Regulation of Glycoconjugate Metabolism in Normal and Transformed

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 15, 2016 | http://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch015

Regulation of Glycoconjugate Metabolism in Normal and Transformed Cells JOSEPH R. MOSKAL, MICHAEL W. LOCKNEY, CHRISTOPHER C. MARVEL, PEGGY A. MASON, and CHARLES C. SWEELEY Department of Biochemistry, Michigan State University, East Lansing MI 48824 STEPHEN T. WARREN and JAMES E. TROSKO Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824 Abstract. TPA and RA have significant effects on glycolipid and glycoprotein biosynthetic enzymes in several cultured cell systems. This suggests that these compounds as well as other "tumor promoters" will be useful in further studies on the regulation and control of glycoconjugate metabolism (metabolic perturbants). Butyrate, TPA and RA appear to exert their effects at different points in the cell cycle. These results could mean that tumor promotion, differentiation and virus infection occur at discrete points in the cell cycle. Membrane glycoconjugates may participate in these processes in a dynamic time-dependent way. Introduction A large body of literature has developed dealing with the chemistry and metabolism of glycosphingolipids and other glycoconjugates (1, 2, 3). However, only recently have research efforts been addressed to the possible interrelationship between the regulation of glycoconjugate metabolism and the control of cell growth and transformation - whether it be the expression of various differentiated functions or transformation by viruses or carcinogens into a "tumorigenic state". Bosmann and Winston (4) were the first ones to examine the possible cell cycle dependence of glycolipid and glycoprotein synthesis. They concluded that glycolipid synthesis occurs almost exclusively in the G2 and M phases while glycoprotein synthesis peaks during the S period. Wolfe and Robbins (5), using radiolabeled palmitate and sugars followed by isolation and thin layer chromatographic characterization, found, however, that simple glycolipids (glucosylceramide, lactosylceramide and G M ^ ganglioside) were synthesized throughout the cell cycle in equal amounts, whereas triglycosylceramide and tetraglycosylceramide were labeled only in the and S phases. Forssman hapten was synthesized throughout the cell cycle but anti-Forssman antibody adhered to cells maximally in the G . and early S phases. They also reported that mitotic cells exposed all detectable antigens and that as cells moved through the cycle much of the Forssman antigen became cryptic.

0-8412-0556-6/80/ 47-128-241 $5.75 / 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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A t this time Chatterjee et a l . (6) reported a maximal incorporation of galactose into the glycolipids of synchronized K B cells during late M and/or early G j phases. They also found a large increase in the levels of gangliosides and total neutral glycolipids during this period. Gahmberg and Hakomori (7), using the cell surface labeling technique of galactose oxidase:sodium borotritiide, reported that monoglycosylceramide through pentaglycosylceramide were labeled maximally during the G ^ phase and minimally during the S phase. Moreover, they found relatively constant amounts of these glycolipids throughout the cell cycle. It was concluded that while glycolipid synthesis occurs in the M - G ^ phase, as cells traverse the mitotic cycle, the exposure of glycolipids at the cell surface varies with the cell cycle. They suggest that a cell surface glycoprotein, "galactoprotein a" (found in confluent cultures of non-transformed cells but at lower levels in virally transformed cells), may be a key factor in the organization of membrane glycolipids and may explain why transformed cell glycolipids show a high rate of labeling throughout the cell cycle. Using techniques such as galactose oxidase:sodium borotritiide reduction and lectin binding to study cell surface changes, Gahmberg and Hakomori (8) have observed that virus-transformed but not control hamster cells contained lacto-N-neotetraosylceramide on the cell surface, that this exposure was cell-cycle specific, and that normal and transformed cells interact with lectins via a different glycoprotein for each cell type. The authors concluded that the binding sites of the lectins were specific glycoproteins and that these interacting proteins are significantly different in normal compared to transformed cells (9). Schnaar et a l . (10) have synthesized polyacrylamide gels with covalently linked carbohydrates to study their interaction with cell membrane glycoconjugates. They report that chicken hepatocytes, in a temperature and calcium dependent way, interact strongly with only N acetylglucosamine-linked polyacrylamide. Orosomucoid, minus sialic acid and galactose, was a potent inhibitor of this interaction. Lingwood et a l . (11) have made monoclonal antibodies directed against various glycolipîHs and glycoproteins and have shown that when bound to temperature-sensitive virally transformed cells they could inhibit the expression of the "oncogenic" state at the permissive temperature. While these studies are not directly pertinent to the regulation of glycoconjugate metabolism they clearly implicate glycoconjugates as playing a significant role in cell metabolism and growth control. Since the first reports of in vitro glycosyltransferase activities (12, 13, lfr) a number of papers have appeared which deal with elucidation of glycolipid biosynthetic pathways in several model systems (15-20). The earliest attempts to implicate glycolipid metabolism in cell transformation came from investigations of the effects of viral transformation. Studies by Brady and Mora (21), Grimes (22), Hakomori (23), Bosmann (24, 25) and Den et a l . (26) have all shown significant differences in cellular glycolipid biosynthetic capability when comparing non-transformed with virally transformed cells. In general, irrespective of the cell line or the virus, a simplification of glycolipid patterns has been observed and often glycosyltransferase activities present in non-transformed cell lines were >

