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Its Metastasizing and Nonmetastasizing Tumors KERMIT L. CARRAWAY, JOHN W. HUGGINS, ANNE P. SHERBLOM, ROBERT W. CHESNUT, ROBERT L. BUCK, SUSAN P. HOWARD, CHARLOTTE L. OWNBY, and CORALIE A. CAROTHERS CARRAWAY Department of Biochemistry, Oklahoma State University, Stillwater, OK 74074

Glycoproteins are ubiquitous components of animal cell surfaces (1) and are generally believed to play important roles in neoplastic diseases (2). Glycoprotein differences between nonmalignant and malignant cells have been demonstrated by such diverse techniques as lectin agglutination (3), glycopeptide analyses (4), gel electrophoresis (5) and enzymatic cell surface labeling (6). The origin of these differences and the roles of the glycoproteins in most cellular phenomena are still unknown. One intriguing possibility is that cell surface glycoproteins may be involved in the mechanism by which tumor cells escape destruction by the immune system of the host. Two very different hypotheses have been presented to explain the role of glycoproteins in this escape from immune surveillance. The first involves the shedding of antigens from the cell surface. These antigens combine with circulating antibodies to form complexes which block antibody-mediated destruction of the tumor cells (7). Kim et al. (8) have extended this hypothesis to suggest that glycoprotein shedding in mammary tumors is related to their metastatic potential. Using a series of mouse mammary tumors selected for variations in ability to metastasize, they found an inverse correlation between metastasis and three parameters: 5'-nucleotidase activity, cell surface glycocalyx staining and immunogenicity of the tumors in rabbits. It was suggested that the loss of the cell surface constituents was due to shedding, but the mechanism was not specified. A second means by which glycoproteins might be involved in "protection" of the tumor cells was suggested by studies on the TA3 mouse mammary tumor. Two sublines of this tumor which are different in their immunological properties have been isolated. The TA3-St subline grows only in the syngeneic host, but the TA3-Ha subline is able to cross histocompatibility barriers (9). This loss of strain specificity is accompanied by a greatly increased amount of a large sialoglycoprotein (10) named 0-8412-0452-7/78/47-080-432$05.00/0 © 1978 American Chemical Society Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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e p i g l y c a n i n . I t was hypothesized that the g l y c o p r o t e i n i n the TA3-Ha s u b l i n e "covers" the h i s t o c o m p a t i b i l i t y antigens at the c e l l surface (11), preventing t h e i r expression and thereby prev e n t i n g the r e c o g n i t i o n of these c e l l s as f o r e i g n to the host. Our own work has been concerned with the c e l l surface g l y coproteins of r a t mammary tumors and the r e l a t i o n s h i p of these g l y c o p r o t e i n s to the p r o p e r t i e s of the tumor c e l l s . In t h i s p r e s e n t a t i o n we s h a l l focus on three questions. 1) Is there a necessary r e l a t i o n s h i p between the expression of c e l l surface g l y c o p r o t e i n s and metastasis of mammary tumors as suggested by Kim et a l . (8)? 2) I s there a r e l a t i o n s h i p between the "form" or environment of the tumor and the c e l l surface g l y c o p r o t e i n s i t expresses? 3) How are the c e l l s u r f a c e p r o p e r t i e s of tumors r e l a t e d to t h e i r b i o l o g i c a l a c t i v i t i e s as tumors? Comparison of membrane g l y c o p r o t e i n s from normal mammary gland, a nonmetastasizing tumor (R3230 AC) and a m e t a s t a s i z i n g tumor (13762). To determine whether there i s a necessary d i f f e r e n c e i n expression of c e l l surface g l y c o p r o t e i n between nonmetastasizing and m e t a s t a s i z i n g mammary tumors, we have examined the R3230 AC and 13762 r a t mammary adenocarcinomas, both grown i n the F i s c h e r 344 r a t . The R3230 AC i s a slow-growing tumor d e r i v e d from a spontaneous carcinoma (12). I t i s hormonally responsive and e x h i b i t s l i t t l e or no m e t a s t a t i c c a p a c i t y under the c o n d i t i o n s used i n our s t u d i e s . The 13762 r a t mammary adenocarcinoma i s a methylcholanthrene-induced s o l i d tumor (13) which i s hormonally responsive and metastasizes widely and h e a v i l y . For comparison of c e l l surface g l y c o p r o t e i n s i t i s necessary to p u r i f y plasma membranes from the t i s s u e samples. P u r i f i c a t i o n of plasma membrane and other s u b c e l l u l a r t i s s u e f r a c t i o n s i s d i f f i c u l t f o r s e v e r a l reasons (14). D i s r u p t i o n of the c e l l s i n the t i s s u e almost n e c e s s a r i l y fragments the plasma membrane. Fragmentation i s not uniform and may r e s u l t i n a heterogeneous d i s t r i b u t i o n of membrane fragments, both i n terms of s i z e and of p h y s i c a l and f u n c t i o n a l a t t r i b u t e s . This complicates the s e p a r a t i o n of p a r t i c u l a r morphological u n i t s because of the v a r i a t i o n of the s i z e s , d e n s i t i e s , compositions and other f a c t o r s upon which s e p a r a t i o n of fragments i s based. Identif i c a t i o n of morphological u n i t s i s a l s o complicated by fragment a t i o n , which destroys morphological c h a r a c t e r i s t i c s and reduces membranes t o sheets or v e s i c l e s . In a d d i t i o n to morphological c r i t e r i a membrane f u n c t i o n a l a c t i v i t i e s (enzymes, antigens) must be used f o r i d e n t i f i c a t i o n of s u b c e l l u l a r components. T h i s comb i n a t i o n of heterogeneity and l o s s of morphological i d e n t i t y makes the p r e p a r a t i o n of coherent, h i g h l y p u r i f i e d s u b c e l l u l a r components very d i f f i c u l t . Most of the work i n t h i s area has been performed on l i v e r . Plasma membranes have been p u r i f i e d by a number of methods, most of which tend to give membranes derived from the b i l e c a n a l i c u l a r regions (15). Mammary t i s s u e presents i t s own p a r t i c u l a r problems.

Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Because of the presence of l a r g e amounts of connective t i s s u e the e l a s t i c i t y of the t i s s u e makes homogenization d i f f i c u l t . The procedure which g i v e s the best r e s u l t s f o r normal l a c t a t i n g mammary t i s s u e i n v o l v e s vigorous homogenization i n a S o r v a l l Omni-mixer and i s o l a t i o n of a microsomal f r a c t i o n by d i f f e r e n t i a l c e n t r i f u g a t i o n (5). Microsomes are f u r t h e r f r a c t i o n a t e d by f l o t a t i o n through a sucrose d e n s i t y gradient ( F i g . 1 ) . 5'-Nucleotidase i s used as a marker f o r p r e l i m i n a r y assessment of plasma membrane p u r i f i c a t i o n , s i n c e t h i s enzyme has been shown to be present predominantly i n the plasma membrane of a number of c e l l types (14), i n c l u d i n g mammary gland and mammary tumors (16, 17). S i g n i f i c a n t p u r i f i c a t i o n of 5'-nucleotidase (>25-fold) was achieved i n both of the l i g h t e r f r a c t i o n s ( F and F ) of the discontinuous g r a d i e n t , and there i s a p a r a l l e l p u r i f i c a t i o n of Na , K -ATPase (5), another plasma membrane marker. S u c c i n i c dehydrogenase (a m i t o c h o n d r i a l enzyme) i s markedly reduced, but NADPH-cytochrome c reductase (an endoplasmic r e t i c u l u m marker enzyme) i s s t i l l present, and g a l a c t o s y l t r a n s f e r a s e i s s i g n i f i c a n t l y concentrated. The l a s t observation suggests G o l g i apparatus contamination (16). Further p u r i f i c a t i o n i s achieved by t r e a t i n g F i and F f r a c t i o n s s e p a r a t e l y with d i g i t o n i n and s u b j e c t i n g these to r e f l o t a t i o n on a s i m i l a r discontinuous gradient c o n t a i n i n g d i g i t o n i n . D i g i t o n i n complexes with c h o l e s t e r o l and would be expected t o s h i f t the c h o l e s t e r o l - e n r i c h e d plasma membrane f r a c t i o n to a higher d e n s i t y (18). Cholesterol-poor f r a c t i o n s such as G o l g i fragments should be unchanged i n d e n s i t y . By t h i s procedure a p o r t i o n of the d i g i t o n i n - t r e a t e d F i s s h i f t e d to F x D F , which shows a 3 - f o l d increase i n n u c l e o t i d a s e a c t i v i t y and i s devoid of g a l a c t o s y l t r a n s f e r a s e and NADPH-cytochrome c reductase (19). C l e a r l y i t has the c h a r a c t e r i s t i c s expected of a h i g h l y p u r i f i e d plasma membrane f r a c t i o n . The F f r a c t i o n behaves d i f f e r e n t l y . Part of i t r e e q u i l i b r a t e s to the Fj. p o s i t i o n upon r e f l o t a t i o n . When t r e a t e d with d i g i t o n i n , part of F i s s h i f t e d to a higher d e n s i t y (F DF ). F D F i s enriched i n 5'-nucleotidase, c h o l e s t e r o l , s i a l i c a c i d and g a l a c t o s y l t r a n s f e r a s e , p r o p e r t i e s suggesting that t h i s s u b f r a c t i o n comes from a plasma membrane c o n t a i n i n g galactosyltransferase. These procedures have been used with s i m i l a r r e s u l t s to p u r i f y plasma membranes from the s o l i d tumors. As shown i n Table I, the 5'-nucleotidase of the m e t a s t a s i z i n g 13762 tumor i s not g r e a t l y diminished when compared t o the nonmetastasizing R3230 AC. S i a l i c a c i d analyses (20) of membrane f r a c t i o n s from these two tumors a l s o f a i l to show any s i g n i f i c a n t d e p l e t i o n i n the m e t a s t a s i z i n g tumor (Table I I ) . S i a l o g l y c o p r o t e i n s a r e analyzed by polyacrylamide g e l e l e c t r o p h o r e s i s i n sodium dodecyl s u l f a t e (SDS). Whereas normal mammary membranes show three major c l a s s e s of g l y c o p r o t e i n s by t h i s technique ( F i g . 2), membranes from the tumor c e l l s have a x

