the membrane glycoproteins of the malignant cell

Clin. Biochem. 13 (5) 191-197 (1980}

The Membrane Glycoproteins of the Malignant Cell

L E O N A R D W A R R E N AND C L A Y T O N A. B U C K

The W i s t a r Ins t i tu te , 36th and Spruce S t ree ts , P h i l a d e l p h i a , Pa. 19104

Experiments are described and reviewed demonstrating that the bound carbohydrates of glycoproteins of many forms of malignant cells differ from their normal counterpart. The difference involves many oligosaccharide groups and is essentially quantitative. The characteristics of the difference are discussed. Despite the consistency of the finding its significance is unknown because the function of bound carbohydrates is largely unknown. Some properties of protein-bound carbohydrates that may be of special relevance to malignancy and other patho- logical processes are considered. The array of structures found in the cell is highly complex but seems to be similar in man, hamster, mouse, chick and fish. On the other hand, the biosynthesis of these structures can be influenced and altered by the environment and by drugs; the cell is tolerant of variation in its bound carbohydrate; microheterogeneity of the carbohydrates is probably the rule rather than the exception; experiments to test the function of bound carbohydrate show only small effects. A role for the bound carbohydrates in evolution is proposed that is consistent with these characteristics. It is also postulated that altered, bound carbohydrates of most glycoprotein does not en- danger the life of the cell but may be responsible for involvement and change of many processes some of which permit the malignant cell to divide persistently and to prosper.

GLYCOPROTEINS OF THE CELL ARE FOUND LARGELY IN or on the surface membrane with their bound oligosaccharides outside the lipid barrier. If they lie on the membrane, they are considered "extr insic" and can probably be removed with solutions of salt. If they t raverse the membrane, part of the polypeptide chain interacts with the membrane lipid through hydrophobic bonding. These glycoproteins are considered "integral" and can be removed from the membrane with detergents . Some glycoprotein is found in all of the membrane systems of the cell; in the membranes of the nucleus, mitockondria, endoplasmic reticulum, Golgi apparatus and lysozomes. Still others, (mucins, plasma proteins etc.) are found in soluble form both in the cell and in purely extracel lular loci.

I t is not unreasonable to assume that bound carbohydrates of the cell surface, which face the outside world, might play an important role in cellular interactions with the environment. They are involved in the in- itial binding of hormones and viruses; carbohydrate- containing proteins may function in intercellular adhesiveness, in cell recognition mechanism, in sperm- egg interactions and probably in growth control (1, 2, 3). Involvement in more specific processes will be discussed later, but it should be apparent that if bound carbohydrates play a part in intercellular adhesiveness and growth control, then their behavior in the cancer cell should be examined.

Changes have been described in the oligosaccharides bound to lipids of the malignant cell (for review, 4, 5). Protein-bound carbohydrates of malignant cells may change because ent i re glycoproteins may decrease,

disappear or increase (see 6). The changes we will discuss in malignancy are brought about by al terat ions of carbohydrate groups on polypeptide chains i.e. shifts in populations of polymeric groups. What do these shifts mean? The answer to this question lies in our understanding of the function of bound carbohydrates. Aside from a few examples (3, 7), little is known about this mat te r and we must admit that at present the significance of this al terat ion in the malignant cell is unclear, despite its widespread occurrence.

PROTEIN-BOUND CARBOHYDRATES IN MALIGNANCY

A change can be seen clearly when the bound carbohydrates of a control cell in culture such as baby hamster kidney (BHK21/C13) are compared with those of its virus-transformed (malignant) counterpart (C13/B4) (8, 9) (Fig. 1). For this purpose one cell type is grown in the presence of 14C-D-glucosamine and the other with 3H-D-glucosamine. Other pairs of I4C and 3H-labeled sugars can also be used. After 48 hr of growth in log phase, the cells are briefly treated with trypsin to free them from their underlying substrate. Approximately 30O/o of the metabolically incorporated isotopic sugar is released in soluble form from the cell surface. Trypsinate from the cell surface and pellet from fractions from the material within the cell are obtained. 14C- and 3H-labeled fractions are combined and treated exhaustively with pronase to remove most of the amino acids from the fragmented glycoproteins. The carbohydrates are not altered by this procedure. The resulting mixed 14C- and 3H-glycopeptides are then fractionated together on a column of Sephadex G50 which separates on the basis of size (and to a lesser extent, shape} of the molecule (Fig. 2). The procedure described has revealed that malignant cells synthesize glycoproteins that bear more, larger carbohydrate groups than do corresponding controls. These early-eluting molecules are called "group A" glycopeptides and consist of a whole family of carbohydrate groups of M r 4200-5500 that are not resolved by Sephadex G50. Other column fractions of glycopeptides, called "B" and "C" (Fig. 2) do not seem to differ from the control. The first peak {tubes 1-5) consists largely of glycosaminoglycans and the last peak (tubes 50-70) consists of very small oligosaccharides, variable in amount that do not appear to be related to malignancy.

Studies with tissue culture cells have shown that the level of group A structures on glycoproteins varies with the state of growth of the cells, being high during the log phase of growth of the cells, and declining to low levels when cells cease to divide (15, 16). Because of this variation, the bound carbohydrates of control and malignant cells are compared, if at all possible, when both cells are in log phase and are dividing at approximately the same rate.