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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absent in their transformed counterpart (27). Furthermore, it has been suggested that this simplification of the oligosaccharide chain upon viral transformation may have important implications with respect to a tumor cell's ability to interact with molecules involved in growth control (28). Studies dealing with cellular transformation to a non-proliferative or "differentiated" state have been reported by a number of investigators. Yeung et a l . (29) have shown a significant elevation in N acetylgalactosaminyltransferase activity in clones of adrenal tumor cells after dibutyryl-cyclic adenosine monophosphate (dBcAMP) treatment. Moskal et a l . (30) and Basu et a l . (31) have also reported significant changes in glycolipid sialyltransferase and galactosyltransferase activities in cultured murine neuroblastoma cells after d B c A M P treatment, as well as between spinner cultured cells and T-flask grown cells. Fishman and coworkers have reported a dramatic morphological change and an elevation in C M P - N e u A c : lactosylceramide sialyltransferase activity after treatment of HeLa cells with butyric acid (32, 33, 34). They found that these effects were dependent on protein synthesis and suggested that the increased levels of II -a-N-acetylneuraminosyllactosylceramide ( G M J were necessary for expression of the morphological changes. Macner et al. (35) also reported a significant elevation in this sialyltransferase in human epithelial carcinoma cells (KB) and observed changes in cell surface labeling patterns and galactosyltransferase (UDPGal-lactosylceramide galactosyltransferase) activity (36), suggesting that butyrate-treated cells could be used to study many aspects of glycoconjugate metabolism. Recently, Presper et a l . (37) have reported two fucosyltransferase activities from h u m a n T l M R - 3 2 7 neuroblastoma cells. They also found that 6-thioguanine but not bromodeoxyuridine-induced differentiation caused a marked elevation in fucose containing glycoconjugates. On the other hand, Dawson et a l . (38) have reported that enkephalins caused a dose-dependent decrease in the incorporation of radiolabeled glucosamine or galactose into glycolipids and glycoproteins in cultured neuroblastoma cells. These investigators suggested that their results may be interpreted in terms of a cyclic A M P mediated process (5). Recently, a class of compounds called "tumor promoters" have emerged that, unlike "differentiating" agents such as butyrate or d B c A M P , stimulate cell proliferation and tumorigenesis. Since tumor promoters alter cell growth patterns in an opposing manner to differentiating agents, they may also alter glycoconjugate metabolism. If this were the case, glycoconjugates could be involved in the regulation of cell growth, and studies with tumor promoters might be a useful approach to elucidate the mechanisms of regulation of glycoconjugate metabolism. The following is a brief review of the "two-stage theory of carcinogenesis" in which tumor promoters play an integral part, and some of the effects tumor promoters have been reported to have on cells. Two-Stage Theory of Carcinogenesis. Historically, the induction of skin tumors was accomplished by repeated applications of a potent carcinogen (39). Berenblum (40), however, found that croton oil, when administered together with a carcinogen, led to more tumors than the carcinogen alone. Mottram (41) then reported that after multiple