2

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+

2

x

3

2

2

2

3

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Figure 1. Schematic for membrane preparations from normal lactating mammary tissue and rat mammary tumor tissue samples

1 F.

I

A

•

A

F.DF,

i V XL

1^.

A

FJDFJ

/ MIGRATION DISTANCE (cm)

Figure 2. Membrane glycoproteins of normal lactating rat mammary tissue. Membrane fractions were prepared as previously described (5,19) and analyzed by electrophoresis on poly aery lamide gels in the presence of SDS (5). Gels were stained by the periodate-Schiff procedure and scanned on a Gilford spectrophotometer with a linear transport device.


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TABLE I Nucleotidase A c t i v i t i e s of Mammary Tumor F r a c t i o n s Fraction

R3230 AC

13762

ymoles/hr•mg p r o t e i n Homogenate FiDF F DF 2

3

3

3.5

4.2

95

126

64

61

TABLE I I S i a l i c A c i d Contents of Tumor C e l l Membranes Homogenate

F

x

F

2

ug/mg p r o t e i n R3230 AC 13762

0.7 0.4

13 19

11 16

f o u r t h c l a s s of g l y c o p r o t e i n s ( F i g . 3) with an apparently lower molecular weight than the other three (5). G l y c o p r o t e i n patterns from the two tumors are very s i m i l a r , though both have patterns more complex than those of membranes from the normal gland. I t i s not p o s s i b l e to a s c e r t a i n a t present whether these s i m i l a r e l e c t r o p h o r e t i c c l a s s e s are the same molecular s p e c i e s , but i t should be f e a s i b l e to o b t a i n a t l e a s t a p a r t i a l answer to that question using immunological techniques. C l e a r l y the d e p l e t i o n of c e l l surface g l y c o p r o t e i n s i s not a u n i v e r s a l c h a r a c t e r i s t i c of plasma membranes i s o l a t e d from m e t a s t a s i z i n g mammary tumors. T h i s does not imply that these tumors do not shed t h e i r surface c o n s t i t u e n t s , nor does i t i n d i c a t e that shedding i s unimportant to metastasis. The conc e n t r a t i o n of any component i n the c e l l r e f l e c t s a balance between s y n t h e t i c and degradative pathways. Since these need to be i n v e s t i g a t e d more c a r e f u l l y , we have examined a s c i t e s c e l l s derived from the m e t a s t a t i c 13762 tumor, because these c e l l s are more e a s i l y manipulated. Glycoproteins of the a s c i t e s form of the 13762 mammary adeno care inoma. The s o l i d 13762 mammary adenocarcinoma has been converted i n t o t r a n s p l a n t a b l e , m e t a s t a t i c a s c i t e s forms MAT-A, MAT-B and


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MAT-C, which we obtained from the Mason Research Laboratory, Worchester, Mass. To serve as models f o r g l y c o p r o t e i n behavior i n the s o l i d tumor, these a s c i t e s tumors must meet two c r i t e r i a . 1) The tumor s u b l i n e s and t h e i r c e l l surface p r o p e r t i e s must be s t a b l e to i n v i v o passage over the p e r i o d of experimentation. 