The increase of group A oligosaccharides has been found in a wide variety of malignant cells of human, rat, hamster, mouse and chicken origin (10, 11). Van Beek et al (12) have found this increase in glycoproteins of the surfaces of human acute and chronic lymphocytic and myelocytic leukemic cells and in cells from individuals with Burkitt's lymphoma but not with infec- tious mononucleosis. The change has been seen in malignant

192 WARREN AND BUCK

Procedure for Fractlonation of GiycopeptidQ=:

| I

i | I ;

1 I I I

I I I I 1 I o I . . . . . . . . . . . . . . . . . . . . . . . . . . I

1 1 1 1 1 I

1 I 1 ~c~. 'r1.c rL¢

Fig. 1 -- Procedures for preparation and analysis of glycopeptides. (Blithe et al. 1980).

cells of fibroblastic, lymphocytic and epithelial origin and in cultured cells that have become malignant spontaneously or by exposure to carcinogenic chemicals, DNA- or RNA-containing oncogenic viruses. Changes have been demonstrated in solid tumors (9).

Glick et al (13, 14) have shown a direct relationship between the relative amount of group A type of bound oligosaccharides in chemically and viral ly t r ans fo rmed cells and the tumorigenicity of the cells in appropriate hosts. Some transformed cells that were non- or poorly tumorigenic did not bear increased amounts of group A structures (14). With the spontaneous acquisition of tumorigenic potential in cells that manifested some in vitro criteria of transformation, the level of group A glycopeptides increased. There appears to be a closer relationship between an increase of group A glycopeptides and tumorgenicity than with in vitro criteria of transformation.

In summary, an impressive range of malignancies undergo characteristic shifts in some of the sugar groups of their glycoproteins. These shifts are independent of the cell type, transforming agent, or species of origin and are directly correlated with the tumorigenic potential of the cell.

In the past few years the glycopeptides have been fur ther fractionated by methods based on affinity for Concanavalin A (17) and on charge (DEAE-Sephadex) (18,19) (Fig. 3). Separation of group A, B and C into approximately 35 fractions in a reproducible manner has been accomplished (Fig. 3) (19). Based on results using columns of Sephadex G50, our original and preliminary conclusion was that the glycoproteins of malignant cells contained more group A (and "pre-A" in tubes 10-16 of Fig. 2) carbohydrate groups and that differences between control and malignant cells though definite and consistent are essentially quantitat ive (9). This conclusion still holds after separation of glycopeptides into many peaks.

It can be seen in Fig. 3, panels a and b, that the "pre A" and Group A glycopeptides are more abundant in the transformed cells compared to controls. On the other hand, the glycopeptides of Group B*(Con A(+) and ( - ) and Group C.Con A(+) are more abundant in the control cells. This can be explained by shifts in populations of oligosaccharides bound to the polypeptide where one group increases at the expense of another. I t should be noted that the shifts involve many glycopeptides.

We have fur ther fractionated radioactive glycopeptides in the discrete peaks seen in Fig. 3 by thin-layer chromatography (20). As many as 5 spots can be detected in Group B-Con A( + ) ,#2. Group A.Con A ( - ) #4 has revealed 2 spots by autoradiography. It is apparent tha t after fractiona- tion by TLC the total array of carbohydrate groups is exten-

ABBREVIATIONS: Con A = Concanavalin A; DEAE = diethyl-aminoethyl; L-fucose = 6-deoxy-L-galactose; sialic acid = N-acetylneuraminic acid; mannose = D-mannose; galactose = D-galactose.

. I iP,~ A

t :13/B~" "II I /

\ /~ /

' 2'0

,B [ C ( O-Glucosamine

; t ~j I--BHK21/CI3

t

S C ~"/

60 80 100

Fraction Number

180

135 t i i

45

Fig. ~ -- Sephadex G50 Chromatography of Glycopeptides. Control hamster cells IBHK21/C13] grown in the presence of SH-D-glucosamine and their virus-transformed, tumorigenic counterpart C13/B~ labeled in culture with ]~C-~glucosamine were treated with t ryps in Both lines of cells grew with approximately the same doubling time 121 hr/. The labeled material removed from the surfaces of the cells were mixed and fur ther digested with pronase. The digest with approximately equal numbers of SH and 14C counts was applied to a column of Sephadex G50 (1 X 100 cm) and eluted (19]. The SH and 1~C in the eluted material in each tube was counted and the data was processed and plotted by computer. The material in tubes 1-5 is largely glycosaminoglyca~, Note the relatively large amount of group A glycopeptide material derived from C13/B~ cells.

sive. However it is probable at this point tha t we are dealing with homogeneous populations of glycopeptides. We can now sense the degree of complexity, of the variety and functioning of bound carbohydrates of the cell. Despite this, we have not yet seen any clear, reproducible qualitative differences in the glycopeptides of control and malignant cells nor is there any evidence for a carbohydrate group peculiar to the malignant cell. In general it appears tha t the polypeptides of malignant cells carry more, larger carbohydrate groups, probably bearing slightly more sialic acid residues on the average than the glycoproteins from control cells.

