgM is poor in mice [676] and IgM does not penetrate well into extravascular tissues [677]; they propose that the widespread occurrence of anti-CSA IgM may be important in immune interactions with circulating tumor cells [674]. In experiments where murine tumor cells in vivo were killed by administered antibody, the addition of guinea pig complement was necessary; antibody given alone was ineffective in producing tumor rejection [678]. However, Irie et al. [679] have shown by immune adherence assays that antigen antibody-complement complexes exist in vivo on the surfaces of a variety of human tumor cells, indicating that humoral responses might contribute to clinically observed tumor regression. Finally, humoral responses may also function to neutralize released tumor viruses. Antibodies reactive to leukemia virus and VEAs have been found in several mouse inbred lines [672,680-682]. (2) Cell-mediated immunity. The efferent cell-mediated arm of host immune surveillance is complex, and much less is known of the cell-cell interactions required in the stimulation and killing phases of these processes. Cell-mediated immunity is thought to be the most important defense against neoplasia [683] based on evidence from neutralization and immune adoptive transfer experiments [521], and the fact that the progress of the disease in many cases has been inversely related to cellmediated reactivity against autochthonous tumors [519,520,684]. The various cellmediated immune mechanisms have been reviewed recently by Cerottini and Brunner [646], Hellstr6m and Hellstr6m [520], Brondz [685] and Herberman [521] and will only be summarized here. There appear to be at least three basic categories of cellmediated reponse depending on the nature of the effector cells [646]: (a) Cytotoxic killing by T-derived cells, probably small lymphocytes. These lymphocytes proliferate and differentiate to blast-like cells during an immune response to tumor associatedantigens. They kill by direct interaction with specific antigens on the target cell surface, the interaction leading to a loss in ionic balance and increased osmotic fragility of the target cell [686,687] without release of soluble cytotoxic factors such as lymphotoxin [688,689]. One effector cell can kill several target cells, and the reaction reaches completion within one hour under optimal conditions in vitro. (b) Antibody-dependent killer or K cells are non-adherent, non-phagocytic, medium

45 to large mononuclear lymphoid cells. They are distinct from B and T cells by velocity sedimentation [690], resistance to anti-Thy-I (0) [691], sensitivity to X-ray irradiation [690] and failure to adsorb on columns derivatized with anti-lg F(ab')2 [692]. K cells contain Fc receptors which interact with IgG antibodies bound to target cells. Target lysis occurs optimally within 10-20 h in vitro, and the lytic process can be inhibited by anti-Ig, aggregated lgG or soluble antigen-antibody complexes. Other K-cells have been identified in mice such as one type which has the characteristics of phagocytic adherent cells of the monocyte-polymorphonuclear leukocyte series [693,694]. (c) Killing by specifically "armed" macrophages. This killing can occur after stimulation by direct interaction through macrophage surface-lg or through cytophilic factors produced by immune T-derived lymphocytes following contact with specific tumor-associated antigens. Armed macrophages which have been activated by direct incubation with target cells or by indirect sensitization with immune spleen cells can exert a cytotoxic effect on tumor cells or even irrelevant cells [695]. Normal macrophages can also be non-specifically activated to "angry" macrophages by agents such as BCG, endotoxin, poly (l).poly (C), and double stranded fungal RNA [696], and after administration of specific antigen can become non-specifically cytotoxic. It should be clear from this brief discussion of cell-mediated immunity that a tumor response in vivo does occur via more than one mechanism. The relative contribution(s) of each of these complex cell killing systems in fighting neoplastic disease is hard to assess because the in vitro methods used to assay each mechanism may have little relationship to the immune response in vivo. There exists the possibility that some of these mechanisms are irrelevant to host tumor rejection, some may be more important than others, or finally, that synergism between these cellmediated (and perhaps also humorai) systems may be important for adequate host protection. In their studies on humoral and cell-mediated syngeneic responses to mouse and hamster MSV-transformed (virus) producer and non-producer tumor cells, McCoy et al. [697] found that humoral antibody was produced largely in response to the producer lines implying a preference for VEA stimulation of humoral activity; transplantation immunity was, in part, produced against non-virion CSAs on non-producer tumor cells. IVB. Tumor escape mechanisms Although an exquisite host defense system has evolved to protect against neoplastic disease, such disease nonetheless occurs with sometimes alarming frequency and manages to evade elaborate host responses that should lead to tumor rejection. A variety of ways in which tumor cells could escape destruction will be discussed here, but it should be kept in mind that other escape pathways not mentioned or unknown at present may be equally important. (1) Surface antigen modulation and antigen loss. Certain cells when treated with specific antisera gradually lose their sensitivity to cytotoxic killing. This process is called surface antigen modulation, and it was first studied by Old et al. [698] using lymphoid cells of the mouse which carried the thymus-leukemia (TL) antigens. They

46 sought to explain why T L - mice could not be protected against TL ÷ leukemias by immunizing them to TL antigens. When the TL ÷ leukemia cells were recovered from these mice, they were phenotypically TL-, but regained their TL ÷ phenotype when returned to nonimmune mice. The reversible loss or modulation of TL antigenicity has been studied in vivo [569] and in vitro [698], and it was found to be a temperaturedependent active process inhibitable by iodoacetemide or actinomycin D. it was proposed to occur by antibody-induced loss from the cell surface of antigen-antibody complexes through endocytosis or shedding. Cell surface TL antigens are known to redistribute rapidly in the presence of anti-TL to form clusters, patches and eventually caps [399,699-701]. Yu and Cohen [702] studied the metabolism of TL antigens on TL ÷ leukemia cells by metabolically labeling the cell surface glycoproteins with 3H-fucose or iodinating using lactoperoxidase techniques. They followed the fate of TL-anti-TL complexes by solubilization and precipitation using rabbit-anti-mouse Ig. Within a few hours after modulation the amount of surface-labeled TL complexes recoverable from the leukemic cells was considerably lower than control cells treated with anti-H-2 suggesting endocytosis and degradation of some (but not all) TL complexes occurred; it also transpired without modifying another antigen, H-2. When the culture medium was examined for shed TL-anti-TL complexes, no significant differences between modulated and control samples could be detected. Yu and Cohen [702] concluded that anti-TL sera induced endocytosis of TL surface antigens which were subsequently catabolized intracellularly, and modulation was not the result of significant surface antigen shedding. A closer look at their data also reveals the existence of a persistent population of TL molecules ( ~ 6 0 % of the total precipitable antigen) at the cell surface after modulation indicating that modulation does not require removal of even a majority of TL antigens. Loor et al. [701] also decided that TL modulation does not correspond to a total depletion of TL from the cell surface. They found that modulation does not correspond to TL cap formation, although anti-TL capped cells are insensitive to complement [578,701]. Stackpole et al. [578] carefully examined the modulation process with anti-TL and anti-TL(Fab') using fluorescent-antibody and immunoelectron microscopy and observed that redistribution of TL antigens into patches and caps is sufficient for modulation. This could be due to stearic hindrance of the tightly packed lg molecules preventing complement approach, or to a relative immobilization of the antibody which does not allow molecular distortions required to initiate the fixation of complement components. Since the correct disposition of two IgG molecules is necessary to fix complement, antigen mobility must be indispensible to start the complement cascade. Therefore, the most complement-sensitive surface state of TL-anti-TL complexes will probably turn out to be small clusters formed by antibody-induced redistribution (Fig. 6). This conclusion is consistent with the known distributional requirements for antibody-complement-dependent killing in other systems [415,704]. Other antigens can undergo modulation after addition of the appropriate antibodies; however, the kinetics of modulation seem to be distinct for each antigen.

.

47

---.-il=-- ESCAPES

~

ESCAPES

KILLED ( ? ) ESCAPES { ? }

'DISPEBSED'

,5

;' ....

REMAINS 'DISPERSED'

Fig. 6. Some possible mechanisms for tumor cell escape from immune surveillance which depend on cell surface antigen dynamics, The usual pathway for antibody-complement-mediated killing (indicated by a box), and several possible alternative pathways are shown with the probable cell fate in the column at the right. Block indicated by: I, low temperature or chemical fixation; 2, local anesthetics or colchicine plus cytochalasin B; 3, metabolic inhibitors.

Takahashi [703,705] studied the modulation o f H-2 alloantigens and surface-lg on mouse lymphoid cells and noted distinct requirements of temperature, time, antibody concentration and cellular metabolism. K n o p f et al. [706] have carefully considered these parameters for surface-lg modulation by anti-lg on myeloma cells. Certain antigens, such as H-2 on most ceils, require indirect antibody sandwich techniques to modulate completely [705], but modulation also occurs directly with anti-H-2 on some cells. Similar to T L modulation, Lesley and H y m a n [707] have concluded that removal o f approximately one-third o f the H-2 antigens from the surfaces of S194 myeloma cells by anti-H-2 during a three hour modulation at 37 °C made greater

48 than 80 ~o of the tumor cells refractory to complement lysis. The fate of H-2 antibody complexes on EL4 leukemia cells has been studied by Lengerov/~ and her collaborators [576]. Capping of H-2 resulted in loss of complement-dependent cytotoxicity similar to TL, and the degree of capping directly correlated with H-2 modulation in agreement with Edidin and Henney [708]. Using antibody coupled to peroxidase, Lengerov~ et al. [576] followed the fate of the complexes ultrastructurally and found that considerable endocytosis occurred within 90 minutes at 37 °C, but this probably represents a minor proportion of the total H-2 antigens. Modulation of Gross-CSA has also been reported on Gross virus-induced leukemia cells in vivo and in vitro by Aoki and Johnson [709]. Gross-CSA ÷ leukemia cells were transplanted into immunized hosts, and upon recovery the tumor cells were found to have undergone modulation and were phenotypically Gross-CSA-. Modulation by anti-Gross-CSA also occurred in vitro; although modulation occurred readily, it did not influence the budding of murine (Gross) leukemia viruses from the cell surface, nor did antiGross-CSA react with Gross leukemia virions [582]. Other CSAs which have been reported to undergo modulation in the presence of specific antisera are present on Burkitt's lymphoma and human melanoma [710]. It has been recently demonstrated that sensitivity to humoral or ceil-mediated killing does not always correlate with cell antigen density [711,712]. Lesley et al. [712] found that most cells behave predictably to antibody-complement-mediated lysis and T-cell-mediated killing from their surface antigen content, but there were notable exceptions. One mouse cell line, P815 mastocytoma, was relatively insensitive to anti-H-2 complement-mediated lysis, although it had a high relative antigen content. However, even cell lines poorly sensitive to antibody-complement lysis, reputedly because of relatively low antigen densities, were sensitive to cell-mediated killing. Edidin and Henney [708] note that even after H-2 modulation and loss of complement sensitivity, DBA/2 mastocytoma cells can remain sensitive to T-cell-mediated killing. These findings suggest that either much lower antigen densities are required for T-cell-mediated attack, or additional unknown determinants are actually involved in the binding and killing phases of the cell-mediated response. Trans-membrane control over the dynamics of surface antigens should be important in determining susceptibility to immune destruction. Influencing modulation or restraining the mobility of surface antigens should aid in escape from host responses. Although scant, the available evidence suggests that trans-membrane events can control the fate of cells during an immune attack. Endocytosis is a transmembrane event which requires metabolically active cell and membrane-associated contractile elements such as microfilaments [713-715]. Obviously, endocytotic removal of surface antigen-antibody complexes would render a cell resistant to subsequent lysis by complement components. Trans-membrane control over the mobility of surface antigens was discussed in Sections IB and llIB, and there is some indirect information on the relationship of cell surface dynamics to immunological events. Segerling et al. [716] found that the resistance of guinea pig hepatoma cells to antibody-complement-mediated lysis was reduced if the cells were pretreated with

49 non-toxic doses of actinomycin D, puromycin, mitomycin C or hydroxyurea (similar to Ferrone et al. [717]). This occurred without an increase in the number of antigenic sites or an increase in complement binding capacity. In addition to the speculations that these drugs impair the effectiveness of cell surface repair mechanisms or synchronize the cells in a certain phase of their growth cycles, active trans-membrane processes could be affected which restrain the mobilities of certain surface receptors. As discussed previously [1,62-70,718], drug inhibition of membrane-associated cytoskeletal elements could result in an uncoupling of trans-membrane anchored surface receptors. This might affect antibody-complement killing in any of three ways: by preventing cap formation, cluster formation or blocking endocytosis. Interestingly, Kurth and Bauer [719] determined that the addition of cyclic AMP to Rous sarcoma virus-transformed mouse D4 tumor cells resulted in a decrease in antibody-complement killing, but only when they examined surface antigens which remained constant and did not increase in number due to cyclic AMP. The interpretation of these results could be based on the well known cyclic AMP-induced increase in membrane-associated cytoskeletal organization (discussed in Section IliA (3)) which could enhance trans-membrane restraints and receptor anchorage. The best evidence on the role of receptor dynamics in antibody-complement-mediated cytotoxicity are the elegant studies of Sundqvist et al. [704] where the interaction between cell surface-bound IgG antibodies and human complement components were examined using microfluorometry. They chose Clq, the Ig-binding molecule in the first complement component, for study because it is responsible for the initial cell surface-complement interaction in conventional complement fixation and cytotoxicity. Sundqvist et al. [704] considered the quantitative and distributional aspects of surface-bound IgG on Clq binding, the influence of multivalent Clq on the redistribution of surface-bound lgG and the distributional requirements of IgG-Clq complexes on cytotoxic killing. They found that the binding of Clq was proportional to the amount of surface-bound IgG and that Clq binding promoted clustering of membrane-bound antibody, both at 0 ° and 37 °C, but failed to cap IgG. Sensitivity to complement lysis was determined at various stages of redistribution of membranebound antibody, and they noted that maximum sensitivity corresponded to clustered or patchy antibody distribution. As diagramed in Fig. 6 several routes probably exist for tumor escape from immune destruction; in this case escape mechanisms are based only on the dynamics of cell surface antigens contained on separate molecules. Tumor cells containing antigens, which remain relatively anchored or fixed in a monomolecularly dispersed distribution, remain refractory to complement fixation (if IgG antibodies are bound) and perhaps also to K-cell interactions although there is no evidence for this latter possibility. Tumor escape can also occur by extensive lateral redistribution of antigens into large patches and caps. The antibody-induced sequestration of antigen-antibody complexes into tight aggregates may prevent antibody molecular distortions required to initiate complement binding. In addition to distributional rearrangements tumor cell escape can occur by antigen shedding (discussed in the next section) and/or endocytosis, both of which removed complement fixing moieties from the cell surface (Fig. 6).