2) The g l y c o p r o t e i n s of the a s c i t e s tumor must be the same as those of the s o l i d tumor. The question of the s t a b i l i t y of the s u b l i n e s proved to be an i n t e r e s t i n g one. During passage of MAT-B and MAT-C s u b l i n e s i n v i v o at weekly i n t e r v a l s over about s i x months, we noted that both were converted to v a r i a n t forms. We s h a l l term these MAT-B1 and MAT-C1 and w i l l discuss t h e i r a l t e r e d c e l l surface p r o p e r t i e s i n a l a t e r s e c t i o n . We suspect that the MAT-A subl i n e a l s o undergoes changes with prolonged passage i n v i v o , but we do not have an appropriate marker to demonstrate these changes. We are maintaining the MAT-B1 and MAT-C1 tumors by t r a n s p l a n t a t i o n and as f r o z e n stock samples f o r f u t u r e uses and comparisons. To analyze c e l l surface g l y c o p r o t e i n s of the a s c i t e s tumors, membranes were prepared by a m o d i f i c a t i o n of the method of Warren j2t a l . (21), using z i n c to " s t a b i l i z e " the c e l l surface membranes. Polyacrylamide g e l e l e c t r o p h o r e s i s of SDSs o l u b i l i z e d membranes was used f o r a n a l y s i s of s i a l o g l y c o p r o t e i n s . A l l a s c i t e s tumor s u b l i n e s showed a major band which b a r e l y penetrated the g e l s , i l l u s t r a t e d i n F i g . 4 f o r the MAT-B1 and MAT-C1 s u b l i n e s . Although SDS e l e c t r o p h o r e s i s i s u n r e l i a b l e f o r molecular weight determinations on g l y c o p r o t e i n s , g e l f i l t r a t i o n experiments a l s o suggest that t h i s s i a l o g l y c o p r o t e i n i s a l a r g e molecule. This g l y c o p r o t e i n i s observed as the major c e l l surface component when i n t a c t c e l l s are l a b e l e d with l a c t o peroxidase and I or with periodate and H-borohydride. These r e s u l t s i n d i c a t e that g l y c o p r o t e i n s i n the s o l i d tumor d i f f e r from those i n a s c i t e s l i n e s derived from i t . However, i t should be noted that the membrane preparations f o r s o l i d and a s c i t e s tumors are d i f f e r e n t . Perhaps the more s t r i n g e n t c o n d i t i o n s of c e l l d i s r u p t i o n used i n s o l i d tumor membrane p r e p a r a t i o n cause breakdown of a l a r g e g l y c o p r o t e i n to give m u l t i p l e smaller g l y c o p r o t e i n s . Two types of experiments argue against t h i s p o s s i b i l i t y . F i r s t , attempts to demonstrate a l a r g e g l y c o p r o t e i n i n the s o l i d 13762 tumor by p e r i o d a t e borohydride l a b e l i n g of the s o l u b i l i z e d t i s s u e followed by SDS e l e c t r o p h o r e s i s were negative. Second, milk f a t globule membranes contain the same three major s i a l o g l y c o p r o t e i n c l a s s e s as the normal mammary gland and s o l i d tumors. Since the milk f a t globule has not been subjected to mechanical d i s r u p t i o n , i t i s u n l i k e l y that the s i m i l a r c l a s s e s of g l y c o p r o t e i n s of normal mammary and the s o l i d tumors are products of the c e l l d i s r u p t i o n . What i s the o r i g i n of t h i s d i f f e r e n c e i n g l y c o p r o t e i n s between s o l i d and a s c i t e s forms of the tumor? Probably i t i s a response to the a l t e r e d environment of the a s c i t e s c e l l s , but 1 2 5