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applications of croton o i l , only one treatment with a carcinogen was necessary to cause tumors. It was later shown that diesters of the diterpene alcohol, phorbol, are the active components of croton oil (42, 43, 44) (Figure 1 shows the general phorbol structure with a list of substituted phorbols and their carcinogenic efficacy). These findings led Boutwell and coworkers to establish the 2-stage protocol for tumor induction in mouse skin and a model system for the 2-stage theory (45, 46). Briefly, it was found that 1) a small single dose of a carcinogen caused no tumor formation in mouse skin (however, the mouse skin is said to be initiated), 2) multiple applications of tumor promotors alone caused no tumor formation, but 3) if a tumor promoter was applied to initiated mouse skin, tumors did arise. Furthermore, if the order of treatments was reversed no tumors were seen (47). Initiation appears to be permanent, since tumors formed when promoter was added as long as one year after initiation (48). More recently, O'Brien et a l . (49) have reported that a single application of tumor promoter (croton oil or 12-0tetradecanoyl-phorbol-13-acetate:TPA) caused a rapid stimulation of ornithine decarboxylase (ODC) activity (2-300-fold induction, reaching maximal levels 4-5 hours after treatment) in mouse skin. Verma and Boutwell (50) later reported that retinoic acid (RA) (the general structure is~shown in Figure 2), when applied with T P A , could completely inhibit the formation of tumors. At about this time O'Brien (51) and Boutwell (52) proposed the twostage theory of carcinogensis. Stated simply, the induction of tumors requires first the "initiation" of a cell by a carcinogen. This process is irreversible and is believed to occur at the genetic level. Following initiation a tumor promoter must be introduced. Promotion is believed to be a reversible phenomenon accompanied by an induction in O D C activity as a key step. The model implicates O D C induction as an essential feature of tumor promotion, based on the following evidence: 1) the degree of induction of enzyme activity (ODC) correlates well with the promoting ability of various concentrations of T P A and other phorbol esters of varying promoter efficacy, 2) retinoic acid inhibits the ability of T P A to induce tumors and also inhibits O D C induction, and 3) tumors produced by T P A treatment have high levels of O D C activity, with malignant tumors possessing higher levels than benign tumors. O'Brien and Diamond (53) have recently reported a bioassay system, based on O D C induction by tumor promoters, to analyze the metabolism of the phorbol diester tumor promoters. Research on the various biochemical systems affected by tumor promoters, in particular T P A , has been recently reviewed by Diamond et al. (54) and Werner and coworkers (55) have reviewed the early effects of phorbol esters on the membranes of cultured cells. The latter group reports that T P A causes permeability changes in 3T3 cell membranes and experimental evidence is cited that phorbol esters interact specifically with a membrane-specific macromolecule rather than passive adsorption by the membrane lipid matrix. One of the earliest observed effects of T P A is a significant modification in the transport of potassium, sodium and phosphate. Lee and Weinstein (56) have found that the addition of phorbol esters immediately stimulated the uptake of 2-deoxyglucose in


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H


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OR

In v i v o Tumor Promoting A c t i v i t y R

1

= R

2

= H

R = CO ( CH ) - ] 2 3 2 1

CH

2

; R

R^I^COfCHgJgCHg R =R =C0CH 1

2

2

6

3

+ + + +

+++ +

3

R-,=R =C0C H

= C 0 C H

5

++

Figure 1. General structure of the tumor-promoting component, phorbol, of croton oil. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is the most potent promoter of the phorbol diesters. Phorbol didecanoate and phorbol dibenzoate, among others, have promoting ability but to a lesser extent than TPA. Phorbol alone has been reported to have no capacity to induce tumors as ODC.

COOH

Figure 2. General structure of retinoic acid (all trans-retinoic acid). Of the many derivatives tested (e.g., retinol, retinyl acetate), none has the "anti-tumor promoter" efficacy, in vivo, as retinoic acid.