50 Loss of antigenic specificities can occur in vivo during tumor development [720]. Antigen loss is not reversible and is probably a selection process where tumor cells carrying certain antigenic specificities are selectively killed by an immune response and variants not expressing the antigen(s) or displaying them in a conformation and/or distribution which is insensitive to humoral and cell-mediated responses survive and grow out [721,722]. Many transplantable tumors that have been passed in other strains or substrains have suppressed antigen phenotypes. (2) Antigen shedding and blocking .factors. Cell plasma membranes are constantly undergoing synthesis and degradation [723-725]. One mechanism of cell surface turnover which seems to be quite normal for cells and very important as a mechanism for tumor cell escape from immune destruction is the shedding of surface antigens and other membrane molecules or their fragments. When Kapeller et al. [414] examined chick embryo fibroblasts in culture, they found that the cells released or shed glucosamine-labeled surface components that were strikingly similar to trypsin removable surface glycopeptides upon chromatography on DEAE-cellulose columns. The natural release of mouse H-2 alloantigens by C3H kidney cells was also investigated [414]. Cone et al. [726] determined that shedding of cell surface proteins occurs at a rapid rate and is dependent on cellular respiration and protein synthesis, although the concentrations of sodium azide and iodoacetamide necessary to inhibit the release of lactoperoxidase iodinated surface proteins were quite high. These researchers found that the components which were naturally shed into the surrounding media by some but not all cell types were similar to the surface material extractable by urea treatment [726]. The actual mechanism of cell surface shedding is unknown, but it could be due to blebing off or release of plasma membrane fragments [727], dissociation of peripheral membrane components or enzymatic hydrolysis ("self-autolysis" [728]) of surface constituents by cell-released proteases and glycosidases. Tumor-associated antigens are spontaneously shed from neoplastic cells in vitro and in vivo [729]. Currie and Alexander [730] found that certain methylcholanthrene-induced fibrosarcomas release appreciable amounts of antigen during in vitro cultivation. The released antigens were, in part, TATAs from the tumor cells, because they were effective in inhibiting the killing of sarcoma cells in vitro by immune lymphoid cells. One tumor cell line (MC-I) was highly immunogenic, rarely metastasized and could be rejected by animals protected by prior immunization using lethally irradiated tumor cells [731]; while another cell line (MC-3) showed low immunogenicity, metastasized frequently to regional lymph nodes and lungs and failed to immunize animals against subsequent tumor challenge [732]. When these two fibrosarcoma lines were grown in vitro, only tissue culture media obtained from MC-3 cultures inhibited autochthonous cell-mediated immunity. In this system shed TATAs are probably acting as blocking facors to cell-mediated immunity. Soluble blocking factors present in the sera of tumor-bearing animals and patients can protect tumor cells from cell-mediated immune destruction. Blocking factors are thought to be soluble tumor-associated antigens [730,731,733,734], anti-

51 bodies against tumor CSAs [519,735,736], antigen-antibody complexes [737-739] or other non-immunological factors [740]. These possibilities and others have been reviewed recently by Heppner [741], Hellstrfm and Hellstr6m [520,684] and Baldwin [545]. In brief, the initial evidence for the existence of serum factors capable of blocking cell-mediated immune reactivity against tumor cells in vitro was obtained with a Moloney virus-induced sarcoma system. Serum from tumor-bearing animals with progressively growing sarcomas blocked sensitized lymph node cells in a colony inhibition assay [735]. The appearance of blocking activity in sera appeared fairly early after tumor inoculation at a time when these solid tumors were not palpable [742] or barely palpable [743]. Initially, Hellstr6m and Hellstr6m [735] proposed that the blocking factors were antibodies. Evidence for specific antibodies as mediators of blocking activity was that the specificity of blocking was similar to the specificity of a humoral response against tumor CEAs [545], blocking factor fractionated as if it were 7 S immunoglobulin, and it could be neutralized by anti-mouse (7 S) lg [735] or blocking activity could be removed from serum by adsorption onto the target tumor cells [735,738]. in addition to an immunoglobulin component there is proof that some blocking factors contain antigen(s) as antibody-antigen complexes. Sj/Sgren et al. [738] separated serum from animals bearing virus- or chemically induced tumors into two fractions ( ~ 100000 molecular weight and 10000-100000 molecular weight) by ultrafiltration at pH 3.1. Neither fraction alone blocked in vitro cell-mediated reactivity against tumor cells, but the combination of these fractions displayed blocking activity. In addition, the lower molecular weight fraction (thought to be antigen(s)) blocked if added at high concentration to the effector cells, but did not block if added to the target cells. Blocking factors have also been eluted from human tumors [737]. After elution the blocking activity was abolished by the fractionation procedure described above into high and low molecular weight mixtures at pH 3.1, and upon remixing at near-neutral pH (in a l : I ratio) the blocking activity reappeared. This time the high molecular weight fraction blocked, but only if added to the target cells prior to addition of the lower molecular weight fraction. All of these results can be reconciled with the concept that there are multiple pathways for blocking cell-mediated immune reactivity. Antibody alone probably blocks T-cell-mediated killing by directly masking or modulating tumor cell receptors (capping, endocytosis, etc.). Antigen alone might block by obstructing recognition sites on the immune effector cells. Antigen-antibody complexes presumably act via both these mechanisms, but their attachment to immune effector cells is thought to be more important than to target cells. Most investigators feel that these last two mechanisms are the most important for blocking in vivo. Serum also contains "unblocking" and "potentiation ~' factors which are thought to be antibodies. During Moloney virus-induced sarcoma tumor regression [744] or after surgical removal of spontaneous neoplastic disease [745], serum sometimes contains unblocking factors which neutralize the effects of blocking factors on cellmediated reactivity. These unblocking factors are related to humoral antibodies directed against tumor CEAs [520]. Potentiation of cell-mediated immunity to

52 tumors can occur in the presence of certain serum factors [746]. Skurzak et al. [745] reported that the serum dilution was critical during in vitro cell-mediated killing assays and that enhancement of tumor cell killing could be obtained at the right dilution. Very dilute serum appears to "arm" the antibody-dependent K-cell response to tumor cells while diluting blocking factors. The in vivo roles of the different cell-mediated immune surveillance mechanisms and their possible synergistic effects are relatively unknown but speculations abound. (3) Tumor enhancement. Up to now the immunosuppressive aspects of immune surveillance against neoplasia have been considered, but there is also evidence indicating that under certain conditions immunostimulation or immune enhancement of tumor growth can occur. The "immune enhancement-inhibition" theory of Prehn [653,747] predicts that a low level of immunity is stimulatory for tumor growth, while a high level is inhibitory. This theory was based on the findings that antibodies against cell surface antigens can stimulate cell growth in vitro [747-479] and in vivo [747,750-752]. The dual role of antibody in immune enhancement-inhibition of cell growth was nicely demonstrated by Shearer et al. [749,753] using cells substitlated with 2,4,6trinitrophenol and purified antibodies against this hapten or anti-whole-cell antibodies. Several mouse and human transformed cell lines were stimulated to grow faster in vitro in the presence of low concentrations of antibody, but were inhibited at higher antibody concentrations. Prehn [654] has examined the immune enhancementinhibition of tumor cells in vivo by mixing varying numbers of immune spleen cells with 3-methylcholanthrene-induced sarcoma tumor cells and inoculating the mixtures subcutaneously into syngeneic thymectomized-irradiated mice. The regimen of thymectomy and X-irradiation prevented skin allograft rejection thus, the effects of the injected immune spleen cells on tumor growth were not complicated by host immunity. Mice given low ratios of immune cells to tumor cells developed significantly larger tumors than controls not receiving spleen cells, but in mice with high spleen: tumor cell ratios inhibition of tumor growth occurred. Studying immune reactivity to circulating tumor emboli, Fidler [754] noted that small numbers of injected sensitized lymphocytes aided in the successful experimental implantation, survival and tumor formation of metastatic BI6 melanoma, whereas high numbers of lymphocytes were inhibitory and prevented experimental metastasis. Also, immunosuppressed mice had lower incidences of experimental metastasis compared to control mice and immune lymphocyte reconstitution one day prior to tumor cell injection increased the amount of pulmonary tumors unless large numbers of lymphocytes were previously administered. Similar experiments have been conducted with humoral immunity against mouse L-929 tumors cells by Fink et al. [752]. Thymectomized, lethally irradiated mice were given syngeneic bone marrow transplants under conditions known to reconstitute their humoral response without reconstituting cell-mediated immunity. Animals given bone marrow cells with rabbit anti-L-929 serum intravenously one day before subcutaneous tumor cell implantation consistently had larger tumors compared to controls, unless a large dose of anti-L-929 was used. Comparable results were obtained in vitro with L-929 cells and anti-L-929

53 TABLE 1I SUMMARY OF IMMUNOLOGICAL RESPONSES TO SV40-TRANSFORMED SARCOMAS IN NEONATAL HAMSTERS* Weeks after infection with SV40

animals with palpable tumors Cytostatic antibody Cell-mediated immunity (in vitro) Cell-mediated immunity (in vivo) Concomitant immunity

0

5

0 -----

0 ~ ~ ---

10

15

20

0 ~5 ~ 80 ÷ ~ -~ ÷ t ± --÷ ÷ ÷ Antibody Antigen enhancement ,--excess Blocking

25 ~ 100 -÷ --~ Metastasis Anergy

* Modified from Coggin et al. [756] with permission. serum [749] indicating that a n t i b o d y e n h a n c e m e n t p r o b a b l y occurs by direct imm u n o g l o b u l i n interaction with the t u m o r cell surface. T h e m e c h a n i s m o f t u m o r e n h a n c e m e n t is u n k n o w n , but s o m e w h a t a n a l o g o u s effects o f a n t i b o d i e s and lectins on cell proliferation in vitro have p r o m p t e d speculation t h a t e n h a n c e m e n t m a y be similar to mitogenic stimulation o f cell g r o w t h [747]. This hypothesis might be testable with F a b antibodies, because l i g a n d - i n d u c e d crosslinking or redistribution o f receptors at the cell surface a p p e a r s to be a requirem e n t for mitogenic triggering [63,755] which p r e s u m a b l y occurs via a t r a n s - m e m b r a n e signaling process. W i t h several routes available for t u m o r escape a n d multiple host i m m u n e defense mechanisms, one might ask how these systems c o o p e r a t e o r c o u n t e r a c t one a n o t h e r in vivo? The answer to this question is not k n o w n at present, but a few investigators have a t t e m p t e d to d e t e r m i n e at least some o f the i m p o r t a n t i m m u n e p a r a m e t e r s d u r i n g t u m o r establishment a n d progression. Coggin et al. [756] have s u m m a r i z e d their extensive studies on h u m o r a l a n d cell-mediated i m m u n e responses to S V 4 0 - t r a n s f o r m e d h a m s t e r s a r c o m a s in vitro a n d in vivo a n d have c o n c l u d e d t h a t host i m m u n e systems are t e m p o r a l l y c o n t r o l l e d o r regulated a n d can be modified by b l o c k i n g factors. In their studies m e w b o r n hamsters were given SV40 virus to induce t u m o r f o r m a t i o n , a n d host i m m u n e functions were then m o n i t o r e d at various times in vitro using a n t i b o d y - c o m p l e m e n t - d e p e n d e n t killing a n d cell-mediated microcytotoxicity assays against a u t o c h t h o n o u s t u m o r cells. Estimates o f in vivo l y m p h node cell cytotoxicity were p e r f o r m e d in the presence o f host serum, while in vitro cell-mediated i m m u n i t y was assayed with washed effector cells to eliminate serum b l o c k i n g factors. Their d a t a have been s u m m a r i z e d in Table II. H u m o r a l i m m u n i t y a p p e a r e d (three weeks p o s t infection) before cell-mediated i m m u n i t y to the a u t o c h t h o n o u s h a m s t e r t u m o r s ; however, h u m o r a l i m m u n i t y was cytostatic a n d only slowed t u m o r g r o w t h and could not prevent ultimate t u m o r progression. One inter-

54 pretation of this observation is that the opposing effects of antibody-enhancement and antibody-dependent complement-mediated cytotoxic killing neutralize one another for several weeks (explaining the long latency period for palpable tumor appearance) with enhancement eventually gaining the upper hand. This might happen if resistant tumor cell variants are selected out of the sensitive population. Alternatively, antibodies against tumor-associated antigens could mask antigenic sites on the tumor cell surface inhibiting the afferent process of the immune response (afferent inhibition). Circulatory antibody titers decreased at around fifteen weeks post-infection, possibly because of the release of excess soluble antigen which might result in antigen-antibody complexes being trapped in the kidneys [777]. Alternatively, the association of a majority of antibody molecules with the growing tumor mass could remove circulating immunoglobulin (see Section IVB (2)). Cell-mediated immunity appears after humoral immunity and then decreases in effectiveness and is unable to stop tumor progression. When washed lymph node cells are assayed in vitro during the period of decreasing cell-mediated cytotoxic effectiveness in vivo, they are strongly cytotoxic suggesting the presence of blocking factors in the animals which could be antibody-antigen complexes initially and later excess antigen as the humoral reponse declines. One inexplicable result that Coggin et al. [756] obtained was that tumor-bearing animals with the impaired immunological conditions described above exhibited concomitant immunity and were able to reject a fresh challenge of tumor cells at a distant site from the primary tumor. They have suggested that the newly implanted secondary tumor cells are not ~coated" with immunoglobulin since the levels of circulating antibody have fallen by the time of implantation, and therefore these secondary tumor cells are susceptible to cell-mediated attack. Coggin et al. [756] concluded that a series of immunologically related interference phenomena are responsible for successful tumor escape from host immune rejection, but it should be stressed that different animal models could yield slightly different results. V. FINAL COMMENT The importance of the cell surface in certain neoplastic phenomena such as growth regulation, cell recognition, cell contact, metastasis, escape from host immune surveillance and many other properties is relatively indisputable. However, none of the many observations on the dynamics and control of surface receptors of normal and tumor cells has been able to explain completely the altered properties of tumor cells. This limitation is probably to be expected because of the complex nature of the phenomena and the systems under investigation. Tumor cells escape many of the controls and social restraints which subjugate normal cells, and they are therefore able to achieve varying degrees of autonomy from their host. This autonomy results in uncontrolled proliferation, but it can also result in aberrant cell-to-cell recognition, allowing malignant tumor cells to escape from the control mechanisms which maintain proper cell position and prevent invasion of surrounding normal tissue. In malignant disease the surface properties of tumor