3


438


Mat - Bi IM-

MIGRATION DISTANCE (cm)

Figure 4. Membrane glycoproteins of the rat mammary adenocarcinoma MAT-B1 and MAT-CI sublines. Membranes were prepared essentially as described previously (17) for MAT-A cells. Electrophoresis and staining were performed as indicated in Figure 2. About 50% more protein was applied to the gel for the MAT-B1 sample.


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the mechanism of change i s u n c l e a r . One p o s s i b i l i t y i s an a l t e r e d gene expression r e s u l t i n g i n the synthesis of d i f f e r e n t p r o t e i n s . A l t e r n a t i v e l y , the change could r e f l e c t an a l t e r e d p o s t - t r a n s l a t i o n a l event such as an a l t e r e d a d d i t i o n of carbohydrate or c r o s s l i n k i n g of the p r o t e i n s . Although we cannot answer these questions a t present, i t should be p o s s i b l e to o b t a i n answers by biochemical and immunological comparisons of g l y c o p r o t e i n s from s o l i d and a s c i t e s tumors. Surface p r o p e r t i e s of the 13762 a s c i t e s tumor c e l l s . The sublines of the 13762 a s c i t e s carcinoma d i f f e r not only from the s o l i d tumor but a l s o from each other. The d i f f e r e n c e i n membrane s i a l o g l y c o p r o t e i n content between MAT-B1 and MAT-C1 c e l l s i s i l l u s t r a t e d i n F i g . 4. The a c t u a l d i f f e r e n c e between the two i s greater than that shown because of the amounts of membranes loaded onto the g e l s . C e l l surface s i a l i c a c i d r e l e a s e d by t r y p s i n or neuraminidase i s about 5 - f o l d greater from the MAT-C1 c e l l s than from MAT-B1 c e l l s , even though the t o t a l c e l l s i a l i c a c i d i s only about 40% g r e a t e r . Labeling s t u d i e s w i t h lactoperoxidase and I or with periodate and Hborohydride a l s o show more major s i a l o g l y c o p r o t e i n i n the MAT-C1 cells. The most s t r i k i n g d i f f e r e n c e between these s u b l i n e s i s t h e i r surface morphology, MAT-C1 c e l l s e x h i b i t i n g a very unusual surface s t r u c t u r e . T h e i r surface i s densely covered with m i c r o v i l l i extending from the c e l l body i n t o h i g h l y branched s t r u c t u r e s ( F i g . 5), whereas MAT-B1 c e l l s have a more normal appearance with fewer m i c r o v i l l i , a l l apparently unbranched (Fig. 6). MAT-B1 and CI c e l l s a l s o have d i f f e r e n t c e l l surface p r o p e r t i e s (Table I I I ) . When t r e a t e d with Concanavalin A (Con A), the MAT-B1 c e l l s r e a d i l y a g g l u t i n a t e . Under the appropriate c o n d i t i o n s of temperature and l e c t i n concentration these B l c e l l s a l s o form patches and caps. The MAT-C1 c e l l s do not respond to the l e c t i n treatment under the same c o n d i t i o n s , i n d i c a t i n g a r e s t r i c t i o n of the m o b i l i t y of the c e l l surface l e c t i n receptor s i t e s . I t i s a l s o noteworthy that the MAT-B1 and MAT-C1 c e l l s have d i f f e r e n t 5'-nucleotidase a c t i v i t i e s . This a c t i v i t y i s e s s e n t i a l l y undetectable i n B l c e l l s , even with a h i g h l y s e n s i t i v e radiochemical assay. In view of the l a r g e d i f f e r e n c e s i n c e l l surface p r o p e r t i e s one might expect s i g n i f i c a n t b i o l o g i c a l d i f f e r e n c e s between the B l and CI tumors. However, we have not found t h i s to be t r u e . The growth and k i l l i n g r a t e s of the two tumors are about the same. Tests f o r s t r a i n s p e c i f i c i t y of the tumors have not shown pronounced d i f f e r e n c e s , and p r e l i m i n a r y s t u d i e s suggest that both s u b l i n e s a r e m e t a s t a t i c . Thus i t appears that d i f f e r e n c e s i n c e l l surface p r o p e r t i e s a r e not n e c e s s a r i l y r e f l e c t e d i n the gross p r o p e r t i e s of the tumors. I t i s i n t e r e s t i n g that the p r o p e r t i e s of the MAT-B1 c e l l s f i t the c r i t e r i a o f Kim et aJL. (8) f o r a m e t a s t a t i c tumor under1 2 5