Changes in the carbohydrates take place in the glycoproteins located in all of the membrane systems of the malignant cell. Control (BHK21/C13) and malignant (C13/B4) cells metabolically labeled with 14C and 3H-D-glucosamine, respec- tively, were fractionated into plasma membrane, smooth endoplasmic reticulum (ER), rough ER, mitochondria, lysozomes and nuclei. Glycopeptides were obtained by digestion with pronase. Comparison of Sephadex G50 pat terns of double-labeled material of each organelle clearly showed an increase of Group A glycopeptides in the malignant cell (21).

Fur ther , in double-label exper iments where individual glycoproteins from control and malignant cells were isolated and their carbohydrates compared, over 80o/o of the purified glycoproteins of malignant cells showed alteration of their carbohydrates(22). These experiments demonstrate tha t the observed changes of glycopeptides in malignancy reflect a shift in populations of carbohydrate groups on individual and probably unchanged polypeptide chains of glycoproteins ra ther than changes in the overall level of specific glycoproteins with special carbohydrate compositions (23, 22). We can also con- clude from the data that the malignant cell suffers structural change in the bound carbohydrate of most of its glycoproteins in every membrane system of the cell.

Speculation on Glycoproteins and Malignancy

Although malignancy probably begins with a discrete, qualitative, structural change in its nucleic acid {mutation), the malignant cell is noted for the quantitative changes from normal of many structures and functions, most of which are probably not relevant to the malignant process but are merely associative. We suggest here that the extensive alterations of

MEMBRANE GLYCOPROTEINS OF THE MALIGNANT CELL 193

'°° ~ ~ r " "

,o~ ~ \ L T ° ' '

im~c,,Oq ~ l l m

: : [ - .......

• o ~ . : , o , y . : : ? o

|Too

l l o o

Hi t o

Fig. 3 -- DEAE-Sephadex chromatography. Glycopeptides from the surfaces of BHK21/C13 cells metabolically labeled with 14C-~glucosamine { J and from C13/B~ cells labelled with 3H-D-glucosamine (----/ were fractionated according to size land shapeJ into groups pre A, A, B & C on a column of Sephadex GSO fFig. 1 & 2J. Each fraction was applied to a column of Sepharose-Concanavalin A and divided into fractions that adhered I + / or did not adhere {--/ to Con A {Fig. lJ. Those fractions with sufficient radioactivity were further fractionated according to charge on columns of D E A E Sephadex. The glycopeptides were eluted first with a linear salt gradient from 0 --* 0.1M sodium borate in 0.01 M pyridine acetate, pH 4.5, then with a second linear salt gradient from 0 ~ 1.0M sodium acetate in 0.1M sodium borate and 0.01 M sodium acetate. / / glycopeptides from {]4C}-labelled control cells; 5---J glycopeptides from I$H/-labeled transformed cells {----/ linear salt gradient based on the molarity of sodium borate {. . . . . . . . J linear salt gradient based on the molarity of sodium acetate {Blithe et al. 1980J.

bound carbohydrates may be the underlying basis for the cascade of involvements of various processes of the cancer cell. The glycoproteins function as enzymes, hormones, receptors and structural and other functional elements. Since changes occur in the carbohydrates of most glycoproteins (> 80O/o), these s tructural alterations may result in myriad shifts of activities tha t would not threa ten the life of the cell since bound carbohydrates seem to operate in a non-acute mode and the cell can tolerate variation in s t ructure of this component. I t is likely tha t they function primarily in an integrative capacity and that they influence only rates of processes or affinities of binding between two molecules. Perhaps the changes in bound carbohydrates of malignant cells affect the ability of cells to adhere to each other, to t ranspor t essential nutr ients into the cell and to sense inhibitory messages from other cells. Altered glycoproteins might, among numerous other effects, be the basis for the cells' persistence to divide. Since so many forms of glycopeptides are involved in the shift in the malignant cell, it is probable tha t no single change in composition will describe the events completely.

It was suggested at one time that the major and perhaps only difference between the glycopeptides of control and malignant cells was due to increased amounts of sialic acid on the glycopeptides of malignant cells. Trea tment of glycopeptides from both types of cells with neuraminidase led to reduction in their size and eliminated any differences in their elution pat- terns from columns of Sephadex G50(24,25). A sialyl t ransferase was described tha t t ransferred sialic from its ac- t i v a t e d form, cy t id ine -5 ' -monophosphos ia l i c acid, to desialylated Group A glycopeptide. Because this t ransferase activity was several fold greater in transformed cells than in control, it seemed likely tha t elevation of this activity might be a key change. Bosmann (26) had also found an elevation in sialyl t ransferase activity in transformed cells, but other researchers have found no real difference (27, 28). The work of Ogata et al (17), Santer and Glick (29) and Blithe et al (19) indicate tha t

the changes in malignancy are complex and involve more than sialic acid. Evidence has been presented that the Group A glycopeptides differ from the B type in tha t they contain an ex- t ra trisaccharide attached to the core, N-acetyl-D-glucosamine- D-galactose-sialic acid (17, 29). Earlier work had shown that there were changes in the mannose core of the larger Group A type of the glycopeptides in growing cells compared to non- growing cells (16). The larger glycopeptides, which are more abundant in growing cells, are probably the same as those tha t are far more abundant in growing malignant cells than in growing normal cells.