55 cells are important not only in tissue invasion, but also in determining the subsequent patterns of cell distribution and establishment of distant metastases [316a,778]. Alterations in trans-membrane communication and control mechanisms which probably maintain somewhat ordered, but dynamic, topographic displays or "patterns" of cell surface receptors on normal cells could contribute to loss of proper cell contact, recognition and positional information in tissues. In addition, abnormal cellular properties of transformed cells such as contact-inhibition, anchorage-dependency, hormonal signaling, agglutinability, etc. are at least in part explainable by alterations in trans-membrane communication and control mechanisms, perhaps mediated through cytoskeletai assemblages [778]. Since proteolytic enzyme treatment appears to be capable of mimicking several transformed cell surface properties in normal cells, the prospect that enzyme-mediated dislocation of trans-membrane controlling mechanisms resulting in a pleiotropic stimulus which initiates a wide range of cell surface and metabolic changes [778] could account for many of the surface alterations accompanying neoplastic transformation has received wide attention [73,121,139, 146,180,197,201,640-643,728]. Unfortunately, a high degree of speculation still surrounds the interpretation of cell surface data in this area, because of the lack of sufficient information. Cell research is still in its infancy and the relevancy of the observations discussed here with respect to the final control of neoplastic disease remains to be demonstrated. ACKNOWLEDGMENTS The excellent and untiring assistance of P. Delmonte, G. Beattie and especially A. Brodginski is gratefully acknowledged! 1 thank my colleagues Drs. J. Robbins and R. Hyman for their comments on parts of the manuscript. Support was provided by contract CB-33879 from the Tumor Immunology Program of the U.S. National Cancer Institute and grants GB-34178 from the Human Cell Biology Program of the U.S. National Science Foundation and CA-15122-1A from the U.S. Public Health Service and BC-211 from the American Cancer Society Inc. REFERENCES 1 Nicolson, G. L. (1976) Biochim. Biophys. Acta 457, 57-108 2 Weiss, L. (1967) The Cell Periphery, Metaslasis and Other Contact Phenomena, Vol. 7 of Frontiers of Biology (Neuberger, A. and Tatum, E. L., eds.), North-Holland, Amsterdam 3 Aaronson, S. A. and Todaro, G. J. (1968) Science 162, 1024-1026 4 Stoker, M. and MacPherson, I. (1961) Virology 14, 359-370 5 Tooze, .I. (1973) Molecular Biology of Tumor Viruses, Cold Spring Harbor Laboratory, New York 6 Todaro, G. J. and Green, H. (1963) J. Cell Biol. 17, 299-313 7 Pollack, R. E., Green, H. and Todaro, G. J. (1968) Proc. Natl. Acad. Sci. U.S. 60, 126-133 8 Dulbecco, R. (1970) Nature 227, 802-806 9 Stoker, M. G. P. (1967) Current Topics in Developmental Biology(Moscona, A. A. and Monroy, A., eds.), pp. 86-100, Academic Press, New York 10 Coman, D. R. (1944) Cancer Res. 4, 625-629

56 11 12 13 14 15 16 17 18 19 20 21 22 23

24 25 26 27 28 29 30

3l 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Gail, M. H. and Boone, C. W. (1972) Exptl. Cell Res. 70, 33-42 Martz, E. and Steinberg, M. S. (1973) J. Cell. Physiol. 81, 25-38 Nicolson, G. L. and Winkelhake, J. L. (1975) Nature 255, 230-232 Winkelhake, J. L. and Nicolson, G. L. (1976) J. Natl. Cancer Inst. 56, 285-291 Vasiliev, J. M. and Gelfand, I. M. (1973) in Locomotion of Tissue Cells, Ciba Foundation Symposium Vol. 14, pp. 311-332, Elsevier-North Holland, Amsterdam Porter, R. K., Todaro, G. J. and Fonte, V. (1973) J. Cell Biol. 59, 633-642 Collard, J. G. and Temmink, J. H. M. (1975) J. Cell Sci. 19, 21-32 Nicolson, G. L. (1974) Int. Rev. Cytol. 39, 89-190 Rapin, A. M. C. and Burger, M. M. (1974) Adv. Cancer Res. 20, 1-91 Lis, H. and Sharon, N. (1973) Annu. Rev. Biochem. 43, 541-574 Holley, R. W. and Kiernan, J. A. (1971) in Growth Control in Cell Cultures (Wolstenholme, G.E.W. and Knight, J. eds.), pp. 3-10, Churchill Livingstone, Edinburgh and London Smith, H. S., Scher, C. D. and Todaro, G. J. (1971) Virology 44, 359-370 Temin, H. M., Pierson, R. W., Jr. and Dulak, N. C. (1972) in Growth, Nutrition and Metabolism of Cells in Culture (Cristafalo, V. 1. and Rothblat, G., eds.), pp. 50-81, Academic Press, New York Macpherson, 1. and Montagnier, L. (1964) Virology 23, 291-294 Shin, S., Freedman, V. H., Risner, R. and Pollack, R. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4435-4439 Wallach, D. F. H. (1972) The Plasma Membrane:Dynamic Perspectives, Genetics and Pathology, Springer-Verlag, New York Oseroff, A. R., Robbins, P. W. and Burger, M. M. (1973) Annu. Rev. Biochem. 42, 647-682 Clarkson, B. and Baserga, R. (1974) The Control of Proliferation in Animal Cells (Ciarkson, B. and Baserga, R., eds.), Cold Spring Harbor Laboratory, New York Pardee, A. B. (1975) Biochim. Biophys. Acta 417, 153-172 Robbins, J. C. and Nicolson, G. L. (1975) in Biology of Tumors: Surfaces, Immunology and Comparative Pathology, Vol. 4 of Cancer: A Comprehensive Treatise (Becker, F. F., ed.), pp. 3-54, Plenum Press, New York Farber, E. (1973) Cancer Res. 33, 2537-2550 Herschman, H. R. (1972) in Membrane Molecular Biology (Fox, C. F. and Keith, A. D., eds.), pp. 471-502, Sinauer Associates, Inc., Stamford, Conn. Nicolson, G. L., Robbins, J. C. and Winkelhake, J. L. (1975) in Cellular Membranes and Tumor Cell Behavior (Walborg, E. F., ed.), pp. 81-127, Williams and Wilkins Co., Baltimore Curtis, A. S. G. (1967) The Cell Surface: Its Molecular Role in Morphogenesis, Academic Press, New York Wallach, D. F. H. and Winzler, R. J. (1974) Evolving Strategies and Tactics in Membrane Research, Springer-Verlag, New York Levine, Y. K. (1972) Prog. Biophys. Mol. Biol. 24, 1-74 Capaldi, R. A. and Green, D. E. (1972) Fehs Lett. 25, 205-209 Singer, S. J. and Nicolson, G. L. (1972) Science 175, 720-731 Steck, T. L. and Fox. C. F. (1972) in Membrane Molecular Biology (Fox, C. F. and Keith, A. D., eds.), pp. 27-75, Sinauer Associates, Inc., Stamford, Conn. Bretscher, M. S. (1973) Science 181,622-629 Chapman, D. (1973) in Biological Membranes (Chapman, D. and Wallach, D. F. H., eds.), pp. 91-144, Academic Press, New York Juliano, R. L. (1973) Biochim. Biophys. Acta 300, 341-378 Green, D. E., Ji, S. and Brucker, R. F. (1973) Bioenergetics 4, 253-284 Edidin, M. (1974) Annu. Rev. Biophys. Bioeng. 3, 179-201 Nicolson, G. L. (1973) Ser. Haematol. 6, 275-291 Singer, S. J. (1974) Annu. Rev. Biochem. 43, 805-833 Singer, S. J. (1974) Adv. Immunol. 19, 1-68 Steck, T. L. (1974) J. Cell Biol. 62, 1-19 Gorter, E. and Grendel, F. (1925) J. Exp. Med. 41,439-443 Danielli, J. F. and Davson, H. (1935) J. Cell. Comp. Physiol. 5, 495-508 Singer, S. J. (1971) in Structure and Function of Biological Membranes (Rothfield, L. I., ed.), pp. 145-222, Academic Press, New York

57 52 Tanford, C. (1973) The Hydrophobic Effect: Formation of Micelles and Biological Membranes, John Wiley, New York 53 Zwall, R. F. A., Roelofsen, B. and Colley, C. M. (1973) Biochim. Biopbys. Acta 300, 159-182 54 de Petris, S. and Raft, M. C. (1973) in Locomotion of Tissue Cells, Elsevier Excerpta Medica, North-Holland, Amsterdam C1BA Fdn. Syrup. 14, 27-52 55 Unanue, E. R., Perkins, W. D. and Karnovsky, M. J. (1972) J. Immunol. 108, 569-572 56 de Petris, S. and Raft, M. C. (1974) Eur. J. lmmunol. 4, 130--137 57 Unanue, E. R. and Karnovsky, M. J. 0973) Transplant. Rev. 14, 184-210 58 Karnovsky, M. J., Unanue, E. R. and Leventhal, M. (1972) J. Exp. Med. 136, 907-930 59 de Petris, S. and Raft, M. C. (1972) Eur. J. Immunol. 2, 523-535 60 Loor, F., Forni, L. and Pernis, B, (1972) Eur. J. Immunol. 2, 203-212 6l Unanue, E. R., Karnovsky, M. J. and Engers, I-t. D. (1973) J. Exp. Med. 137, 675-689 62 Yahara, I. and Edelman, G. M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 608-612 63 Edelman, G. M., Yahara, I. and Wang, J. L. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1442-1446 64 de Petris, S. (1974) Nature 250, 54--56 65 Poste, G., Papahadjopoulos, D., Jacobson, K. and Vail, W. J. (1975) Biochim. Biophys. Acta 394, 520-539 66 Ryan, G. B., Borysenko, J. Z. and Karnovsky, M. J. (1974) J. Cell BioL 62, 351-356 67 Poste, G., Papahadiopoulos, D. and Nicolson, G. L. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4430--4434 68 Yahara, 1. and Edelman, G. M. (1973) Nature 236, 152-155 69 Yahara, 1. and Edelman, G. M. (1973) Exp. Cell Res. 81, 143-155 70 Yahara, I. and Edelman, G. M. (1975) Exp. Cell Res. 91, 125-142 71 Allison, A. C. (1973) in Locomotion of Tissue Cells, Elsevier-North Holland, Amsterdam, CIBA Fdn. Symp. 14, 109-146 72 Durham, A. C. H. (1974) Cell 2, 123-136 73 Hynes, R. O. (1976) Biochim. Biophys. Acta, in the press 74 Boone, C. W. (1975) Science 188, 68-70 75 Nigam, V. N. and Cantero, A. (1972) Adv. Cancer Res. 16, 1-96 76 Barker, S. A., Stacey, M., Tipper, D. J. and Kirkham, J. H. (1959) Nature 184, BA68-BA69 77 Mabry, E. W. and Carubelli, R. (1972) Experientia 28, 182-183 78 Bryant, M. L., Stoner, G. D. and Metzger, R. P. (1974) Biochim. Biophys Acta 343, 226-231 79 Grimes, W. J. (1973) Biochemistry 12, 990-996 80 Perdue, J. F., Kletzien, R. and Wray, V. L. (1972) Biochim. Biophys. Acta 266, 505-510 81 Ohta, N., Pardee, A. B., McAuslan, B. R. and Burger, M. M. (1968) Biochim. Biophys. Acta 158, 98-102 82 Weiss, L. (1973) J. Natl. Cancer Inst. 50, 3-19 83 Makita, A. and Shimojo, H. (1973) Biochim. Biophys. Acta 304, 571-574 84 Satoh, C., Duff, R., Rapp, F. and Davidson, E. A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 54-56 85 Saito, H. and Uzman, B. G. (1971) Biochem. Biophys. Res. Commun. 43, 723-728 86 Roblin, R. O., Albert, S. O., Gelb, N. A. and Black, P. H. (1975) Biochemistry 14, 347--457 87 Kraemer, P. and Tobey, R. A. (1972) J. Cell Biol. 55, 713-717 88 Poste, G., Greenham, L. W., Mallucci, L., Reeve, P. and Alexander, D. J. (1973) Exp. Cell Res. 78, 303-313 89 Temin, H. M. (1965) J. Natl. Cancer Inst. 35, 679-693 90 Bosmann, H. B., Case, K. R. and Morgan, H. R. (1974) Exp. Cell Res. 83, 15-24 91 Bosmann, H. B., Lockwood, T. and Morgan, H. R. (1974) Exp. Cell Res. 83, 25-30 92 Terry, A. H. and Culp, L. A. (1974) Biochemistry 13,414--425 93 Chiarugi, V. P., Vannucchi, S. and Urbano, P. (1974) Biochim. Biophsy. Acta 345, 283-293 94 Hamerman, D., Todaro, G. J. and Green, H. (1956) Biochim. Biophsy. Acta 101, 343-351 95 lshirnoto, N., Temin, H. M. and Strominger, J. L. (1966) J. Biol. Chem. 241, 2052-2057 96 Kojima, J., Nakamura, N., Kanatanai, M. and Ohmori, K. (1975) Cancer Res. 35, 542-547 97 Buddecke, E. and Sittner, B. (1966) Z. Klin. Chem. 4, 157-163 98 Christensen Lou, H. O., Clausen, J. and Bierring, F. (1965) J. Neurochem. 12, 619-627 99 Haven, F. L. and Bloor, W. R. (1956) Adv. Cancer Res. 4, 237-243 100 Vlodavsky, I. and Sachs, L. (1974) Nature 250, 67-68 101 Inbar, M. and Shinitzky, M. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2128-2130

58 102 103 104 105 106 107 108 109 I10 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129