3

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Figure 5. Scanning electron micrograph of MAT-C1 cells. Cells were removed from the animal, fixed in 3% glutaraldehyde in 0.1M sucrose-OJM cacodylate, postfixed with 2% osmium tetr oxide, dehydrated in ethanol, dried in a Polar on critical point dryer, coated with gold/palladium, and examined in a JEOL JSM-35 Scanning Electron Microscope. Note the extensive microvilli, whose branched structures can be readily observed at the periphery of the cell. Magnification, 8160.


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Figure 6.

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Scanning electron micrograph of MAT-B1 cells prepared as noted for Figure 5. Magnification, 8840.


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TABLE I I I C e l l Surface P r o p e r t i e s of MAT-B1 and MAT-C1 Mammary A s c i t e s C e l l s

Property Agglutination Patching and Capping 5 -Nucleotidase, umoles/hr*mg p r o t e i n

MAT-B1

MAT-C1

-HHH-H-H-

-

0

2.0

1

going c e l l surface shedding. On the other hand the MAT-C1 c e l l s show s t r i k i n g s i m i l a r i t i e s to the TA3-Ha c e l l s (11). Thus these two s u b l i n e s , derived from the same s o l i d tumor, may represent two d i f f e r e n t modes of escaping the immune system of the host. Discussion The r e s u l t s presented permit us to supply t e n t a t i v e answers to the questions r a i s e d at the beginning of t h i s a r t i c l e . 1) There does not appear to be a necessary c o r r e l a t i o n i n mammary tumors between metastasis and g l y c o p r o t e i n shedding. However, both g l y c o p r o t e i n expression and metastasis are very complex phenomena, so t h e i r r e l a t i o n s h i p s may a l s o be complex. 2) Tumor environment apparently a f f e c t s g l y c o p r o t e i n expression, as i n d i c a t e d by the d i f f e r e n c e s between the a s c i t e s and s o l i d tumors. These r e s u l t s p o i n t out the problems i n v o l v e d i n e x t r a p o l a t i n g s t u d i e s on a s c i t e s c e l l s to conclusions about s o l i d tumors. However, the s t u d i e s on a s c i t e s c e l l s have t h e i r own inherent v a l u e . In the process of metastasis i n d i v i d u a l tumor c e l l s or small aggregates d i s s o c i a t e from the primary tumor i n t o the body f l u i d s . The s i m i l a r i t y of these d i s s o c i a t e d c e l l s to a s c i t e s c e l l s should be obvious. Since tumors l o s e many c e l l s , the c a p a c i t y of a tumor f o r metastasis must depend on the a b i l i t y of the d i s s o c i a t e d c e l l s to s u r v i v e i n the c i r c u l a t i o n and become e s t a b l i s h e d at a new s i t e . The a b i l i t y of a tumor c e l l to a l t e r i t s surface r e a d i l y i n response to a change i n environment might be a u s e f u l a t t r i b u t e f o r the m e t a s t a t i c tumor. Since the 13762 tumor has t h i s c a p a b i l i t y , f u r t h e r i n v e s t i g a t i o n of the r e l a t i o n s h i p s between the g l y c o p r o t e i n s of the s o l i d and a s c i t e s tumors may provide i n s i g h t i n t o the requirements f o r such changes. 3) The s t u d i e s on the MAT-B1 and MAT-C1 c e l l s c l e a r l y show that c e l l s with very d i f f e r e n t c e l l surface p r o p e r t i e s can be very s i m i l a r b i o l o g i c a l l y . We suspect that these r e s u l t s r e f l e c t the f a c t that d i f f e r e n t tumor c e l l s may use d i f f e r e n t ways of s u r v i v i n g the defense system of the