Since the changes involve many oligosaccharides on many glycoproteins, the defect in malignancy is probably one of general carbohydrate biosynthetic machinery since both cellular glycoproteins of transformed cells (30) and viral glycoproteins produced by the transformed cells (31) bear increased amounts of Group A type of glycopeptides.

A difference in the s t ructure of glycoproteins of normal and malignant cells has been described which is perhaps the most consistent structural difference known. Unfortunately, we do not know at present how it occurs, nor, more importantly, how relevant it is in malignancy in terms of creating and arming the malignant cell once the appropriate genetic changes have taken place. We are at a loss because the basic functions of bound carbohydrates are not known despite some information about specific sugars in certain sites tha t have specific functions. The fact is that in most experiments in which protein- bound carbohydrate is altered to tes t its role in a process, no change of any magnitude is observed. The bound sugars do not seem to do anything. Are they vestigial like a human appendix? This does not seem to be a promising s ta r t for implicating carbohydrates in the complicated disorder of malignancy.

However certain relevant characteristics of protein-bound carbohydrates will now be considered that may help us to exploit them for the diagnosis and therapy of malignancy.

Characteristics of Protein-bound Carbohydrates

1. Complexity. The closer we look at the number of species of protein-bound carbohydrate groups in a cell, the larger the number grows. Our routine procedure permits us to identify about 35 peaks containing glycopeptides of M r 2000-5500 {19}. Other carbohydrate structures, mostly glycosaminoglycans and very large glycopeptides are found in the peak of material excluded from Sephadex G50. There appear to be glycopeptides of M r approximately 5500-10,000 that are present on the glycoproteins of embryonic ( teratocarcinoma) cells t ha t decrease sharply in amount but do not disappear al together when cells differentiate {32, 33). There are also some glycopeptides smaller than those tha t elute from Sephadex G50 in the group C area (Fig. 2). Recently, we have described purified membrane hybrid glycoproteins from control and virus- transformed baby hamster kidney cells that contain oligosaccharides linked to OH of serine and/or threonine and to the amide N of asparagine and in addition bear short segments of glycosylaminoglycan covalently linked to the polypeptide {34). As mentioned previously further separation of materials in A, B and C areas (Fig. 2) using thin-layer chromatography have revealed as many as 2 to 5 spots from samples eluting as sharp peaks from columns of DEAE-Sephadex. In summary, there is clearly a formidable array of carbohydrate polymers, perhaps 60 to 100 or more, tha t can modify the polypeptide chain. 2. Glycopeptides in nature. We have been fractionating the glycopeptides of other species of cells in culture. Our work to date reveals remarkable similarity in the DEAE-Sephadex elution pat terns of material from human, hamster, mouse, chick and fish cells (Blithe et a~ unpublished}. Although some quan- t i tat ive and qualitative differences between these cells are seen, it appears that the populations of bound carbohydrates though very complex, may be to a large extent common to vertebrate , sialic acid-containing species and that these structures have been stable for hundreds of millions of years. 3. Microheterogeneity. In the course of our work, we have examined the glycopeptides from crude "trypsinates", and in pellet fractions where complex arrays of isotopically labelled molecules from many glycoproteins have been resolved. It was felt tha t if glycopeptides could be obtained from purified,

194 WARREN AND BUCK

a Group A'Con A (-) 200

100 ~ - ~

0

112 b Group B'Con A (+1

rJ 75

Q_ 37

0 I I I I I I I I I I = = = = = I

c Group B'Con A (-)

30O :E q

150 ':" "

0 20 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 160 180

Tube number Fig. 4 -- DEAE-Sephadex chromatography of glycopeptides obtained from a single puri f ied glycoproteins of hamster cells, BHK21/C13 (Al-3) (34). The glycopeptides had been subdivided on the basis of Sephadex G50 elution (size and shape) and binding affinity for Con A into Group A =Con A 5), Group B=Con A/-) and Group B=Con A(+). There was not enough Group A .Con A( + ) or Group C material for column chromatography. Each group was subjected to ion exchange chromatograpgy on DEAE-Sephadex A25 as described in the legend of Fig. 3 (19).

(oJ Group A glycopeptides not adherent to Con A. (b] Group B glycopeptides adherent to Con A. (c) Group B glycopeptides not adherent to Con A.

homogeneous labeled glycoproteins, the array would be greatly simplified because a homogeneous glycoprotein might bear only a few carbohydrate groups. Cells were grown in the presence of 14C-D-glucosamine, and glycoproteins were purified to homogeneity where fingerprinting revealed single amino terminal peptide groups. After t rea tment with pronase, purification of glycopeptides revealed a complex pat tern of glycopeptides (Fig. 4)(22,34) with as many as 10 to 15 peaks (34). From the approximate M r of the purified glycoprotein and the numerous glycopeptides, it can be calculated that there is too much carbohydrate to be accommodated on a single polypeptide chain. Our conclusion is that the glycoproteins may be homogeneous in their polypeptides but not in their carbohydrate groups. Microheterogeneity has been known for some time and it has been found in ovalbumin (35), t ranspor t proteins (36, 37) and many other glycoproteins (for reviews see 35 and 38). Our experience suggests to us tha t microheterogeneity of various degrees may be found in most if not all glycoproteins depending on how carefully one looks for it. I t should be noted that very few complex glycopeptides have been isolated which upon analysis yields ratios of sugars tha t are truly integral. Non-integral ratios inescapably signify

t/;I t 340 ' ~ 271 !