130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146

147 148 149

lnbar, M. and Shinitzky, M. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4229--4231 Quigley, J. P., Rifkin, D. B. and Reich, E. (1971) Virology 46, 106-116 Figard, P. H. and Levine, A. S. (1966) Biochim. Biophys. Acta 125, 428-434 Yau, T. M. and Weber, M. J. (1972) Biochem. Biophys. Res. Commun. 49, 114-120 Hakomori, S.-1., Kijimoto, S. and Siddiqui, B. (1972) in Membrane Research (C. F. Fox, ed.), pp. 253-277, Academic Press, Inc., New York Hakomori, S-I. (1973) Adv. Cancer Res. 18, 265-315 Brady, R. O. and Fishman, P. H. (1974) Biochim. Biophys. Acta 355, 121-148 Hakomori, S-I., Saito, T. and Vogt, P. K. (1971) Virology 44, 609-621 Hakomori, S-1. (1970) Proc. Natl. Acad. Sci. U.S. 67, 1741-1747 Gahmberg, C. G. and Hakomori, S.-i. (1975) J. Biol. Chem. 250, 2447-2451 Yogeeswaran, G., Sheinin, R., Wherrett, J. R. and Murray, R. K. (1972) J. Biol. Chem. 247, 5146-5158 Steiner, S., Brennan, P. J. and Melnick, J. L. (1973) Nat. New Biol. 245, 19-21 Gahmberg, C. G. and Hakomori, S-I. (1974) Biochem. Biophys. Res. Commun. 59, 283-291 Hakomori, S-I. (1975) Biochim. Biophys. Acta 417, 55-89 Patt, L. M. and Grimes, W. J. (1974) J. Biol. Chem. 249, 4157-4165 Den, H., Sela, B-A., Roseman, S. and Sachs, L. (1974) J. Biol. Chem. 249, 659-661 Fishman, P. H., Brady, R. O., Bradley, R. M., Aaronson, S. A. and Todaro, G. J. (I 974) Proc. Natl. Acad. Sci. U.S. 71,298-301 Yogeeswaran, G., Laine, R. A. and Hakomori, S. (1974) Biochem. Biophys. Res. Commun. 59, 591-599 Critchley, D. R. and MacPherson, 1. (1973) Biochim. Biophys. Acta 296, 145-159 Hynes, R. O. (1974) Cell 1, 147-156 Phillips, D. R. and Morrison, M. (1971) FEBS Lett. 18, 95-97 Steck, T. L. (1972) J. Mol. Biol. 66, 295-305 Steck, T. L. and Dawson, G. (1974) J. Biol. Chem. 249, 2135-2142 Maizel, J. V., Jr. (1971) Methods in Virology 5, 179-246 Stone, K. R., Smith, R. E. and Joklik, W. K. (1974) Virology 58, 86-100 Segrest, J. P. and Jackson, R. L, (1972) Methods Enzymol. 28, 54-63 Hynes, R. O. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3170-3174 Wickus, G. G., Branton, P. E. and Robbins, P. W. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 541-546, Cold Spring Harbor Laboratory, New York Robbins, P. W. (1975) Am. J. Clin. Pathol. 63, 671-676 Gahmberg, C. G. and Hakomori, S.-I. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3329-3333 Gahmberg, C. G., Kiehn, D. and Hakomori, S.-I. (1974) Nature 248, 413-415 Yamada, K. M. and Weston, J. A. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3492-3496 Hynes, R. O. and Bye, J. M. (1974) Cell 3, 113-120 Pearlstein, E. and Waterfield, M. D. (1974) Biochim. Biophys. Acta 362, 1-12 Hunt, R. C., Gold, E. and Brown, J. C. (1976) Biochim. Biophys. Acta 413, 453-458 Hynes, R. O. and Humphreys, K. C. (1974) J. Cell Biol. 62, 4 3 8 4 4 8 Poste, G. (1972) Exp. Cell Res. 73, 319-328 Poste, G. and Weiss, L. (1976) in Fundamental Aspects of Metastasis (Weiss, L., ed.), ASP Biological and Medical Press, B.V., Amsterdam Gahmberg, C. G., Kiehn, D. and Hakomori, S.-1. (1974) Nat. New Biol. 248,413-415 Hogg, N. M. (1974) Proc. Natl. Acad. Sci. U.S. 71,489-492 Wickus, G. G. and Robbins, P. W. (1973) Nat. New Biol. 245, 64-67 Vaheri, A. and Ruoslahti, E. (1974) Int. J. Cancer 13, 579-586 Bussell, R. H. and Robinson, W. S. (1973) J. Virol. 12, 320-327 Hynes, R. O. and Wyke, J. A. (1975) Virology 64, 492-504 Hynes, R. O., Wyke, J. A., Bye, J. M., Humphryes, K. C. and Pearlstein, E. S. ((1975) in Proteases and Biological Control (Reich, E., Shaw, E., and Rifkin, D. B., eds.), pp. 931-944, Cold Spring Harbor Laboratory, New York Robbins, P. W. (1975) Am. J. Clin. Pathol. 63, 671-676 Rieber, M. and Irwin, J. C. (1974) Cancer Res. 34, 3469-3473 Weston, J. A. and Hendricks, K. I_. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3727-3731

59 150 Graham, J. M., Hynes, R. O., Davidson, E. A. and Bainton, D. F. (1975) Cell 4, 353-365 151 Mallucci, L., Wells, V. and Young, M. R. (1975) submitted for publication 152 Vaheri, A., Ruoslahti, E., Linder, E., Wartiovaara, J., Keski-Oja, J., Kuusela, P. and Saksela, O. (1976) J. Supramol. Struct., in the press 153 Keski-Oja, J., Vaheri, A. and Ruoslahti, E. (1976) Int. J. Cancer, in the press 154 Wartiovaara, J., Linder, E., Ruoslahti, E. and Vaheri, A. (1974) J. Exp. Med. 140, 1522-1533 155 Ruoslahti, E., Vaheri, A., Kuusela, P. and Linder, E. (1973) Biochim. Biophys. Acta 322, 352-358 156 Ruoslahti, E. and Vaheri, A. (1974) Nature 248,789-791 157 Vaheri, A. (1975) personal communication 158 Vaheri, A., Ruoslahti, E., Westermark, B. and Pont~n, J. (1976) J. Exp. Med. 143, 64-72 159 Critchley, D. R. (1974) Cell 3, 121-125 160 Kuusela, P., Ruoslahti, E. and Vaheri, A. (1975) Biochim. Biophys. Acta 379, 295-303 161 Warren, L., Fuhrer, J. P. and Buck, C. A. (1973) Fed. Proc. 32, 80-85 162 Glick, M. C., Rabinowitz, Z. and Sachs, L. (1974) J. Virol. 13, 967-974 163 Buck, C. A., Glick, M. C. and Warren. L. (1970) Biochemistry 9, 4567-4576 164 Sakiyama, H. and Burge, B. W. (1972) Biochemistry I1, 1366-1377 165 Meezen, E., Wu, H. C., Black, P. H. and Robbins, P. W. (1969) Biochemistry 8, 2518-2524 166 Lai, M. M. and Duesberg, P. H. (1972) Virology 50, 359-372 167 Warren, L., Critchley, D. and MacPberson, I. (1972) Nature 235, 275-278 168 Van Beck, W. P., Smets, L. A. and Emmelot, P. (1973) Cancer Res. 33, 2913-2922 169 Warren, L., Fuhrer, J. P. and Buck, C. A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1838-1843 170 Glick, M. C., Rabinowitz, Z. and Sachs, L. (1973) Biochemistry 12, 4864-4869 171 Voyles, B. A. and Moskowitz, M. (1974) Biochim. Biophys. Acta 351, 178-181 172 Chiarugi, V. P. and Urbano, P. (1972) J. Gen. Virol. 14, 133-140 173 De Long, R. P., Coman, D. R. and Zeidman, 1. (1950) Cancer 3, 718-721 174 Zeidman, I. (1957) Cancer Res. 17, 157-162 175 Bosmann, H. B. and Hall, T. C. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1833-1837 176 Kijimoto, S. and Hakomori, S.-I. (1971) Biochem. Biophys. Res. Commun. 44, 557-563 177 Bosmann, H. B. (1972) Biochim. Biophys. Acta 264, 339-343 178 Schengrund, C.-L., Lausch, R. N. and Rosenberg, A. (1973) J. Biol. Chem. 248, 4424-4428 179 Cumar, F. A., Brady, R. O., Kolodny, E. H., McFarland, V. W, and Mora, P. T. (1970) Proc. Natl. Acad. Sci. U.S. 67, 757-764 180 Schnebli, H. P. (1972)Schweiz. Med. Wochenschr. 102, 1194-1197 181 Sylv6n, B. and Bois-Svensson, I. (1965) Cancer Res, 25, 458-468 182 Sylv6n, B., Snellman, O. and Str/iuli, P. (1974) Virchows Arch. B. Cell Path. 17, 97-112 183 Lipkin, G. and Knecht, M. E. (1974) Proc. Natl. Acad. Sci. U.S. 71. 849-853 184 Sylv6n, B. and Malmgren, H. (1957) Acta Radiol. (suppl) 154, I-124 185 Grabowska, M. (1959) Nature 183, 1186-1187 186 Robertson, D. M. and Williams, D. C. (1969) Nature 221,259-260 187 Taylor, A. C., Levy, B. M. and Simpson, J. W. (1970) Nature 228, 366-367 188 Riley, W. B., Jr. and Peacock, E. E., Jr. (1967) Proc. Soc. Exp. Biol. Med. 124, 207-210 189 Dresden, M. H., Heilman, S. A. and Schmidt, J. D. (1972) Cancer Res. 32, 993-996 190 Hashimoto, K., Yamanishi, Y., Maeyens, E., Dabbous, M. K. and Kanzaki, T. (1973) Cancer Res. 33, 2790-2801 191 Yamanishi, T., Maeyens, E., Dabbous, M. K., Ohyama, H. and Hashimoto, K. (1973) Cancer Res. 33, 2507-2512 192 Strauch, L. (1972) in Tissue ! nteracti ons in Carcinogenesis (Tarin, D., ed.), pp. 399-434, Academic Press, New York 193 Unkeless, J., Dano, K., Kellerman, G. M. and Reich, E. (1974) J. Biol. Chem. 249, 4295-4305 194 Christman, J. K. and Acs, G. (1974) Biochim. Biophys. Acta 340, 339-347 195 Ossowski, L., Unkeless, J. C., Tob. ia, A., Quigley, J. P., Rifkin, D. B. and Reich, E. (1973) J. Exp. Med. 137, 113-126 196 Unkeless, J. C., Tobia, A., Ossowski, L., Quigley, J. P., Rifkin, D. B. and Reich, E. (1973) 3. Exp. Med. 137, 85-111 197 Weber, M. J., llale, A. H. and Roll, D. E. (1975) in Proteases in Biological Control (E. Shaw, E. Reich, D. Rifkin, eds.) pp. 925-930, Cold Spring Harbor Laboratory, New York

60 198 199 200 20l 202 203 204 205

Goldberg, A. R. (1974) Cell 2, 95-102 Rifkin, D. B., Loeb, J. N., Moore, G. and Reich, E. (1974) J. Exp. Med. 139, 1317-1328 Chen, L. B. and Buchanan, J. M. (1975) Proc. Natl. Acad. Sci. U.S. 72. 1132-1136 Ossowski, L., Quigley, J. P. and Reich, E. (1974) J. Biol. Chem. 249, 4312-4320 Chou, I.-N., Black, P. H. and Roblin, R. O. (1974) Nature 250, 739-741 Quigley, J, P., Ossowski, L. and Reich, E. (1974) J. Biol. Chem. 249, 4306-4311 Ossowski, L., Quigley, J. P., Kellerman, G. M. and Reich, E. (1973) J. Exp. Med. 138, 1056-1064 Nicolson, G. L., Winkelhake, J. L. and Nussey, A. C. (1976) in Fundamental Aspects of Metastasis (Weiss, L., ed.), pp. 307-319, ASP Biological and Medical Press, B.V., Amsterdam, The Netherlands 206 Fidler, I. J. (1973) Nat. New Biol. 242, 148-149 206a Fidler, I. J. and Nicolson, G. L. (1976) J. Natl. Cancer Inst., in the press 207 Bosmann, H. B., Bieber, G. F., Brown, A. E., Case, K. R., Gersten, D. M., Kimmerer, T. W. and Lione, A. (1973) Nature 246, 487-489 208 Sefton, B. M. and Rubin, H. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3154-3157 209 Kletzien, R. F. and Perdue, J. F. (1974) J. Biol. Chem. 249, 3383-3387 210 Rosenblith, J. Z., Ukena, T. E., Yin, H. H., Berlin, R. D. and Karnovsky, M. J, (1973) Proc. Natl. Acad. Sci. U.S. 70, 1625-1629 211 Nicolson, G. L. (1972) Nat. New Biol. 239, 193-197 212 Burger, M. M. (1971) Nature 231, 125-126 213 Hakomori, S.-I., Teather, C. and Andrews, H. (1968) Biochem. Biophys. Res. Commun 33, 563-568 214 Burger, M. M. (1970) Nature 227, 170--171 215 Sefton, B. M. and Rubin, H. (1970) Nature 227, 843-845 216 Poste, G. (1975) in Concanavalin A (Chowdhury, T. K. and Weiss, A. K., eds.), Plenum Press, New York, Adv. Exp. Med. Biol. 55, 153-172 217 Dorsey, J. K. and Roth, S. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 533-539, Cold Spring Harbor Laboratory, New York 218 Hsu, A.-F., Baynes, J. W. and Heath, E. C. (1974) Proc. Natl. Acad. Sci. U.S. 71,2391-2395 219 Pernis, B., Forni, L. and Amante, L. (1971) Ann. N.Y. Acad. Sci. 190, 420-431 220 Parkhouse, R. M. E. (1973) Transplant. Rev. 14, 131-145 221 Melchers, F. and Andersson, J. (1973) Transplant. Rev. 14, 76-130 222 Vitetta, E. S. and Uhr. J. W. (1973) Transplant. Rev. 14, 50-75 223 Melchers, F. and Knopf, P. M. (1967) Cold Spring Harb. Syrup. Quant. Biol. 32, 255-262 224 Knopf, P. M. (1973) Transplant. Rev. 14, 146-162 225 Nicolson, G. L. and Singer, S. J. (1974) J. Cell Biol. 60, 236-248 226 Hirano, H., Parkhouse, B., Nicolson, G. L., Lennox, E. S. and Singer, S. J. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2945-2949 227 Bennett, G., Leblond, C. P. and Haddad, A. (1974) J. Cell Biol. 60, 258-284 228 Roseman, S. (1970) Chem. Phys. Lipids 5, 270-300 229 Roth, S. (1973) Q. Rev. Biol. 48, 541-563 230 Deppert, W., Werchau, H. and Walter, G. (1974) Proc. Natl. Acad. Sci. U.S. 71,3068-3072 231 Bosmann, H. B. (1972)Biochem. Biophys. Res. Commun. 49, 1256-1262 232 Den, H., Schultz, A. M., Basu, M. and Roseman, S. (1971) J. Biol. Chem. 246, 2721-2723 233 Roth, S. and White, D. (1972) Proc. Natl, Acad. Sci. U.S. 69, 485-489 234 Steiner, S., Courtney, R. J. and Melnick, J. L. (1973) Cancer 33, 2402-2407 235 Hatanaka, M. (1974) Biochim. Biophys. Acta 355, 77-104 236 Foster, D. O. and Pardee, A. B. (1969) J. Biol. Chem. 244, 2675-2681 237 Isselbacher, K. J. (1972) Proc. Natl. Acad. Sci. U.S. 69, 585-589 238 Cunningham, D. D. and Pardee, A. B. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1049-1056 239 Vaheri, A., Rouslahti, E. and Nordling, S. (1972) Nature (London) 238, 211-212 240 Rozengurt, E. and Jim6nez de Asfa, L. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3609-3612 241 Seifert, W. and Rudland, P. S. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4920~924 242 Rudland, P. S., Gospodarowicz, D. and Seifert, W. (1974) Nature 250, 741-742, 773-774 243 Abell, C. W. and Monahan, T. M. (1973) J. Cell Biol. 59, 549-558 244 Bannai, S. and Sheppard, J. R. (1974) Nature 250, 62-64 245 Pardee, A., Jim6nez de Asfia, L. and Rozengurt, E. (1974) in Control of Proliferation in Animal

61

246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 26:l 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290