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host. However, i t i s a l s o p o s s i b l e that the mechanisms of tumor s u r v i v a l are too s u b t l e to be detected by the methodologies used i n our s t u d i e s . The s t u d i e s presented here have r a i s e d more questions than they have answered. The d i f f e r e n c e s between the MAT-B1 and MAT-C1 c e l l s are p a r t i c u l a r l y i n t r i g u i n g . The abundance and complexity of the m i c r o v i l l i at the surface of the CI c e l l i n d i c a t e the presence of an extensive submembrane c y t o s k e l e t a l network, s i n c e the m i c r o v i l l i c o n t a i n m i c r o f i l a m e n t s . One explanation f o r the extensive, branched m i c r o v i l l i i s that there i s an imbalance i n the turnover of plasma membrane compared to c y t o s k e l e t a l m a t e r i a l . However, these c e l l s a l s o cont a i n an excess of surface s i a l o g l y c o p r o t e i n . Is t h i s a c o i n c i d e n t or a r e s u l t of some type of coupling between the submembrane c y t o s k e l e t o n and extramembrane g l y c o p r o t e i n ? This question assumes greater importance i n view of the p o s s i b l e r o l e s of c e l l surface component r e d i s t r i b u t i o n s i n v a r i o u s c e l l u l a r responses. Our p r e l i m i n a r y s t u d i e s i n d i c a t e that the s i a l o g l y c o p r o t e i n i s not s o l e l y r e s p o n s i b l e f o r i n h i b i t i o n of Con A receptor movements i n the CI c e l l s . There a l s o appears to be a c y t o s k e l e t a l involvement. Regardless of the exact mechanism by which receptor movement i s repressed, the absence of m o b i l i t y may be an important f a c e t i n escape from immune d e s t r u c t i o n . I f m o b i l i t y of the antigens of the target c e l l i s a p r e r e q u i s i t e f o r i t s d e s t r u c t i o n , the immobilization of the e n t i r e surface would be an e f f e c t i v e defense. T h i s might a l s o be considered as an a l t e r n a t i v e mechanism f o r the l a c k of s t r a i n s p e c i f i c i t y of the TA3-HA c e l l s . The decreased nucleotidase and c e l l surface g l y c o p r o t e i n s of the MAT-B1 c e l l s are a l s o i n t r i g u i n g . Can t h i s r e a l l y be explained by a "shedding" mechanism? Or has the c a p a c i t y to synthesize these molecules been l o s t ? I t i s necessary to examine the dynamics of the surfaces of these c e l l s i n order to understand t h e i r behavior. Such s t u d i e s are complex but could provide i n s i g h t i n t o the r e l a t i o n s h i p of c e l l s t r u c t u r e and n e o p l a s t i c behavior. In attempting to understand tumor c e l l behavior i n r e l a t i o n s h i p to i t s c e l l surface p r o p e r t i e s , an important concept i s that tumor c e l l s appear h i g h l y adaptable. Perhaps t h i s a d a p t a b i l i t y i s the key to the success of the tumor i n s u r v i v i n g a h o s t i l e environment and i s the cause of much of the f r u s t r a t i o n i n t r y i n g to e s t a b l i s h d e f i n i t e r e l a t i o n s h i p s between cancer and p a r t i c u l a r c e l l p r o p e r t i e s . This a d a p t a b i l i t y i s not l i m i t e d to c e l l surface p r o p e r t i e s , as evidenced by the a b i l i t y of tumors to develop drug r e s i s t a n c e . However, the c o n t r o l of the c e l l surface would a s s i s t the c e l l i n meeting the r i g o r s of i t s environment. Studies on c e l l surface v a r i a t i o n s should help us understand the r e l a t i o n s h i p of c e l l surface to tumor s u r v i v a l and the more general question of a d a p t a b i l i t y of tumor c e l l s .