° .7 .2 /

227113 ~ 18190

0 0 0 20 4O 60 O0

TUBE NUMBER Fig. 5 -- Sephadex G50 chromatography. Double-label experi- ment in which one f lask of C13/B4 cells were grown for 16 hr at pH 6.5 in the presence of ~4C-D-glucosamine (- - . . . J and another f lask of cells was grown at pH Z2 in the presence of SH-D- glucosamine. (-- --L Materials were removed from the surfaces of the cells with trypsin, equal amounts of z4C and 3H radioac- t iv i ty were combined and exhaust ively digested with pronase. The digest was chromatographed on a column of Sephadex G50 as described in the legend of Fig. 2. There appears to be much less group A & B material formed at pH 6.5 but much more small glycopeptide (probably 1-3 sugars] accumulated at this pH.

heterogeneity and a complexity of polSulations tha t the analyst would ra ther forget.

4. Variability of structure. We have described changes in the s t ructure of protein-bound carbohydrates. These changes can take place at certain points in the growth cycle of the cell (15, 16, 39, 40) or when malignant cells are grown in the presence of butyrate (41), cyclic AMP (42}, and when the cell becomes malignant. Radical alterations in the carbohydrates are found when cells are exposed to small amounts of ethidum bromide (43), and changes must certainly occur in cells that are mutant in enzymes involved in the biosynthesis of mono- and oligosaccharides (44). Shifts in populations of glycopeptides have been found in a mouse cell defective in its ability to acetylate glucosamine (48). Differences in bound carbohydrates can be found in cells grown at different pH values (Fig. 5). I t is probable that biosynthesis of these s t ructures is responsive to environmental influences (availability of substrate, s tate of the sugar transferases, metabolic state, NADH/NAD ratio, pH, ca- tion concentrations, etc.), because their s t ructures are not dic- tated by a template. 5. Function~ Some involvement of certain sugars in specific processes have been described (1, 3, 7). On the whole, however, most work in which the s t ructure of protein-bound carbohydrate is altered to induce changes in the function of glycoproteins has been disappointing. The enzymatic removal of various sugars from the surface of the cell is clearly not lethal. Perhaps the tests have not been sufficiently s t r ingent because in most experiments only terminal sialic acid has been removed and the underlying sugars left intact. Nevertheless, our impression is that the bound carbohydrates are not acute in their functioning and the cell can tolerate wide variation in the makeup of its bound carbohydrates. On the other hand the cell cannot do without bound carbohydrates. Tunicamycin which prevents the formation of oligosaccharide groups that are t ransferred to the polypeptide chain of glycoproteins and thus arrests the formation of glycoproteins (45, 46) is highly toxic to cells (47). I t is also known that many mutants of carbohydrate and glycoprotein metabolism, if they survive at all without missing nutrients, are seriously impaired (48).

A Possible Role for Bound Carbohydrates in Evolution

The characteristics of bound carbohydrates briefly outlined seem to give rise to a paradox. On the one hand they are probably very ancient and universal in living organisms, possibly


predating the origin of life. They are present in significant amounts and in complex arrays tha t have been stable for vast periods of time. These would suggest that they are of critical importance. On the other hand microheterogeneity of carbohydrate s t ructure of glycoproteins may be the rule not the exception, the carbohydrate s t ructure can be changed by altering the environment or with drugs and the cell is tolerant of these changes {within limits}. More often than not only small changes in function of glycoproteins are effected by altering their carbohydrates. The following hypothesis is presented to resolve these contrast ing elements. Whatever the function of a glycoprotein a single species of molecule is not involved because numerous combinations of carbohydrate groups are probably resident on the polypeptide chain. If the glycoprotein must operate in different environments there are always polypeptide chains bearing certain combinations of carbohydrate groups that operate optimally. This would con- s t i tute a preadaptive advantage where small differences in function of the various molecular species in various environments would be a significant factor over evolutionary periods of time. The carbohydrate groups might operate here as covalently bound allosteric effectors controlling conforma- tion and mobility of polypeptide chains. By contrast, in the known examples of response where removal of a sugar leads to the appearance or disappearance of a function, bound carbohydrates may have become a component of specific mechanisms, perhaps late and unpredictably in the course of evolution.

In support of the above notion, glycoproteins are most abundant where the cell has least control of the e n v i r o n m e n t - o n the cell surface (50 to 70% of the total glycoprotein of the cell) or in the intercellular compartment. There is considerably less bound carbohydrate within the cell and least in the nucleus separated from the outside by three membrane systems where control might be expected to be maximal. Most metabolic and intracellular enzymes do not bear carbohydrate except for the lysozomal enzymes which process material from the outside. What has been postulated here for glycosylation can apply with some variation to other forms of posttranslational modification.