Cells (Clarkson, B. and Baserga, R. eds.), pp. 547-562, Cold Spring Harbor Laboratory, New York Pastan, 1. and Johnson, G. S. (1974) Adv. Cancer Res. 19, 303-330 Grimes, W. J. and Schroeder, J. L. (1973) J. Cell Biol. 56, 487-491 Kletzien, R. F. and Perdue, J. F. (1974) J. Biol. Chem. 249, 3375-3382 Jim6nez de Asfla, L., Rozengurt, E. and Dulbecco, R. (1974) Proc. Natl. Acad. Sci. U.S. 71, 96-98 Paul, D. (1973) Biochem. Biophys. Res. Commun. 53, 745-753 Holley, R. W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2840-2841 Holley, R. W. (1974) in Control of Proliferation in Animal Cells (B. Clarkson and R. Baserga, eds.), pp. 13-18, Cold Spring Harbor Laboratory, New York Hale, A. H., Winkelhake, J. L. and Weber, M. J. (1975) J. Cell Biol. 64, 398-407 Ambrose, E. J., James, A. M. and Lowick, J. H. B. (1956) Nature 177, 567-577 Lowick, J. H. B., Purdom, L., James, A. M. and Ambrose, E. J. (1961) J. R. Microscop. Soc. 80, 47-57 Simon-Reuss, 1., Cook, G. M. W., Seaman, G. V. F. and Heard, D. H. (1964) Cancer Res. 24 2038-2043 Doljanski, F. and Eisenberg, S. (1963) in Cell Electrophoresis (Ambrose, E. J., ed.), pp. 78-84, Churchill, Ltd., London Mehrishi, J. N. and Thomson, A. E. R. (1968) Nature 219, 1080-1081 Thomson, A. E. R. and Mehrishi, H. N. (1969) Eur. J. Cancer 5, 195-206 Schubert, J. C. F., Walther, F., Holzberg, E., Pascher, G. and Zeiller, K. (1973) Klin. Wochenschr. 51,327-332 Vassar, P. S. (1963) Lab. Invest. 12, 1072-1077 Patinkin, D., Schlesinger, M. and Doljanski, R. (1970) Cancer Res. 30, 489-497 Lichtman, M. A. and Weed, R. I (1970) Blood 35, 12-22 Martinez-Palomo, A. (1970) Int. Rev. Cytol. 29, 29-76 Defendi, V. and Gasic, G. (1963) J. Cell Comp. Physiol. 62, 23-26 Martinez-Palomo, A., Braislovsky, C. and Bernhard, W. (1969) Cancer Res. 29, 925-937 Morgan, H. R. (1968) J. Virol 2, 1133-1146 Shigematsu, T. and Domochowski, L. (1973) Cancer 31, 165-174 Vorbrodt, A. and Koprowski, H. (1969) J. Natl. Cancer Inst. 43, 1241-1244 Dermer, G. B., Lue, J. and Neustein, H. B. (1974) Cancer Res. 34, 31-38 Mallucci, L., Poste, G. and Wells, V. (1972) Nat. New Biol. 235, 222-223 Poste, G. (1973) Exp. Cell Res. 77, 264-270 McNutt, N. S. and Weinstein, R. S. (1967) Science 156, 597-599 McNutt, N. S., Culp, L. A. and Black, P. H. (1971) J. Cell Biol. 50, 691-708 Perdue, J. F. (1973) J. Cell Biol. 58,265-283 McNutt, N. S., Culp, L. A. and Black, P. H. (1973) J. Cell Biol. 56, 412-428 Pinto da Silva, P. and Gilula, N. B. (1972) Exp. Cell Res. 71,393-401 Pinto da Silva, P. and Martinez-Palomo, A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 572-576 Scott, R. E., Furcht, L. T. and Kersey, J. H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3631-3635 Barnett, R. E., Furcht, L. T. and Scott, R. E. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1992-1994 Mclntyre, J. A., Gilula, N. B. and Karnovsky, M. J. (1974) J. Cell Biol. 60, 192-203 Torpier, G., Montagnier, L., Biquard, J.-M. and Vigier, P. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1695-1698 Pollack, R., Osborn, M. and Weber, K. (1975) Proc. Natl. Acad Sci. U.S. 72, 994-998 Wickus, G., Gruenstein, E., Robbins, P. W. and Rich, A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 746-749 Abercrombie, M. (1965) in Cells and Tissues in Culture (Willmer, E. N., ed.), Vol. 1, pp. 177-202, Academic Press, New York Harris, A. (1974) in Cell Communication (R. P. Cox, ed.), pp. 147-185, John Wiley & Sons, New York Abercrombie, M. and Heaysman, J. E. M. (1953) Exp. Cell Res. 5, 111-131 Abercrombie, M. and Heaysman, J. E. M. (1954) Exp. Cell Res. 6, 293-306 Abercrombie, M. and Ambrose, E. J. (1958) Exp. Cell Res. 15, 332-345 Abercrombie, M., Heaysman, J. E. M. and Karthauser, H. M. (1957) Exp. Cell Res. 13,276-291

62 291 292 293 294 295 296 297 298 299

Coman, D. R. (1961) Cancer Res. 21, 1436-1438 Harris, A. (1973) Develop. Biol. 35, 97-114 Vesel~, P. and Weiss, R. A. (1973) Int. J. Cancer 11, 64--76 Curtis, A. S. G. and Varde, M. (1964) J. Natl. Cancer Inst. 33, 15-26 Furshpan, E. J. and Potter, D. D. (1968) Current Topics in Develop. Biol. 3, 95-127 Trinkhaus, J. P., Betchaku, T. and Krulikowski, L. S. (1971) Exp. Cell Res. 64, 291-300 Curtis, A. S. G. (1964) J. Cell Biol. 20, 199-215 Macieira-Coelho, A. (1967) Exp. Cell Res. 47, 193-200 Lipton, A., Klinger, I., Paul, D. and Holley, R. W. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2799-2801 300 Paul, D., Lipton, A. and Klinger, 1. (1971) Proc. Natl. Acad. Sci. U.S. 68, 645-648 301 Holley, R. W. (1975) Nature 258, 487-490 302 Todaro, G. J., Lazar, G. K. and Green, H. (1965) J. Cell Comp. Physiol. 66, 325-334 303 Temin, H. M. (1966) J. Natl. Cancer Inst. 37, 167-175 304 Oppenheimer, S. B., Edidin, M., Orr, C. W. and Roseman, S. (1969) Proc. Natl. Acad. Sci. U.S. 63, 1395-1402 305 Oppenheimer, S. B. (1973) Exp. Cell Res. 77, 175-182 306 Chipowsky, S., Lee, Y. C. and Roseman, S. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2309-2312 307 Coman, D. R. (1944) Cancer Res. 4, 625-629 308 Dorsey, J. K. and Roth, S. (1973) Develop. Biol. 33, 249-256 309 Roth, S., McGuire, E. J. and Roseman, S. (1971) J. Cell Biol. 51, 525-535 310 Walther, B. T., Ohman, R. and Roseman, S. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1569-1573 311 Curtis, A. S. G. and Greaves, M. F. (1965) J. Embryol. Exp. Morph. 13, 309-326 312 Weiss, L. (1958) Exp. Cell Res. 14, 80-83 313 Fidler, 1. J. (1975) Cancer Res. 35, 218-224 314 Gasic, G. J., Gasic, T. B., Galanti, N., Johnson, T. and Murphy, S. (1973) Int. J. Cancer I1, 704-718 315 Nicolson, G. L. and Winkelhake, J. L. (1975) in Cell Surfaces and Malignancy (Mora, P., ed.), Fogarty International Center Proc. 28, U.S. Government Printing Office, Washington, D.C., in the press 316 Nicolson, G. L. and Smith, J., in preparation 316a Nicolson, G. L., Birdwell, C. R., Brunson, K. W. and Robbins, J. C. (1976) J. Supramol. Struct., in the press 317 Nicolson, G. L. (1974) in Control of Proliferation in Animal Cell Surfaces (Clarkson, B. and Baserga, R., eds.), pp. 251-270, Cold Spring Harbor Laboratory, New York 318 Burger, M. M. (1973) Fed. Proc. 32, 91-101 319 Aub, J. C., Sanford, B. H. and Cote, M. N. (1965) Proc. Natl. Acad. Sci. U.S. 54, 396-399 320 Aub, J. C., Sanford, B. H. and Wang, L. (1965) Proc. Natl. Acad. Sci. U.S. 54, 400-402 321 Burger, M. M. and Goldberg, A. R. (1967) Proc. Natl. Acad. Sci. U.S. 57, 359-366 322 Pollack, R. E. and Burger, M. M. (1969) Proc. Natl. Acad. Sci. U.S. 62, 1074-1076 323 Burger, M. M. (1969) Proc. Natl. Acad. Sci. U.S. 62, 994-1001 324 Burger, M. M. (1970) in Permeability and Function in Biological Membranes (Bolis, L., Katchalsky, A., Keynes, R. and Loewenstein, W., eds.), pp. 107-120, North Holland, Amsterdam 325 Benjamin, T. L. and Burger, M. M. (1970) Proc. Natl. Acad. Sci. U.S. 67, 929-934 326 Ben-Bassat, H., lnbar, M. and Sachs, L. (1970) Virology 40, 854-859 327 Sheppard, J. R., Levine, A. J. and Burger, M. M. (1971) Science 172, 1345-1346 328 Inbar, M. and Sachs, L. (1969) Proc. Natl. Acad. Sci. U.S. 63, 1418-1425 329 Lehman, J. M. and Sheppard, J. R. (1972) Virology 49, 339-341 330 Salzberg, S. and Green, M. (1974) J. Virol. 13, 1001-1004 331 Chaudhuri, S., Koprowska, 1. and Rowinski, J. (1975) Cancer Res. 35, 2350-2354 332 Sivak, A. and Wolman, S. R. (1972) In Vitro 8, 1-5 333 Gantt, R. R., Martin, J. R. and Evans, V. J. (1969) J. Natl. Cancer Inst. 42, 369-373 334 Berman, L. D. (1975) Int. J. Cancer 15, 973-979 335 Glimelius, B., Westermark, B. and Pont6n, J. (1974) Int. J. Cancer 14, 314-325 336 Glimelius, B., Nilsson, K. and Pont6n, J. (1975) Int. J. Cancer 15, 888-896 337 Van Nest, G. A. and Grimes, W. J. (1974) Cancer Res. 34, 1408-1412 338 Burger, M. M. and Martin, G. S. (1972) Nat. New Biol. 237, 9-12

63 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389

Rabinowitz, Z. and Sachs, L. (1969) Virology 38, 336-342 Rabinowitz, Z. and Sachs, L. (1969) Virology 38, 343-346 Inbar, M., Rabinowitz, Z. and Sachs, L. (1969) Int. J. Cancer 4, 690-696 Pollack, R., Wolman, S. and Vogel, A. (1970) Nature 228, 938-970 Culp, L. A., Grimes, W. J. and Black, P. H. (1971) J. Cell Biol. 50, 682-690 Ozanne, B. and Sambrook, J. (1971) in Biology of Oncogenic Viruses (Silvestri, L., ed.), pp. 248-257, North Holland, Amsterdam Culp, L. A. and Black, P. H. (1972) J. Virol. 9, 611-620 Ozanne, B. (1973) J. Virol. 12, 79-89 Wright, J. A. (1973) J. Cell Biol. 56, 666-675 Aaronson, S. A. and Todaro, G. J. (1968) J. Cell. Physiol. 72, 141-148 Weber, 3. (1973) J. Cell. Physiol. 81, 49-54 Inbar, M., Ben-Bassat, H. and Sachs, L. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2748-2751 De Micco, Ph. and Berebbi, M. (1972) Int. J. Cancer 10, 249-253 Hozumi, M. Miyake, S., Mizunoe, F., Sugimura, T., Irie, R. F., Koyama, K., Tomita, M. and Ukita, T. (1972) Int. J. Cancer 9, 393-401 Smets, L. A. and Broekhuysen-Davies, J. (1972) Eur. J. Cancer 8, 541-548 Dent, P. B. and Hillcoat, B. L. (1972) J. Natl. Cancer Inst. 49, 373-377 Nicolson, G. L. (1975) in Concanavalin A (Chowdhury, T. K. and Weiss, A. K., eds.), Plenum Press, New York, Adv. Exp. Med. Biol. 55, 153-172 Trowbridge, 1. S. and Hilborn, D. A. (1974) Nature 250, 304-307 Ozanne, B. and Sambrook, J. (1971) Nat. New Biol. 232, 156-160 Cline, M. J. and Livingston, D. C. (1971) Nat. New Biol. 232, 155-156 Sela, B., Lis, H., Sharon, N. and Sachs, L. (1971) Biochim. Biophys. Acta 249, 564-568 Ben-Bassat, H., lnbar, M. and Sachs, L. (1971) J. Memb. Biol. 6, 183-194 Arndt-Jovin, D. J. and Berg, P. (1971) J. Virol. 8,716-721 Inbar, M., Ben-Bassat, H. and Sachs, L. (1971) J. Memb. Biol. 6, 195-209 Phillips, P. G., Furmanski, P. and Lubin, M. (1974) Exp. Cell Res. 86, 301-308 Nicolson, G. L. (1973) J. Natl. Cancer Inst. 50, 1443-1451 Nicolson, G. L. and Lacorbiere, M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1672-1676 Barbarese, E., Sauerwein, H. and Simkins, H. (1973) J. Memb. Biol. 13, 129-142 Nicolson, G. L., Lacorbiere, M. and Hunter, T. R. (1975) Cancer Res. 35, 144-155 Nicolson, G. L., Lacorbiere, M. and Eckhart, W. (1975) Biochemistry 14, 172-179 Noonon, K. D. and Burger, M. M. (1973) J. Biol. Chem. 248,428~4292 Noonon, K. D. and Burger, M. M. (1973) J. Cell Biol. 59, 134-142 Noonan, K. D., Renger, H. C., Basilico, C. and Burger, M. M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 347-349 Oliver, J. M., Ukena, T. E. and Berlin, R. D. (1974) Proc. Natl. Acad. Sci. U.S. 71,394-398 Edelson, P. J. and Cohn, Z. A. (1974) J. Exp. Med. 140, 1364-1386 Nicolson, G. L. (1974) Nature 251, 628-630 Nicolson, G. L. (1971) Nat. New Biol. 233, 244-246 Martinez-Palomo, A., Wicker, R. and Bernhard, W. (1972) Int. J. Cancer 9, 676-683 Bretton, R., Wicker, R. and Bernhard, W. (1972) Int. J. Cancer 10, 397-409 Comoglio, P. M. and Guglielmone, R. (1972) Febs. Lett. 27, 256-258 Inbar, M. and Sachs, L. (1973) Febs. Lett. 32, 124-128 Inbar, M., Ben-Bassat, H. and Sachs, L. (1973) Int. J. Cancer 12, 93-99 Nicolson, G. L. (1973) Nat. New Biol. 243,218-220 Comoglio, P. M., Guglielmone, R. and Forni, G. (1971) Giron. Acad. Med. 134, 1-6 lnbar, M., Ben-Bassat, H., Sachs, L., Huet, Ch. and Oseroff, A. R. (1973) Biochim. Biophys. Acta 31 I, 594-599 Sachs, L., lnbar, M. and Shinitzky, M. (1974) in Control of Proliferation in Animal Cells (B. Clarkson and R. Baserga, eds.), pp. 283-296, Cold Spring Harbor Laboratory, New York Roth, J. and Thoss, K. (1974) Experientia 30, 414 Wagner, M. and Wagner, B. (1973) Z. lmmun. Forsch. 146, 274-282 Klein, P. A. and Adams, W. R. (1972) J. Virol. 10, 844-854 Huet, Ch. and Bernhard, W. (1974) Int. J. Cancer 13, 227-239 Roth, J. (1974) Int. J. Cancer 14, 762-770