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cultured cells. This enhancement peaked after 90 minutes, persisted as long as three hours and was temperature dependent. These results support the idea that transport mechanisms rather than effects on intracellular metabolism were responsible for these observations. Another interesting observation reported by these investigators was that tumor promoting phorbols inhibit epidermal growth factor (EGF) binding to cell surface receptors (57), suggesting that the E G F receptor may be the binding site for T P A . Thus, it appears that the plasma membrane plays a very important part in the 2-stage theory of carcinogenesis. In an attempt to further elucidate how glycoconjugates and cellular transformation are linked, we began a study of the effects of some of the compounds involved in tumor promotion. Previous efforts with "differentiating" agents set the stage for similar studies with compounds directly implicated in cellular proliferation and tumorigenesis. In these studies, then, the effects of tumor promoters on cell morphology, gly cocon jugate metabolism and composition, and cell cycling activity are reported, and the results are discussed in terms of how tumor promoters might affect cell metabolism. Materials A l l materials were obtained from the following sources: human epidermoid carcinoma (KB) cells from The American Type Culture Collection (Rockville, MD.); nontransformed (NIL 8) and virally-transformed (NIL 8HSV) cells were a gift from D r . P.W. Robbins (Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology); modified Eagle's medium, calf serum, trypsin, and fetuin from Grand Island Biological Company (Detroit, ML); radiolabeled C M P sialic acid, UDP-galactose, sialic acid and DL-ornithine from New England Nuclear (Boston, MA.); unlabeled UDP-galactose from Sigma (Si. Louis, MO.). Lactosylceramide was purified from canine intestine. II α - Ν - a c e t y l n e u r a m i n o s y l - l a c t o s y l c e r a m i d e (GM^) and mixed gangliosides were from Supelco, Inc., (Bellefonte, PA.) and high performance thin layer chromatography (HPTLC) plates (silica gel 60; without fluorescent indicator) were from EM laboratories (Elmsford, N.Y.). Methods C e l l Culture. K B cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 3 7 ° C in the presence of modified Eagle's medium containing calf serum (10%), penicillin (100 Mg/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. A l l drugs were administered with fresh media 2k hours after subculture in the following concentrations: T P A , 1.6 μΜ; R A , 1.6 μΜ; butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% E D T A and stored at - 2 0 ° C in 0.32 M sucrose.


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Glycolipid Sialyltranferase Assays. Complete incubation mixtures contained the following components (in micromoles), in a final volume of 0.05 ml: lactosylceramide, 0.05; Triton CF-54-Tween 80 (2:1 w/w), 200//g; C a c o d y l a t e - H Ç l buffer, pH 6.5, 10; magnesium chloride, 0.1; C M P - N e u A c (1.5 χ 10 cpm//imole), 0.05; and 0.2 to 0.6 mg of protein. Enzyme reactions were incubated for 60 min at 3 7 ° C , terminated by the addition of 0.6 //mole of E D T A (pH 7.0) and assayed by the double chromatographic procedure of Basu (16). Glycoprotein Galactosyltransferase Assays. Complete incubation mixtures contained the following components (in micromoles, unless otherwise stated) in a final volume of 0.05 ml: MES buffer, pH 6.7, 12.5; manganese chloride, 0.5; 0.5% Triton X-100; desialized (mild acid hydrolysis) and degalactosylated (58) fetuin, 125 ^g; UDP-galactose (specific activity, 8.8 χ 10 cpm~7Mmole), 0.0025; protein, 1-50 μ g . Incubations were carried out for 60 min at 3 7 ° C and were terminated by the addition of 5 ml of cold 5% phosphotungstic acid in 0.5 M HC1. Precipitates were collected on millipore filters ( 0 Λ 5 micron pore size), washed twice in the acid mixture, and dissolved in 1% SDS-0.1N N a O H . After neutralization with IN HC1, samples were counted by liquid scintillation spectrometry using 10 ml of a Toluene Triton X-100 based liquid scintillation cocktail. Ornithine Decarboxylase Assays. The double-chamber assay system of Moskal and Basu (59) was used to measure enzyme activity in the form of L C] carbon dioxI3e evolution. The assay conditions of O'Brien and Diamond (60) were used and consisted of the following components (in micromoles, unless otherwise stated) in a total volume of 100 μΐ; sodium phosphate buffer, pH 7.2, 5.0; E D T A , 1.0; dithiothreitol, 5.0; py/idoxal5 -monophosphate, 0.2; L-ornithine (specific activity 0.5 χ 10 c p m / jumole), 0.1 and protein, 0.1-0.5 mg. Incubations were carried out at 3 7 ° C for 60 min, and the reactions were terminated by the addition of 200 μΐ of 2M sodium citrate followed by a post-incubation period of 3 hours at 3 7 ° C to insure maximal release of radiolabeled carbon dioxide. Scanning Electron Microscopy. K B cells were synchronized by the following procedure: 2mM thymidine was added for 20 hr followed by release for 8 hr and shaking for mitotic cells. After mitotic selection, cells were briefly suspended in and triturated with P B S - E D T A (0.05%) before transfer to flasks with fresh media. Synchronized or drug-treated cells were washed three times with PBS (calcium and magnesium free) and fixed for 30 min in 3% glutaraldehyde (EM grade, Polysciences, Warrington, PA.) in 50 mM cacodylate-HCl buffer, pH 7.2. The glutaraldehyde was removed by washing the cells three times with cacodylate buffer and dehydration with graded ethanol-water solutions was performed followed by several washes with absolute ethanol. Critical point drying of the samples was carried out in a Bomar S P C 900/EX apparatus (Bomar C o . , Tacomia, WA.) using carbon dioxide as the carrier gas. Gold coating of the samples was done using a Film-Vac Mini Coater (Englewood, N.J.) to a density of 200 A . A n ISI super III scanning electron microscope was used (International Scientific Instruments, Santa Clara, C A . ) . 6