In pathology, these same small differences found naturally or induced by altered environments operating over the long term, tolerated by the host but ultimately result ing in breakdown, could be the basis of chronic diseases {arteriosclerosis, long-term effects of diabetes, ageing), silent and undetected for long periods of time.

significantly elevated (56). An increase in the serum level of an isozyme of galactosyltransferase has been shown to correlate well with malignancy in humans (57). Fur ther experience is needed to determine whether these enzymes, which in general are not simple to assay, will be clinically useful for the diagnosis and monitoring of patients during t rea tment and in remission.

From our previous discussion, it would seem that precise and specific forms of bound carbohydrates of glycoproteins are not acutely essential cell consti tutents which, if tampered with, would lead to the death of the cell. This would not satisfy one of the requirements of the coventional rationale of tumor therapy which is to find a difference in some essential process between the normal and malignant cell and to exploit this difference in order to kill the malignant cell.

The increase in Group A glycopeptides in the malignant cell does appear to be a remarkably consistent occurrence. However, the ability of cells to tolerate shifts in their bound carbohydrates and the lack of acute function ascribable to this component do not inspire hope that this particular change in bound carbohydrate can be exploited unless the situation is considered in a different way. Let us postulate that the most important characteristic of the malignant cell, persistent cell division, is closely associated with and perhaps requires that more, larger sugar groups be attached to its glycoproteins. In other words greatly elevated Group A glycopeptide levels, nor- mally associated with the growth of the cell, are in some way a part of the expression of malignant behavior. If the level could be lowered to "normal", perhaps persis tent cell division would cease. As stated previously, the populations of oligosac- charities of a glycoprotein are not synthesized on a template. Their synthesis probably depends on the metabolic s tate of the cell, and various drugs can alter synthesis. The therapeutic rationale is to alter the synthesis of bound carbohydrates so that fewer Group A glycopeptides are made, and perhaps the malignant cell will calm down and the tumor will become silent. The aim of this approach is more modest than that of the conven- tional approach. It is not to eliminate and cure, with its atten- dant damage to the host, but ra ther to ar res t the incessant growth of the tumor without its eradication. Hopefully, the agents that might affect oligosaccharide biosynthesis would be relatively gentle in their side effects as normal cells can probably tolerate lowering of the level of Group A glycopeptides on their glycoproteins.

ACKNOWLEDGEMENTS

Implications for Diagnosis and Therapy

Although the change in glycoprotein carbohydrates in malignancy :s very widely found, it is doubtful that this will be useful in di. jnosis. The natural variation of the Group A glycopeptides during growth, though of smaller magnitude in the normal cell as compared to the increase found in the malignant cell, will always undermine confidence in the test. Ap- propriate control cells for a tumor are often not available or even defined. For a meaningful comparison, detailed knowledge of the s tate of growth of control and malignant cells during isotopic labeling is essential, and this is not always ob- tainable.

As previously mentioned, sialyl t ransferase activity of virus- transformed cells was found to be higher than in the corresponding controls (49, 30). Others have found no difference or a decrease in activity in transformed cells (27, 28). Differences in results may be due to the sugar acceptor used in the assay.

There are several studies in which sialyl transferases in the plasma or serum of animals (51, 53} and humans (50, 52) bearing tumors were compared to controls. The level of activities in serum from those with tumors is clearly elevated. Presumably the increased level of enzymes arises from the shedding by the tumor. The function of these enzymes in the serum is unknown, and it is especially puzzling because activated sugars are probably present in the serum in extremely small concentrations. Similarly, tumor patients manifest elevated levels of serum fucosyl transferases (54, 55). Galactosyl t ransferase is elevated several-fold in human ovarian tumors compared to normal ovarian tissue, and the serum level of this enzyme is

Much of t h e w o r k d e s c r i b e d in t h i s p a p e r w as done w i t h t h e s u p p o r t of t h e fo l lowing g ran t s " CA-19130, A C S BC 275, C A - 1 0 8 1 5 a n d T r a i n i n g G r a n t #T32-CA-09171 a w a r d e d to t h e W i s t a r I n s t i t u t e .

REFERENCES

i. Cook, G.M.W. and Stoddart, P.W. Surface carbohydrates of the eukaryotic cell, Academic Press, New York, {1973}.

2. Nicolson, G.L. Trans-membrane control of the receptors on normal and malignant cells. Biochim. Biophys. Acta. 458, 1-57, (1976}.

3. Hughes, R.C. Membrane Glycoproteins, Butterworths, London, (1976).

4. Hakomori, S. Structures and organization of cell surface glycolipid dependency on cell growth and malignant transformation. Biochim. Biophys. Act,- 417, 55-89, (1975).

5. Critchley, D.R. Glycolipids as membrane receptors important in growth regulation. Chapter 3. In Surfaces of Nor- mal and Malignant Cells., ed. R.O. Hynes, J. Wiley and Sons, New York, pp 63-101, (1979).

6. Hynes, R.O. Proteins and Glycoproteins. Chapter 4. In Surfaces of Normal and Malignant Cells, ed. R.O. Hynes, J. Wiley and Sons, New York, pp. 103-148, (1979).

7. Rosenberg, H. and Schengrund, C.L. eds. Biological Roles of Sialic Acid, Plenum Press, New York, (1976).

8. Buck, C.A., Glick, M.C. and Warren, L. A Comparative study of glycoproteins from the surface of control and Rous sarcoma virus-transformed hamster cells. Bio- chemistry 9, 4567-4576, (1970}.