64 390 Roth, J., Neupert, G. and Bolck, F. (1975) Exp. Path. 10, 143-155 391 Rowlatt, C., Wicker, R. and Bernhard, W. (1973) Int. J. Cancer 11,314-326 392 Garrido, J., Burglen, M.-J., Samolyk, D., Wicker, R. and Bernhard, W. (1974) Cancer Res. 34, 230-243 393 Roth, J., Wagner, M. and Meyer, H. W. (1974) Path. Eur, 9, 37-42 394 Inbar, M., Ben-Bassat, H., Fibach, E. and Sachs, L. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2577-2581 395 Comoglio, P. M. and Filogamo, G. (1973) J. Cell Sci. 13,415-420 396 Huet, Ch. and Bernadac, A. (1975) Biochim. Biophys. Acta 394, 605~19 397 Roth, J. submitted 398 de Petris, S. and Raft, M. C. (1973) Nat. New Biol. 241,257-259 399 Stackpole, C. W., DeMilio, L. T., Jacobson, J. B., Hfimmerling, U. and Lardis, M. P. (1974) J. Cell. Physiol. 83, 441-448 400 de Petris, S., Raft, M. C. and Mallucci, L. (1973) Nat. New Biol. 244, 275-278 401 Raft, M. C., de Petris, S. and Mallucci, L. (1974) in Control of Proliferation in Animal Cell Surfaces (Clarkson, B. and Baserga, R., eds.), pp. 271-282, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 402 Huet, Ch., Lonchampt, M., Huet, M. and Bernadac, A. (1974) Biochim. Biophys. Acta 365, 28-39 403 Cunningham, B. A., Wang, J. L., Pflumm, M. M. and Edelman, G. M. (1972) Biochemistry I I, 3233-3239 404 Huet, Ch. (1976) J. Microscopic (Paris), in press 405 Torpier, G. and Montagnier, L. (1973) Int. J. Cancer 11,604-615 406 Smith, S. B. and Revel, J.-P. (1972) Develop. Biol. 27, 434-441 407 Collard, J. G. and Temmink, J. H. M. (1974) Exp. Cell Res. 86, 81-86 408 Roth, J., Meyer, H. W., Neupert, H. and Bolck, F. (1973) Exp. Pathol. 8, 19-30 409 Bretton, R., Ternynck, T. and Avrameas, S. (1972) Exp. Cell Res. 71, 145-155 410 Temmink, J. H. M., Collard, J. G., Spits, H. and Roos, E. (1975) Exp. Cell Res. 92, 307-322 411 Nicolson, G. L. (1976) in Advanced Techniques in. Biological Electron Microscopy (Koehler, J. K., ed.), Vol. 2, Springer-Verlag, New York, in the press 412 Henkart, P. A. and Fisher, R. 1. (1975) J. lmmunol. 114, 710-714 413 Cuatrecasas, P. (1973) Biochemistry 12, 1312-1323 414 Kapeller, M., Gal-Oz, R., Grover, N. B. and Doljanski, F. (1973) Exp. Cell Res. 79, 152-158 415 Nicolson, G. L. (1972) in Membrane Research (C. F. Fox, ed,), pp. 53-70, Academic Press, New York 416 Huet, Ch. and Herzberg, M. (1973) J. Ultrastruc. Res. 42, 186-199 417 Pollack, W. (1965) Ann. N. Y. Acad. Sci. 127, 892-900 418 Pollack, W., Hager, H. J., Reckel, R., Toren, D. A. and Singher, H. O. (1965) Transfusion 5, 158-183 419 Mehrishi, J. N. (1972) Prog. Biophys. Mol. Biol. 25, 1-70 420 Yamada, T. and Yamada, M. (1973) Nature 244, 297-299 421 Nicolson, G. L. (1973) J. Cell Biol. 57, 383-387 422 Zarling, J. and Tevethia, S. (1971) Virology 45, 313-316 423 Poste, G. and Reeve, P. (1972) Nat. New Biol. 237, 113-114 424 Becht, H., Rott, R. and Klenk, H. D. (1972) J. Gen. Virol. 14, 1-8 425 Birdwell, C. R. and Strauss, J. H. (1973) J. Virol. 1 I, 502-507 426 Poste, G. and Reeve, P. (1974) Nature 247, 469-471 427 Reeve, P., Poste, G. and Alexander, D. J. (1975) in Negative Strand Viruses (Barry, R., and Mahy, W. J., eds.), Academic Press, New York, in the press 428 Shinitzky, M., Inbar, M. and Sachs, L. (1973) FEBS Lett. 34, 247-250 429 Inbar, M., Shinitzky, M. and Sachs, L. (1973) J. Mol. Biol. 81, 245-253 430 Rutishauser, U. and Sachs, L. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2456-2460 431 Rutishauser, U. and Sachs, L. (1975) J. Cell Biol. 65, 247-257 432 Noonan, K. D. and Burger, M. M. (1970) Nature 228, 512-515 433 Steinberg, M. S. and Gipner, 1 A. (1973) Nat. New Biol. 241,249-251 434 Porter, R. R. (1959) Biochem. J. 73, 119-126 435 Nicolson, G. L. (1973) unpublished observation

65 436 Daniel, A. and Stenberg, M. personal communication 437 Gunther, G. R., Wang, J. L., Yahara, 1., Cunningham, B. A. and Edelman, G. M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1012-1016 438 Lotan, R., Lis, H., Rosenwasser, A., Novogrodsky, A. and Sharon, N. (1973) Biochem. Biophys. Res. Commun. 1347-1355 439 Gordon, J. A. and Marquardt, M. D. (1974) Biochim. Biophys. Acta 332, 136-144 440 Rittenhouse, H. G., Williams, R. E., Wisnieski, B. and Fox, C. F. (1974) Biochem. Biophys. Res. Commun. 58, 222-228 441 Hoyer, L. W. and Trabold, N. C. (1970) J. Clin. Invest. 49, 87-95 442 Inoue, M. (1974) J. Cell Sci. 14, 197-202 443 Vlodavsky, I. and Sachs, L. (1975) Exp. Cell Res. 93, 111-119 444 Schnebli, H. P. and B/ichi, T. (1975) Exp. Cell Res. 91,175-183 445 Novogrodsky, A. and Katchalski, E. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2515-2518 446 Pardoe, G. 1., Uhlenbruck, G. and Bird, G. W. G. (1970) Immunology 18, 73-83 447 Hughes, R. C., Palmer, P. D. and Sanford, B. H. (1973) J. Immunol. !11, 1071-1080 448 Berlin, R. D. (1975) in Concanavalin A (Chowdhury, T. K. and Weiss, A. K., eds.), Plenum Press, New York, Adv. Exp. Med. Biol. 55, 173-186 449 Gu6rin, C., Zachowski, A., Prigent, B., Paraf, A., Dunia, 1., Diawara, M.-A. and Benedetti, E. L. (1974) Proc. Natl. Acad. Sci. U.S. 71, !14-117 450 Garrido, J. (1975) Exp. Cell Res. 94, 159-175 451 Edidin, M. and Weiss, A. (1974) In Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 213-220, Cold Spring Harbor Laboratory, New York 452 Frye, L. D. and Edidin, M. (1970) J. Cell Sci. 7, 319-335 453 Rutishauser, U. and Sachs, L. (1975) J. Biol. Chem. 66, 76-85 454 Rutishauser, U., Yahara, 1. and Edelman, G. M. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1149-1153 455 Yin, H. H., Ukena, T. E. and Berlin, R. D. (1972) Science 178, 867-868 456 O'Neill, C. H. (1968) J. Cell Sci. 3, 405-422 457 Weiss, L. (1967) J. Cell Biol. 33, 341-347 458 Weiss, L. and Subjeck, J. R. (1974) Int. J. Cancer 13, 143-150 459 Weiss, L. and Subjeck, J. R. (1974) J. Cell Sci. 14, 215-223 460 Grinnell, F., Tobleman, M. Q. and Hackenbrock, C. R. (1975) J. Cell Biol. 66, 470-479 461 Willingham, M. C. and Pastan, I. (1974) J. Cell Biol. 63, 288-294 462 Willingham, M. C. and Pastan, 1. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1263-1267 463 Hsie, A. W., Jones, C. and Puck, T. T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1648-1652 464 Hsie, A. W. and Puck, T. T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 358-361 465 Johnson, G. S., D'Armiento, M. and Carchman, R. A. (1974) Exp. Cell Res. 85, 47-56 466 Sheppard, J. R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1316-1320 467 Puck, T. T., Waldren, C. A. and Hsie, A. W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1943-1947 468 Yin, H. H. and Berlin, R. D. (1975) J. Cell. Physiol. 85, 627-634 469 Collard, J. G., Temmink, J. M. H. and Smets, L. A. (1975) in Concanavalin A (Chowdhury, T. K. and Weiss, A. K., eds.), Plenum Press, New York, Adv. Exp. Med. Biol. 55, 221-244 470 Collard, J. G. and Temmink, J. H. M. (1975) J. Cell Sci. 19, 21-32 470a Collard, J. G. and Temmink, J. H. M. (1976) J. Cell Biol. 68, 101-112 471 Loor, F. (1973) Exp. Cell Res. 82, 415-425 472 Loor, F. and H~gg, L.-B. (1975) Eur. J. Immunol. 5, 854-865 473 O'Neill, C. H. and Burnett, M. E. (1974) Exp. Cell Res. 83, 247-254 474 Steiner, S., Altenbrug, B. and Melnick, J. L. (1974) J. Natl. Cancer. Inst. 52, 617-620 475 Nicolson, G. L. (1973) J. Supramol. Struct. 1,410-416 476 Nicolson, G. L. and Painter, R. G. (1973) J. Cell Biol. 59, 395-406 477 Elgsaeter, A, and Branton, D. (1974) J. Cell Biol. 63, 1018-1036 478 Tilney, L. G. (1975) J. Cell. Biol. 66, 508-520 479 Weisenberg, R. C., Borisy, G. G. and Taylor, E. W. (1968) Biochemistry 7, 4466-4479 480 Wilson, L. (1970) Biochemistry 9, 4999-5007 481 Owellen, R. J., Owens, A. H. and Donigian, C. (1972) Biochem. Biophys. Res. Commun. 47, 685-691 482 Berlin, R. D. and Ukena, T. E. (1972) Nat. New Biol. 238, 120-122 483 Rittenhouse, H. G., Williams, R. E. and Fox, C. F. (1975) submitted

66 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532

Kaneko, 1., Satoh, H. and Ukita, T. (1973) Biochem. Biophys. Res. Commun. 50, 1087-1094 Vlodavsky, 1., lnbar, M. and Sachs, L (1973) Proc. Natl. Acad. Sci. U.S. 70, 1780-1784 Barnett, R. E., Furcht, L. T. and Scott, R. E. (1974) Proc. Natl. Acad. Sci. U.S. 72, 1217 Gaffney, B. J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 664-668 Gaffney, B. J., Branton, P. E., Wickus, G. G. and Hirschberg, C. B. (1974) in Viral Transformation and Endogenous Viruses (A. S. Kaplan, ed.), pp. 97-115, Academic Press, New York Yau, T. M., Buckman, T., Hole, A. N. and Weber, M. J. (1976) Biochemistry, in the press Quigley, J. P., Rifkin, D. B. and Reich, E. (1972) Virology 50, 550-557 Albrecht-Buhler, G. (1973) Exp. Cell Res. 78, 67-70 Albrecht-Buhler, G. and Soloman, F. (1974) Exp. Cell Res. 85, 225-233 Shinitzky, M. and lnbar, M. (1974) J. Mol. Biol. 85, 603-615 lnbar, M. and Shinitzky, M. (1975) Eur. J. Immunol 5, 166-170 Humphries, G. M. K. and McConnell, H. M. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2483-2487 Wood, R. and Falch, J. (1974) Lipids 8, 702-710 Jacobs, R. A. and Majerus, P. W. (1973) J. Biol. Chem. 248, 8392-8401 Wisnieski, B. J., Williams, R. E. and Fox, C. F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3669-3673 Williams, R. E., Wisnieski, B. J., Rittenhouse, H. G. and Fox, C. F. (1974) Biochemistry 13, 1969-1977 Horwitz, A. F., Hatten, M. E. and Burger, M. M. (1974) Proc. Natl. Acad. Sci. U.S. 71,3115-3119 Jansons, V. K. and Burger, M. M. (1973) Biochim. Biophys. Acta 291, 127-135 Dievard, J. C. and Bourrillon, R. (1974) Biochim. Biophys. Acta 345, 198-2t2 Otten, J., Johnson, G. S. and Pastan, I. (1972) J. Biol. Chem. 247, 7082-7087 Otten, J., Johnson, G. S. and Pastan, I. (1971) Biochem. Biophys. Res. Commun. 44, 1192-1198 Sheppard, J. R. (1972) Nat. New Biol. 236, 14-16 Lazarides, E. and Weber, K. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2268-2272 Goldman, R. D., Lazarides, E., Pollack, R. and Weber, K. (1975) Exp. Cell Res. 90, 333-350 Gruenstein, E., Rich, A. and Weihing, R. R. (1975) J. Cell Biol. 64, 223-234 Ostland, R. E., Pastan, !. and Adelstein, R. S. (1974) J. Biol. Chem. 249, 3903-3907 Pollard, T. D. and Weihing, R. R. (1974) CRC Critical Rev. Biochem. 2, 1-65 lshikawa, H., Bischoff, R. and Holtzer, H. (1969) J. Cell Biol. 43, 312-328 Spooner, B. S., Yamada, K. M. and Wessells, N. K. (1971) J. Cell Biol. 49, 595-613 Weber, K., Lazarides, E., Goldman, R. D., Vogel, A. and Pollack, R. (1974) Cold Spring Harb. Syrup. Quant. Biol. 39, 363-369 Wessells, N. K., Spooner, B. S. and Luduena, M. A. (1973) in Locomotion of Tissue Culture Cells, Excerpta Medica, North-Holland, Amsterdam, C1BA Fdn, Syrup. 14, 53-77 Goldberg, N. D., Haddox, M. K., Hartle, D. K. and Hadden, J. W. (1973) Fifth International Congress of Pharmacology, pp. 146-169, Karger, Basel, Switzerland Sheppard, J. R. and Bannai, S. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 571-579, Cold Spring Harbor Laboratory, New York Kram, R. and Tomkins, G. M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1659-1663 Old, L. J. and Boyse, E, A. (1964) Annu. Rev. Med. 15, 167-186 Hellstr~Sm, K. E. and Hellstr6m, I. (1969) Adv. Cancer Res. 12, 167-223 Hellstr/Sm, K. E. and Hellstrt~m, 1. (1974) Adv. lmmunol. 18, 209-277 Herberman, R. B. (1974) Adv. Cancer Res. 19, 207-263 Klein, G. (1966) Annu. Rev. Microbiol. 20, 223-252 Law, L. W. (1970)Transplant. Proc. 2, 117-132 Coggin, J. H., Jr. and Anderson, N. G. (1974) Adv. Cancer Res. 19, 105-165 Coggin, J. H., Jr. and Anderson, N. G. (1974) in Developmental Aspects of Carcinogenesis and Immunity (the 32rid Symposium of the Society for Developmental Biology), pp. 173-185, Academic Press, Inc., New York Coggin, J. H., Jr., Ambrose, K. R. and Anderson, N. G. (1973) Adv. Exp. Med. Biol. 29,483-490 Baldwin, R. W., Embleton, M. J., Price, M. R. and Vose, B. M. (1974) Transplant. Rev. 20, 77-99 Gold, P., Gold, M. and Freeman, S. O. (1968) Cancer Res. 28, 1331-1334 Gold, P., Krupey, J. and Ansari, H. (1970) J, Natl. Cancer Inst. 45, 219-225 Von Kleist, S. and Burtin, P. (1969) Int. J. Cancer 4, 874-879 Burtin, P., Buffe, D., Von Kleist, S., Wolff, E. and Wolff, E. (1970) int. J. Cancer 5, 88-95 Gold, P. and Freedman, S. (1965) J. Exp. Med. 121,439462