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Acknowledgements This is contribution P-490 of the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma. This research was conducted in cooperation with the USDA, Agricultural Research Service, Southern Region and supported by the National Cancer Institute (NO-l-CB-33910 and CA 19985), the American Cancer Society (BC-246), a Presidential Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station. Literature Cited 1. Hughes, R. C. (1976) in Membrane Glycoproteins, Butterworths, London. 2. Emmelot, P. (1973) Eur. J. Cancer 9, 319-333. 3. Nicolson, G. L. (1974) Int. Rev. Cytol. 39, 89-190. 4. Buck, C. A. Glick, M. C., and Warren, L. (1970) Biochemistry 9, 4567-4576. 5. Shin, B. C., Ebner, K. E . , Hudson, B. G., and Carraway, K. L. (1975) Cancer Res. 35, 1135-1140. 6. Hynes, R. 0. (1974) Cell 1, 147-156. 7. Hellström, K. E . , and Helström, I. (1974) Adv. Immunol. 18, 209-277. 8. Kim, U., Baumler, A., Carruthers, C., and Bielat, K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1012-1016. 9. Friberg, S., Jr. (1972) J. Natl. Cancer Inst. 48, 14631476. 10. Codington, J. F., Sanford, B. H., and Jeanloz, R. W. (1973) J . Natl. Cancer Inst. 51, 585-591. 11. Miller, S. C., Hay, E. D., and Codington, J. F. (1977) J. Cell Biol. 72, 511-529. 12. Hilf, R., Michel, I., and Bell, C. (1967) Recent Prog. Hormone Res. 23, 229-289. 13. Segaloff, A. (1966) Recent Prog. Hormone Res. 22, 351-379. 14. DePierre, J. W., and Karnovsky, M. L. (1973) J. Cell Biol. 56, 275-303. 15. Steck, T. L . , and Wallach, D. F. H. (1970) Methods in Cancer Res. 5, 93-153. 16. Keenan, T. W., Morré, D. J., and Huang, C. M. (1974) in Lactation: A Comprehensive Treatise, (B. L. Larson and U. R. Smith, eds.), Vol. 2, Academic Press, New York, pp. 191-233. 17. Carraway, K. L . , Fogle, D. D., Chesnut, R. W. Huggins, J. W., and Carraway, C. A. C. (1976) J. Biol. Chem. 251, 6173-6178. 18. Beaufay, H., and Amar-Costesec, A. (1976) Methods in Membrane Biol. 6, 1-100. 19. Huggins, J . W., and Carraway, K. L. (1976) J. Supramol. Struct. 5, 59-63.


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Warren, L. (1959) J. Biol. Chem. 234, 1971-1975. Warren, L . , Glick, M. C., and Nass, M. K. (1966) J. Cell. Physiol. 68, 269-287.

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