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Light Microscopy. Cells were photographed using an Olympus (model IMT) inverted microscope. Thymidine Uptake Studies. Tritiated thymidine (52 mCi/jimole; 0.1 juCi/ml) was added to cells for 60 min at 3 7 ° C . Cells were then washed with PBS, incubated at 4 ° C for 15 min in the presence of ice cold 5% T C A , rinsed with T C A and scraped from the flasks with a rubber policeman. Cells were again washed with PBS, and solubilized in 0.1N NaOH overnight. Aliquots were assayed for protein (62) and radioactivity (scintillation fluid: 100 ml Biosolve (Beckman, Fullerton, C A . ) , 7g of PPO and 0.6 g of POPOP per liter of toluene). Results and Discussion Glycoconjugate Metabolism Studies. C M P - N e u A c : lactosylceramide sialyltransferase activity: This particular sialyltransferase was assayed because of the dramatic induction in activity reported by Fishman et a l . (32) and Mâcher et a l . (35) after treatment with butyrate, a drug with putative anti-tumor properties (63). Thus, gangliosides (in particular GM^) may play an important role in growth control and differentiation and sialyltransferase could be a pivotal enzyme in the regulation of such processes. Table I gives the results of the incubation of K B cells in log phase growth with butyrate, T P A or R A as described in Methods. Butyrate caused an approximately 5-fold increase in K B sialyltransferase, as expected. T P A treatment also resulted in a 5-fold elevation of sialyltransferase activity. The most dramatic elevation in enzyme activity, however, was seen when cells were treated with R A , in which case a 10 to 15 fold increase in activity was observed. We have also seen similar increases in sialyltransferase activity after R A treatment of both non-transformed and virally transformed hamster embryo cells (NIL). However, sialyltransferase activity in the non-transformed NIL cells was slightly decreased after T P A treatment (64).

Table I. The Effect of Various Compounds Implicated in the 2-Stage Theory of Carcinogenesis on CMP-NeuAc:lactosylceramide Sialyltransferase Activity in Human Epithelial Carcinoma with (KB) Cells CONDITION

CONTROL

SIALYLTRANSFERASE ACTIVITY* (pmoles/mg protein/hr)

% CONTROL

477

BUTYRATE

2549

TPA

2605

546

6726

1410

RA

534

*Minus endogenous values CMP-NeuAc:lactosylceramide sialyltransferase


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Glycoprotein Galactosyltransferase Assays. In order to further investigate the effects of T P A and R A on glycoconjugate metabolism, UDP-galactose:DSG-fetuin galactosyltransferase activity was assayed. Table II shows the results of this study. The effects of T P A and R A on

Table II. The Effect of Various Compounds Implicated in the 2-Stage Theory of Carcinogenesis on UDP-galactose:DSG-fetuin Galactosyltransferase Activity in Human Epithelial Carcinoma (KB) Cells CONDITION

G A L A C T O S Y L T R A N S F E R A S E A C T I V I T Y *

Regulation of Glycoconjugate Metabolism in Normal and Transformed

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