67 533 534 535 536

Chu, T. M., Reynoso, G. and Jansen, J. H. (1972) Nature 238, 152-153 Martin, F. and Martin, S. M. (1970) Int. J. Cancer 6, 352-360 Pusztaszeri, G. and Mach, J.-P. (1973) lmmunochemistry 10, 197-204 Thomson, D. M. P., Krupey, J., Freedman, S. O. and Gold. P. (1969) Proc. Natl. Acad. Sci. U.S. 64, 161-167 537 Terry, W. D., Henkart, P. A., Coligan, J. E. and Todd, C. W. (1974) Transplant. Rev. 20, 100-129 538 Krupey, J., Gold, P. and Freedman, S. (1968) J. Exp. Med. 128, 387-398 539 Terry, W. D., Henkart, P. A., Coligan, J. E. and Todd, C. W. (1972) J. Exp. Med. 136, 200-204 540 Rule, A. H. and Goleski-Reilly, C. (1974) Cancer Res. 34, 2083-2087 541 Coligan, J. E., Henkart, P. A., Todd. C. W. and Terry, W. D. (1973) lmmunochemistry 10, 591-599 542 Abelev, G. 1. (1974) Transplant. Rev. 20, 3-37 543 Ruoslahti, E. and Seppfil~i, M. (1972) Nature 235, 161-162 544 Ruoslahti, E., Pihko, H. and Sepp~ilfi, M. (1974) Transplant. Rev. 20, 38-60 545 Baldwin, R. W. (1973) Adv. Cancer Res. 18, 1-76 546 Butel, J. S., Tevethia, S. S. and Melnick, J. L. (1972) Adv. Cancer Res. 15, 1-55 547 Morton, D. L., Miller, G. F. and Wood, D. A. (1969) J. Natl. Cancer Inst. 42, 289-301 548 Tevethia, S. S., Diamandopoulos, G. T., Rapp, F. and Enders, J. F. (1968) J. lmmunol. 101, 1192-1198 549 Aoki, T., Stephenson, J. R. and Aaronson, S. A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 742-746 550 Aoki, T., Stephenson, J. R., Aaronson, S. A. and Hsu, K. C. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3445-3449 551 Old, L. J., Boyse, E. A. and Stockert, E. (1965) Cancer Res. 25, 813-819 552 Khera, K. S., Ashkenazi, A., Rapp, F. and Melnick, J. L. (1963) J. Immunol. 91,604-613 553 Aoki, T. and Sibal, L. R. (1975) in Biology of Tumors: Surfaces, Immunology and Comparative Physiology; Vol. 4 of Cancer: A Comprehensive Treatise (Becker, F. F., ed.), pp. 159-183, Plenum Press, New York 554 Aoki, T. and Sibal, L. R. (1975) Cancer Res. Monogr., in press. 555 Bauer, H. (1974) Adv. Cancer Res. 20, 275-341 556 Yoshiki, T., Mellors, R. C. and Hardy, W. D., Jr. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1878-1882 557 Yoshiki, T., Mellors, R. C., Hardy, W. D., Jr. and Fleissman, E. (1974) J. Exp. Med. 139, 925-942 558 Ferrer, J. F. (1973) Int. J. Cancer 12, 378-388 559 Kennel, S. J., Del Villano, B. C., Levy, R. L. and Lerner, R. A. (1973) Virology 55, 464-475 560 Del Villano, B. C., Nave, B., Croker, B. P., Lerner, R. A. and Dixon, F. J. (1975) J. Exp. Med. 141, 172-187 561 Obata, Y., lkeda, H., Stockert, E. and Boyse, E. A. (1975) J. Exp. Med. 141, 188-197 562 Ikeda, H., Pincus, T., Yoshiki, T., Strand, M., August, 3. T., Boyse, E. A. and Mellors, R. C. (1974) J. Virol 14, 1274-1280 563 Grant, J. P., Bigner, D. D., Fischinger, P. J. and Bolognesi, D. P. (1974) Proc. Natl. Acad. Sci. U.S. 71, 5037-5041 564 Kurth, R. (1975) Nature 256, 613-614 565 Lengerovfi, A. (1972) Adv. Cancer Res. 16, 235-271 566 Ting, C.-C. and Herberman, R. B. (1971) Nat. New Biol. 232, 118-120 567 Boyse, E. A. and Old, L. J. (1969) Annu. Rev. Genetics 3, 269-290 568 Stockert, E., Old, L. J. and Boyse, E. A. (1971) J. Exp. Med. 133, 1334-1355 569 Boyse, E. A., Stockert, E. and Old, L. J. (1967) Proc. Natl. Acad. Sci. U.S. 58,954-957 570 Herberman, R. B. and Aoki, T. (1972) J. Exp. Med. 136, 94-111 571 Tung, J.-S., Vitetta, E. S., Fleissner, E. and Boyse, E. A. (1975) 3. Exp. Med. 141, 198-205 572 Aoki, T., H/immerling, U., de Harven, E., Boyse, E. A. and Old, L. J. (1969) J. Exp. Med. 130, 979-1001 573 Davis, W. C. (1972) Science 175, 1006-1008 574 Davis, W. C., Alspaugh, M. A., Stimpfling, J. H. and Walford, R. L. (1971) Tissue Antigens 1, 89-93 575 Edidin, M. and Weiss, A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2456-2459 576 Lengerov~, A., Pokornfi, Z., Viklick)~, V. and Zelne2~, V. (1972) Tissue Antigens 2, 332-340

68 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

Lengerov~t, A. and Pi~knicova, J. (1973) Eur. J. Cancer 9, 471-475 Stackpole, C. W., Jacobson, J. B. and Lardis, M. P. (1974) J. Exp. Med. 140, 939-953 Yefenof, E. and Klein, G. (1974) Exp. Cell Res. 88,217-224 Mintz, U. and Sachs, L. (1975) Int. J. Cancer 15, 253-259 Leonard, E. J. (1973) J. Immunol. 110, 1167-1169 Aoki, T., Boyse, E. A., Old, L. J., de Harven, E., H~immerling, U. and Wood, H. A. (1970) Proc. Natl. Acad. Sci. U.S. 65, 569-576 Phillips, E. R. and Perdue, J. F. (1974) J. Cell Biol. 61,743-756 Phillips, E. R. and Perdue, J. F. (1976) J. Cell Sci., in the press Gelderblom, H., Bauer, H. and Graft, T. (1972) Virology 47, 416-425 Gelderblom, H. and Bauer, H. (1973) Int. J. Cancer 11,466-472 Rutter, G. and Mannweiler, K. (1973) Arch. Virusforsch. 43, 169-172 Aoki, T. (1973) Bibl. Haemat. 39, 307-315 Aoki, T., Potter, M. and Sturm, M. M. (1973) J. Natl. Cancer Inst. 51, 1609-1617 Aoki, T. and Takahashi, T. (1972) J. Exp. Med. 135, 443-457 Hinuma, Y., Suzuki, M. and Sairenji T. (1975) Int. J. Cancer 15, 799-805 Ehrnst, A. and Sundqvist, K.-G. (1975) Cell 5, 351-359 Aisenberg, A. C. and Long, J. C. (1975) Am. J. Med. 58,300-306 Grey, H. M., Rabellino, E. and Pirofsky, B. (1971) J. Clin. Invest. 50, 2368-2375 Knapp, W., Schuit, H. R. E., Bolhuis, R. L. H. and Hijmans, W. (1974) Clin. Exp. immunol. 16, 541-552 Cohen, H. J. and Gilbertsen, B. B. (1975) J. Clin. Invest. 55, 84-93 Palencia, C., Baumgartner, G. and Knapp, W. (1975) Eur. J. Cancer 11, 193-197 Menne, H. D. and Flad, H.-D. (1973) Clin. Exp. Immunol. 14, 57-67 S~illstr6m, J. F., Juhlin, R., and Stenkvist, B. (1974) Acta Cytol. 18, 392-398 Turner, R. S. and Burger, M. M. (1974) Ann. N. Y. Acad. Sci. 234, 332-347 Erlandson. R. A. and de Harven, E. (1971) J. Cell Sci. 8,353-397 Porter, K., Prescott, D. and Frye, J. (1973) J. Cell Biol. 57, 815-836 Porter, K. R. and Fonte, V. G. (1973) in Scanning Electron Microscopy: Proceedings of the Workshop on Scanning Electron Microscopy in Pathology, pp. 683-688, 1TT Research Institute, Chicago Paweletz, N. and Schroeter, D. (1974) Cytobiologie 8, 238-246 Fox, T. O., Sheppard, J. R. and Burger, M. M. (1971) Proc. Natl. Acad. Sci. U.S. 68,244-247 Shoham, J. and Sachs, L. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2479-2482 Shoham, J. and Sachs, L. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 297-304, Cold Spring Harbor Laboratory, New York Mallucci, L. (1971) Nat. New Biol. 233, 241-244 Noonan, K. D., Levine, A. J. and Burger, M. M. (1973) J. Cell Biol. 58,491-497 Nicolson, G. L. and Paul, D. (1974) unpublished observations Smets, L. A. (1973) Nat. New Biol, 245, 113-115 Kuhns, W. J. and Bramson, S. (1968) Nature 219, 938-939 Thomas, D. B. (1971) Nature 233, 317-321 Cikes, M. (1970) .I. Natl. Cancer Inst. 45, 979-988 Cikes, M. and Friberg, S., Jr. (1971) Proc. Natl. Acad. Sci. U.S. 68, 566-569 Pasternak, D. A., Warmsley, A. M. H. and Thomas, D. B. (1971) J. Cell Biol. 50, 562-564 Buell, D. N. and Fahey, J. L. (1969) Science 164, 1524-1525 Burger, M. M. (1972) in Membrane Research (Fox, C. F., ed.), pp. 241-252, Academic Press, New York Stadler, J. K. and Adelberg, E. A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1929-1933 Kraemer, P. M. and Tobey, R. A. (1972) J. Cell Biol. 55, 713-717 Hakomori, S.-I. and Kijimoto, S. (1972) Nat. New Biol. 239, 87-88 Goto, M., Kataoka, Y., Goto, K., Yodoyama, T. and Sato, H. (1972) G a n n 63, 505-506 Furcht, L. T. and Scott, R. E. (1974) Exp. Cell Res. 88, 311-318 Yamada, K. M. and Weston, J. A. (1975) Cell 5, 75-81 Bose, S. K. and Zlotnick, B. J. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2374-2378 Plagemann, P. G. W. (1973) J. Cell. Physiol. 82, 421-434 Robbins, P. W. and MacPherson, I. A. (1971) Proc. Roy. Soc. Lond. B 177, 49-58

69 628 Sakiyama, H., Gross, S. K. and Robbins, P. W. (1972) Proc. Natl. Acad. Sci, U.S. 69, 872-876 629 Fishman, P. H., McFarland, V. W., Morat, P. T. and Brady, R. O. (1972) Biochem. Biophys. Res. Commun. 48, 48-57 630 Yogeeswaran, G. and Hakomori, S.-I. (1975) Biochemistry 14, 2151-2156 631 Gahmberg, C. G. and Hakomori, S.-I. (1975) J. Biol. Chem. 250, 2438-2446 632 Bosmann, H. B. and Winston, R. A. (1970) J. Cell Biol. 45, 23-33 633 Wolf, B. A. and Robbins, P. W. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.) pp. 473-480, Cold Spring Harbor Laboratory, New York 634 Warmsley, A. M., Phillips, B. and Pasternak, C. A. (1970) Biochem. J. 120, 683-688 635 Critchley, D, R., Chandrabose, K. A., Graham, J. M. and MacPherson, I. (1974) in Control of Proliferation in Animal cells (Clarkson, B. and Baserga, R., eds.), pp. 481-493, Cold Spring Harbor Laboratory, New York 636 Glick, M. C., Gerner, E. W. and Warren, L. (1971) J. Cell. Physiol. 77, 1-6 637 Glick, M. C. and Buck, C. (1973) Biochemistry 12, 185-190 638 Onodera, K. and Sheinin, R. (1970) J. Cell Sci. 7, 337-355 639 Glynn, R. D., Thrash, C. R. and Cunningham, D. D. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2676-2677 640 Levine, A. J. and Burger, M. M. (1972) J. Theoret. Biol. 37, 435-446 641 Reich, E., Rifkin, D. B. and Shaw, E. (1975) Proteases and Biological Control (Reich, E., Rifkin, D. B. and Shaw, E., eds.), Cold Spring Harbor Conferences on Cell Proliferation, Vol. 2, Cold Spring Harbor Laboratory, New York 642 Talmadge, K. W., Noonan, K. D. and Burger, M. M. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds), pp. 313-325, Cold Spring Harbor Laboratory, New York 643 Schnebli, H.-P. and Burger, M. M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3825-3827 644 Alexander, P. (1968) Prog. Exp. Tumor Res. 10, 22-71 645 Smith, R. T. and Landy, M. (1971) Immune Surveillance, Academic Press, New York 646 Cerottini, J.-C. and Brunner, K. T. (1974) Adv. lmmunol. 18, 67-132 647 Burnet, F. M. (1970) Prog. Exp. Tumor Res. 13, 1-27 648 Miller, J. F. A. P., Grant, G. A. and Roe, F. J. C. (1963) Nature 199, 920-922 649 Yohn, D. S., Funk, C. A., Kalnins, V. 1. and Grace, .1. T. (1965) J. Natl. Cancer Inst. 35, 617-624 650 Allison, A. C. (1970) Proc. R. Soc. Med. 63, 1077-1080 651 Good, R. A. and Finstad, J. (1969) Natl. Cancer Inst. Monogr. 31, 41-58 652 Good, R. A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1026-1032 653 Prehn, R. T. (1971) J. Reticuloendothel. Soc. 10, 1-12 654 Prehn, R. T. (1972) Science 176, 170-171 655 Stutman, O. (1974) Science 181,534-536 656 Hewitt, H. G. (1976) in Fundamental Aspects of Metastasis (Weiss, L., ed.), ASP Biological and Medical Press, B.V., Amsterdam, in the press 657 Rygaard, J. and Poulsen, C. O. (1974) Transplantation 17, 135-136 658 Weston, B..1. (1973) in Contemporary Topics in Immunology (Davies, A. J. S. and Carter, R. L., eds.), Vol. 2, pp. 237-252, Plenum Press, New York 659 Allison, A. C. (1970) Br. Med. J. 4, 419-420 660 Ting, R. C. and Law, L. B. (1967) Frog. Exp. Tumor Res. 9, 165-191 661 McKhann, C. F. and Vagarlamoody, S. M. (1971) Transplant, Rev. 7, 55-77 662 Zbar, B., Bernstein, 1. D., Bartlett, G. L., Hanna, M. G., Jr. and Rapp, H. J. (1972) J. Natl. Cancer Inst. 49, 119-130 663 Eilber, F. R. and Morton, D. L. (197lJ in Progress in Immunology (First International Congress on Immunology) (Amos, B., ed.), pp. 951-957, Academic Press, New York 664 Ran, M. and Witz, I. P. (1970) Int. J. Cancer 6, 361-372 665 Witz, 1. P., Yagi, Y. and Pressman, D. (1967) Cancer Res. 27, 2295-2299 666 Sobczak, E. and De Vaux Saint Cyr, C. L. (1971) Int. J. Cancer 8, 47-52 667 Klein, G., Clifford, P., Henle, G., Henle, W., Geering, G. and Old, L. J. (1969) Int. J. Cancer 4, 416-421 668 Morton, D. L., Malmgren, R. A., Holmes, E. C. and Ketcham. A.S. (1968) Surgery 65, 223-240 669 Muna, N. M., Marcus, S. and Smart, C. (1969) Cancer 23, 88-93 670 Witz, I. P., Kinamon, S., Ran, M. and Klein, G. (1974) Clin. Exp. lmmunol. 16, 321-333