196 WARREN AND BUCK

9 Warren, L., Buck, C.A. and Tuszynski, G.P. Glycopeptide changes and malignant transformation. A possible role for carbohydrate in malignant behavior. Biochim. Biophys. Acts 516, 97-127, (1978).

10. Buck, C.A., Glick, M.C. and Warren, L. Glycopeptides from the surface of control and virus-transformed cells. Science 172a, 169-171, (1971).

11. Van Beck, W.P., Smets, L.A. and Emmelot, P. Increased sialic acid density in surface glycoproteins of transformed and malignant cells. A general phenomenon7 Cancer Res. 33, 2913-2922, (1973).

12. Van Beck, W.P., Smets, L.A. and Emmelot, P. Changed surface glycoprotein as a marker of malignancy in human leukemic cells. Nature 253, 457-460, (1975).

13. Glick, M.C., Rabinowitz, Z. and Sachs, L. Surface membrane glycopeptides correlated with tumorigenesis. Biochemistry 12, 4864-4869, (1973).

14. Giick, M.C., Rabinowitz, Z. and Sachs, L. Surface n~em- brahe glycopeptides which coincide with virus transformation and tumorigenesis. J. ViroL 13, 967-074, (1974).

15. Buck, C.A., Glick, M.C., and Warren, L. Effect of growth on the glycoproteins from the surface of control and Rous sarcoma virus-transformed hamster cells. Biochemistry 10, 2176-2180, (1971).

16. Muramatsu, T., Atkinson, P.H., Nathenson, S.G. and Ceccarini, G. Cell surface glycopeptides: growth dependent changes in the carbohydrate-peptide linkage region. J. MoL BioL 80, 781-799, (1973).

17. Ogata, S.I., Muramatsu, T., Kobata, A. A New structural characteristics of the large glycopeptides from transformed cells. Nature 259, 580-582, (1976).

18. Glick, M.C. Membrane glycoproteins from virus- transformed hamster fibroblasts and the normal counterpart. Biochemistry 18, 2525-2532, (1979).

19. Blithe, D,L., Buck, C.A. and Warren, L. Comparison of glycopeptides from control and virus-transformed BHK fibroblasts. Biochemistry 19, 3386-3395, (1980).

20. Holmes, E.W. & O'Brien, J.S. Separation of glycoprotein- derived oligosaccharides by thin-layer chromatography. Anal Methods 93, 167-170, (1979).

21. Buck, C.A., Fuhrer, J.P., Soslau, G. and Warren, L. Mem- brane glycopeptides from subcellular fractions of control and virus-transformed cells. J. BioL Chem. 249, 1541-1550, (1974).

22. Tuszynski, G.P., Baker, S.R., Fuhrer, J.P., Buck, C.A. and Warren, L. Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus- transformed hamster fibroblasts. J. BioL Chem. 253, 6092-6099, (1979).

23. Van Nest, G.A. and Grimes, W.J. A comparison of membrane components of normal and transformed Balb/c cells. Biochemistry 16, 2902-2908, (1977).

24. Warren, L., Fuhrer, J.P. and Buck, C.A. Surface glycoproteins of normal and transformed cells. A difference deter- mined by sialic acid and a growth-dependent sialyltransferase. Proc. NatL Acad. Sci. U.S.A. 69, 1838-1842, (1972).

25. Warren, L., Fuhrer, J.P., Buck, C.A. Surface glycoproteins of cells before and after transformation by oncogenic viruses. Fed. Proc. 32, 80-85, (1973).

26. Bosmann, H.B. A sialyl transferase activity in normal and RNA and DNA virus-transformed cells utilizing desialylated trypsinized cell plasma membrane external surface glycoproteins as exogenous acceptors. Biochem. Biophys. Res. Commun. 49, 1256-1262, (1972).

27. Grimes, W.J. Sialic acid transferase and sialic acid levels in normal and transformed cells. Biochemistry 9, 5083-5092, (1970).

28. Grimes, W.J. Glycosyl transferase and sialic acid levels of normal and transformed cells. Biochemistry 12, 990-996, (1973).

29. Santer, U.V. and Glick, M.C. Partial structures of a membrane glycopeptide from virus-transformed hamster cells. Biochemistry 18, 2533-2540, (1979).

30. Warren, L., Crtichley, D.R. and Macpherson, I. Surface glycoproteins and glycolipids of chicken embryo cells transformed by a temperature-sensitive mutant of Rous sarcoma virus. Nature 235, 275-278, (1972).

31. Lai, M.M.C. and Duesberg, P.H. Differences between the envelope glycoproteins and glycopeptides of avian tumor viruses released from transformed and from normal cells. Virology 50, 259-272, (1972).

32. Muramatsu, T., Gachelin, G., Nicolas, J.F., Condamine, H., Jackob, H. and Jacob, F. Carbohydrate structure and cell differentiation: Unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells. Proc. NatL Acad. Sci. U.S.A. 75, 2315-2319, (1978).

33. Rasilo, M.-L., Wartiovaara, J. and Renkonen, O. Mannose containing glycopeptides of cells derived from human teratocarcinoma (line PA 1). Can. J. Biochem. 58, 384-393, (1980).