70 671 Bodurtha, A. J., Chee, D. O., Laucius, J. F., Mastrangelo, M. J. and Prehn, R. T. (1975) Cancer Res. 35, 189-193 672 Aoki, T., Boyse, E. A. and Old, L. J. (1966) Cancer Res. 26, 1415-1420 673 Sato, H., Boyse, E. A., Aoki, T., lritani, C. and Old, L. J. (1973) J. Exp. Med. 138, 593-606 674 Martin, S. E. and Martin, W. J. (1975) Int. J. Cancer 15,658-664 675 Martin, W. J. and Martin, S. E. (1974) Nature 249, 564-565 676 Silver, D. M. and Winn, H. J. (1973) J. Immunol. 111, 1281-1284 677 Barth, W. F., Wochner, R. D., Waldman, T. A. and Fahey, J. L. (1964) J. Clin. Invest. 43, 1036-1048 678 Negroni, G. and Hunter, E. (1973) J. Natl. Cancer Inst. 51,265-268 679 lrie, K., lrie, R. F. and Morton, D. L. (1974) Science 186, 454--456 680 Nowinski, R. C. and Haehler, S. L. (1974)Science 185, 869-871 681 Batzing, B., Yurconic, M. and Hanna, M. G. (1974)J. Natl. Cancer Inst. 52, 117-132 682 Aaronson, S. A. and Stephenson, J. R. (1974) Proof. Natl. Acad. Sci. U.S. 71, 1957-1961 683 Burnet, F. M. (1971) Transplant. Rev. 7, 3-25 684 HellstrCSm, K. E. and Hellstr/Sm, I. (1972) Annu. Rev. Med. 23, 19-38 685 Brondz, B. D. (1972)Transplant. Rev. 10, 112-151 686 Allison, A. C. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds), pp. 447-459, Cold Spring Harbor Laboratory, New York 687 Ferluga, J. and Allison, A. C. (1974) Nature 250, 673-675 688 Kolb, W. P. and Granger, G. A. (1968) Proc. Natl. Acad. Sci. U.S. 61, 1250-1255 689 Kolb, W. P. and Granger, G. A. (1970) Cell. lmmunol. I, 122-132 690 Trinchieri, G., Bauman, P., de Marchi, M. and T/SkSs, Z. (1975) J. Immunol. 115, 249-255 691 Van Boxel, J. A., Stobo, J., Paul, W. E. and Green, i. (1972) Science) 175, 194--196 692 Perlmann, P., Wigzell, H., Golstein, P., Lamon, E. W., Larsson,/~., O'Toole, C., Perlmann, H. and Svedmyr, E. A. J. (1974) Adv. Biosci, 12, 71-85 693 Greenberg, A. H., Shen, L. and Roitt, 1. M. (1973) Clin. Exp. Imrnunol. 15, 251-259 694 Greenberg, A. H., Shen, L., Walker, L., Arnaiz-Villena, A. and Roitt, 1. M. (1975) Eur. J. lmmunol. 5, 47~478 695 Evans, R. and Alexander, P. (1972) Nature 236, 168-179 696 Alexander, P. and Evans, R. (1971) Nat. New Biol. 232, 76-78 697 McCoy, J. L., Ring, R. C., McCoy, N. T., Reisher, J. I., Chan, S. P. and Law, L. W. (1974) Int. J. Cancer 13, 731-741 698 Old, L. J., Stockert, E., Boyse, E. A. and Kim, J. (1968) J. Exp. Med. 127, 523-539 699 Taylor, R. B., Duffus, W. P. H., Raft, M. C. and de Petri, S. (1971) Nat. New Biol. 233, 225-229 700 Stackpole, C. W., Jacobson, J. B. and Lardis, M. P. (1974) Nature 248,232-234 701 Loor, F., Block, N. and Little, J. R. (1975) Cell. lmmunol. 17, 351-365 702 Yu, A. and Cohen, E. P, (1974) J. lmmunol. 112, 1296-1307 703 Takahashi, T., Old, L. J., Melntire, K. R. and Boyse, E. A. (1971) J. Exp. Med. 134, 815-832 704 Sundqvist, K. G., Svehag, S. E. and Thorstensson, R. T. (1974) Scand. J. Immunol. 3, 237-250 705 Takahashi, T. (1971) Transplant. Proc. 3, 1217-1220 706 Knopf, P., Destree, A. and Hyman, R. (1973) Eur. J. lmmunol. 3, 251-259 707 Lesley, J. and Hyman, R. (1974) Eur. J. lmmunol. 4, 732-739 708 Edidin, M. and Henney, C. S. (1973) Nat. New Biol. 246, 47-49 709 Aoki, T. and Johnson, P. A. (1972) J. Natl. Cancer Inst. 49, 183-192 710 Aoki, T., Geering, G., Beth, E. and Old, L. J. (1971) in Recent Advances in Human Tumor Virology and Immunology (Nakahara, W., Nishioka, K., Hirayama, T. and Ito, Y., eds.), pp. 425-429, University Park Press, Baltimore 711 Ohanian, S., Borsos, T. and Rapp, H. (1973) J. Natl. Cancer Inst. 50, 1313-1320 712 Lesley, J., Hyman, R. and Dennert, G. (1974) J. Natl. Cancer Inst. 53, 1759-1765 713 Allison, A. C., Davies, P. and de Petris, S. (1971) Nature 232, 153-155 714 Wagner, R., Rosenberg, M. and Estensen, R. (1971) J. Cell Biol. 50, 804-817 715 Williams, J. A. and Wolff, J. (1971) Endocrinology 88,206-217 716 Segerling, M., Ohanian, S. H. and Borsos, T. (1974) J. Natl. Cancer Inst. 53, 1411-1413 717 Ferrone, S., Pellegrino, M. A., Dierieh, M. P. and Reisfeld, R. A. (1974) Tissue Antigens 4, 275-282 718 Berlin, R. D., Oliver, J. M., Ukena, T. E. and Yin, H. H. (1974) Nature 247, 45-46

71 719 Kurth, R. and Bauer, H. (1973) Nat. New Biol. 243, 243-245 720 Klein, J. (1975) in Biology of the Mouse Histocompatibility-2 Complex, pp. 305-326, SpringerVerlag, Berlin 721 Herzenberg, L. A., Herzenberg, L. A., Goodlin, R. C. and Rivera, E. C. (1967) J. Exp. Med. 126, 701-713 722 Klein, E., Klein, G. and Hellstr6m, K. E. (1960) J. Natl. Cancer Inst. 25, 271-294 723 Warren, L. and Glick, M. C. (1968) .1. Cell Biol. 37, 729-746 724 Nachman, R. L., Ferris, B. and Hirsch, J. G. (1971) J. Exp. Med. 133, 807-820 725 Evans, W. H. and Gurd, J. W. (1971) Biochem. J. 125, 615-624 726 Cone, R. E., Marchalonis, J. J. and Rolley, R. T. (1971) J. Exp. Med. 134, 1373-1384 727 Nowotny, A., Grohsman, J., Abdelnoor, A., Rote, N., Yang, C. and Waltersdorff, R. (1974) Eur. J. Immunol. 4, 73-78 728 Poste, G. (1971) Exp. Cell Res. 67, 11-16 729 Alexander, P. (1974) Cancer Res. 34, 2077-2082 730 Currie, G. A. and Alexander, P. (1974) Br. J. Cancer 29, 72-75 731 Thompson, D. M. P., Steele, K. and Alexander, P. (1973) Br. J. Cancer 27, 27-34 732 Currie, G. A. and Gage, J. O. (1973) Br. J. Cancer 28, 136-146 733 Currie, G. A. and Bashan, C. (1972) Br. J. Cancer 26, 427-438 734 Baldwin, R. W., Embleton, M. J., Jones, J. S. P. and Langman, M. J. S. (1973) Int. J. Cancer 12, 73-83 735 Hells,r6m, 1. and Hellstr6m, K. E. (1969) Int. J. Cancer 4, 587-600 736 Hellstr6m, I., Hellstr6m, K. E., Evans, C. A., Heppner, G. H., Pierce, G. E. and Yang, J. P. S. (1969) Proc. Natl. Acad. Sci. U.S. 62, 362-368 737 Sj6gren, H. O., Hellstr6m, I., Bansal, S. C., Warner, G. A. and Hellstr6m, K. E. (1972) Int. J. Cancer 9, 274-283 738 Sj6gren, H. O., Hellstr6m, I., Bansal, S. C. and Hellstr6m, K. E. (1971) Proc. Natl. Acad. Sci. 68, 1372-1375 739 Baldwin, R. W., Price, M. R. and Robins, R. A. (1972) Nat. New Biol. 238, 185-187 740 Cooperband, S. R., Bondevik, H., Schmid, K. and Mannick, J. A. (1968) Science 159, 1243-1244 741 Heppner, G. H. (1972) Ser. Haematol. 5, 41-67 742 Hellstr6m, 1., Hellstr6m, K. E. and Sj6gren, H. O. (1970) Cell. Immunol. 1, 18-30 743 Sj6gren, H. O. and Borum, K. (1971) Cancer Res. 31,890-900 744 Hellstr6m, I. and Hellstr6m, K. E. (1970) Int. J. Cancer 5, 195-201 745 Skurzak, H. M., Klein, E., Yoshida, T. O. and Lamon, E. W. (1972) J. Exp. Med. 135, 997-1002 746 Hellstr6m, I., Hellstr6m, K. E., Sj6gren, H. O. and Warner, G. A. (1971) Int. J. Cancer 8, 185-191 747 Prehn, R. T. and Lapp6, M. A. (1971) Transplant. Rev. 7, 26-54 748 Sell, S. and Gell, P. G. H. (1965) J. Exp. Med. 122, 423-439 749 Shearer, W. T., Philpott, G. W. and Parker, C. W. (1973) Science 182, 1357-1359 750 Hutchin, P., Amos, D. B. and Prioleau, W. H. (1967) Transplantation 5, 68-75 751 Duff, R., Doller, E. and Rapp, F. (1973) Science 180, 79-81 752 Fink, M. P., Parker, C. W. and Shearer, W. T. (1975) Nature 255, 404-405 753 Shearer, W. T., Philpott, G. W. and Parker, C. W. (1974) J. Exp. Med. 139, 367-379 754 Fidler, I. J. (1974) Cancer Res. 34, 491-498 755 Edelman, G. M. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, eds.), pp. 357-377, Cold Spring Harbor Laboratory, New York 756 Coggin, J. G., Jr.. Ambrose, K. R., Dierlam, P. J. and Anderson, N. G. (1974) Cancer Res. 34, 2092-2101 757 Goetz, 1. E., Weinstein, C. and Roberts, E. (1972) Cancer Res. 32, 2469-2474 758 Borek, C., Grob, M. and Burger, M. M. (1973) Exp. Cell Res. 77, 207-215 759 Latner, A. L., Longstaff, E. and Pradhan, K. (1973) Br. J. Cancer 27, 460-464 760 Schnebli, H.-P. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 327-337, Cold Spring Harbor Laboratory, New York 761 Weber, M. J. (1975) Cell 5, 253-261 762 Chou, I.-N., Black, P. H. and Roblin, R. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga, R., eds.), pp. 339-350, Cold Spring Harbor Laboratory, New York 763 Tomita, M., Kurokawa, T., Osawa, T., Sakurai, Y. and Ukita, T. (1972) Gann 63,269-271

72 764 765 766 767 768 769 770 77l 772 773 774 775 776 777

Moore, E. G. and Temin, H. M. (1971) Nature 231, 117-118 Biddle, F., Cronin, A. P. and Sanders, F. K. (1970) Cytobios 5, 9-17 Inbar, M., Ben-Bassat, H. and Sachs, L. (1972) Nal. New Biol. 236, 34,16 Nicolson, G. L. and Blaustein, J. (1972) Biochim. Biophys. Acta 266, 543-547 Sela, B., Lis, H,, Sharon, N. and Sachs, L. (1970) J. Memb. Biol. 3, 267-279 Becker, F. F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4307-4311 Vesel~, P., Entlicher, G. and Kocourek, J. (1972) Experientia 28, 1085-1086 Ben-Bassat, H., Goldblum, N., Manny, N. and Sachs, L. (1974) Int. J. Cancer 14, 367-371 Eckhart, W., Dulbecco, R. and Burger, M. M. (1971) Proc. Natl. Acad. Sci. U.S. 68, 283-286 Sakiyama, H. and Robbins, P. W. (1973) Fed. Proc. 32, 86-90 Kapeller, M. and Doljanski, F. (1972) Nat. New Biol. 235, 184-185 De Salle, L., Munakata, N., Pauli, R. M. and Strauss, B. S. (1972) Cancer Res. 32, 2463-2468 Aoki, T. (1973) in Immunological Aspects of Neoplasia (Hearsh, E., ed.), in the press Oldstone, M. B. A., Tishon, A., Tonietti, G. and Dixoo, F. J. (1972) Clin. lmmunol, lmmunopath. 1, 6-14 778 Nicolson, G. L. and Poste, G. (1976) New Engl. J. Med., in the press