34. Baker, S.R., Blithe, D.L., Buck, CA. and Warren, L. Glycosaminoglycans and other carbohydrate groups bound to proteins of control and transformed cells. J. Blot Chem., in press, (1980).

35. Montgomery, R. Heterogeneity of the Carbohydrate Groups of Glycoproteins in GLYCOPROTEINS. A. Gott- schalk, ed. Elsevier, Amsterdam, Part A, pp 518-527, (1972}.

36. Gorga, F.R., Baldwin, S.A. and Lienhard, G.E. The monosaccharide transporter from human erythrocytes is heterogeneously glycosylated. Biochem. Biophys. Res. Commu~ 91, 955-961, (1979).

37. Yu, J. and Steck, T.L. Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane. J. BioL Chem. 250, 9170-9175, (1975).

38. Spiro, R.G. GLYCOPROTEINS, in Advances in Protein Chemistry, Academic Press, New York 27, 349-467, (1973).

39. Glick, M.C. and Buck, C.A. Glycoproteins from the surface of metaphase cells. Biochemistry 12, 85-90, (1973).

40. Ceccarini, C., Muramatsu, T., Tsang, J. and Atkinson, P.H. Growth-dependent alterations in oligomannosyl cores of glycoproteins. Proc. NatL Acad. Sci~ U.S.A. 72, 3139-3143, (1975).

41. Simmons, J.F., Fishman, P.H., Friese, L. and Brady, O. Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in Hela cells. J. Cell BioL 66, 414-424, (1975).

42. Van Veen, J., Noonan, K.D. and Roberts, R.M. A correla- tion between membrane glycopeptides composition and losses in concanavalin A agglutinability induced by db- cAMP in chinese hamster ovary cells. Exp. Cell Res. 103, 405-413, (1976).

43. Soslau, G., Fuhrer, J.P., Nass, M.M.K. and Warren, L. The effect of ethidium bromide on the membrane glycopeptides in control and virus-transformed Cells. J. BioL Chem. 249, 3014-3020, (1975).

44. Li, E. and Kornfeld, S. Structure of the altered oligosaccharides present in glycoproteins from a clone of chinese ham s t e r ovary cel ls de f i c i en t in N-ace ty l - glucosaminyltransferase activity. J. BioL Chem. 253, 6426-6431, (1978).

45. Takatsuki, A. and Tamura, G. Effect of Tunicamycin on the synthesis of macromolecules in cultures of chick embryo fibroblasts infected with Newcastle disease virus. J. Antibiot. 24, 785-794, (1971).

46. Tkacz, J. S. and Lampen, J.O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf liver microsomes. Biochem. Biophys. Res. Commun. 65, 248-257, (1975).

47. Damsky, C.H., Levy-Benshimol, A., Buck, C.A. and War- ren, L. Effect of Tunicamycin on the synthesis intracellular transport and shedding of membrane glycoproteins in BHK cells. Exper. Cell Res. 119, 1-13, (1979).

48. Pouyssegur, J. and Pastan, I. Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. J. BioL Chem. 252, 1639-1646, (1977).

49. Bossman, H.B., Hagopian, A. and Eylar, E.H. Membrane glycoprotein biosynthesis: Changes in levels of glycolsyl transferases in fibroblasts transformed by oncogenic viruses. J. Cell Physio£ 72, 81-92, (1968).

50. Warren, L., Fuhrer, J.P., Buck, C.A. and Walborg, E.F. Jr. Membranes glycoproteins in normal and virus- transformed cells. In Membrane Transformation in Neoplasia. J. Schultz and R.E. Block eds. New York, Academic Press, pp 1-21, (1974).


51. Bosmann, H.B. and Hilf, R. Elevation in serum glycoprotein: N-acetylneuraminic acid transferase in rats bearing mammary tumors. F E B S Lett . 44, 313-316, (1974).

52. Kessel, D. and Allen, J. Elevated plasma sialyltransferase in the cancer patient. Cancer Res. 35, 670-672, (1975).

53. Bernacki, R.J. and Kim, U. Concomitant elevation in serum sialyltransferase activity and sialic acid content in rats with metastasizing mammary tumors. Science 195, 577-579, (1977).

54. Bauer, C.H., Kottingen, E.K. and Reutter, W. Elevated activity of a-2 and a-3 fucosyltransferases in human serum as a new indicator of malignancy. Biochem. Biophys. Res.

Commun. 76, 488-494, (1977). 55. Khilanani, P., Chou, T.H., Ratanatharathorn, V. and

Kessel, D. Evaluation of two plasma fucosyltransferases as marker enzymes in non-Hodgkin's lymphoma. Cancer 41, 701-725, (1978).

56. Battacharya, M., Chatterjee, S.K. and Varlow, J.J. Uridine-5'-diphosphate galactose: glycoprotein galactosyltransferase activity in the ovarian cancer patient. Cancer Res. 36, 2096-2101, (1976).

57. Weiser, M.M., Podolsky, D.K. and Isselbacher, K.J. Cancer-associated isoenzyme of galactosyl transferase. Proc. NatL Acad. Sci. U.S.A. 73, 1319-1322, (1976).

the membrane glycoproteins of the malignant cell

Documents