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[CANCER RESEARCH 37, 4250-4255, December 1977] Inhibition of L-Fucose Incorporation into Glycoprotein of Sarcoma 180 Ascites Cells by 6-Thioguanine1 John Stephen Lazo, Kou M. Hwang,2 and Alan C. Sartorelli Department Connecticut of Pharmacology 06510 and Developmental Therapeutics Program, Comprehensive SUMMARY The incorporation of [3H]fucose into glycoproteins of Sarcoma 180 cells in vitro was decreased within 2 hr of exposure to 6-thioguanine (6-TG); 50% inhibition was pro duced by 8 /nM 6-TG. Under identical conditions, no de crease in [3H]glucosamine incorporation into acid-insoluble material was detected. Similarly, [3H]fucose incorporation, but not [14C]glucosamine incorporation into glycoproteins of Sarcoma 180 ascites cells in vivo, was significantly reduced 1 to 6 hr after 6-TG treatment (20 mg/kg) of mice bearing 6-day implants of this neoplasm; the maximum depression of fucose utilization occurred at 2 hr after drug treatment. The decrease in [3H]fucose incorporation into glycoprotein was dose dependent and reached a maximum reduction of 77% of control incorporation 2 hr after 6-TG (10 mg/kg). The radioactivity from fucose found in acid-soluble extracts of Sarcoma 180 cells was decreased by 45% after 6-TG (10 mg/kg). In contrast, this concentration of 6-TG did not decrease the level of radioactivity from [3H]fucose found in acid-soluble extracts of a subline of Sarcoma 180 resist ant to 6-TG and produced only a 38% decrease in fucose incorporation into the acid-insoluble fraction of this neo plasm. The inhibition of [3H]fucose incorporation into gly coproteins of Sarcoma 180 produced by 6-TG appeared to be due to a drug-induced decrease in the formation of guanosine diphosphate-[3H]fucose and a concomitant de crease in the intracellular content of guanosine diphosphate-fucose. These data suggest that 6-TG exerts, as a metabolic lesion, a suppression of guanine nucleotide sugar biosynthesis. Since fucose is largely a terminal car bohydrate of glycoproteins and glycolipids of the plasma membrane, 6-TG may alter membrane composition. This phenomenon may be associated with the cytotoxicity of 6TG to neoplastic cells. INTRODUCTION The antimetabolite 6-TG3 is a clinically useful drug in the treatment of the acute leukemias, largely in combination with other drugs (7, 22). The available evidence indicates 1 Supported in part by Grants CA-02817 and CA-16359 from the National Cancer Institute, USPHS. 2 Present address: Section of Immunology, Department of Developmental Therapeutics, M. D. Anderson Hospital, University of Texas System Cancer Center, Houston, Tex. 77025. 3 The abbreviations used are: 6-TG, 6-thioguanine; TCA, trichloroacetic acid. Received 4250 February 7, 1977; accepted August 23, 1977. Cancer Center, Yale University School of Medicine, New Haven, that the antineoplastic activity of 6-TG requires its conver sion to the nucleotide form (4). Nevertheless, the precise metabolic lesion(s) responsible for its cytotoxicity remains unclear. Several sites of action have been proposed, includ ing inhibition of purine nucleotide biosynthesis de novo (23), inhibition of purine ribonucleotide interconversion (19), inhibition of protein synthesis (9), and incorporation into RNA and DNA following conversion to the nucleotide form (17, 18). Although the available evidence favors the incorporation of 6-TG into DNA as being critical to the cytotoxic action of this drug (26), the mechanism by which the incorporated analog exerts its cytotoxic effects has not been delineated (22), and an exception to the importance of this lesion to antineoplastic activity exists (24). Conse quently, it is possible that other biochemical alterations caused by 6-TG might be involved in its cytotoxic mode of action. Previous studies from our laboratory have shown that the exposure of Sarcoma 180 to 6-TG in vitro results in cell surface alterations detected by concanavalin A agglutina tion (11, 12). To gain evidence for the role of membrane lesions in the cytotoxic action of 6-TG, we have determined the effects of 6-TG on fucose metabolism in a susceptible transplanted neoplasm. The findings demonstrate that ex posure of Sarcoma 180 cells to 6-TG for as little as 1 hr in vivo results in an inhibition of [3H]fucose but not [14C]glucosamine incorporation into cell glycoproteins. Such an inhibition of the incorporation of a terminal carbo hydrate may be involved in the modification of surface architecture caused by this drug. The inhibition by 6-TG of fucose utilization for glycoprotein formation appears to be due largely to a decreased activation of fucose to GDPfucose in drug-treated cells. MATERIALS AND METHODS 6-TG was generously provided by Dr. George H. Hitchings of the Burroughs Wellcome Research Laboratories (Re search Triangle Park, N. C.). [14C]Glucosamme, [3H]glucosamine, [3H]fucose, and GDP-['"C]fucose were pur chased from New England Nuclear, (Boston, Mass.). Fischer's medium and horse serum were obtained from Grand Island Biological Co., (Grand Island, N. Y.). Sarcoma 180 ascites cells were collected from the ascitic fluids of female CD-1 mice (Charles River Breeding Labora tories, North Wilmington, Mass.) 6 to 8 days after i.p. inoculation of mice with 6 x 106 tumor cells. In studies of the in vivo effects of 6-TG, animals were given injections of CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 6-7G Effects on Fucose Incorporation 5 to 8.8 /jCi (0.07 to 0.12 ^g) of [3H]fucose and/or 2.5 /¿Ci the use of a column (2.5 x 17 cm) of Sephadex G-25 (9.5 /¿g)of [14C]glucosamine per mouse, and 30 min were (Pharmacia, Piscataway, N. J.) and 50 mw ammonium acetate as eluent. The effluent containing GDP-fucose was allowed for incorporation unless otherwise stated. Contam of 0.1 M TCA, inating erythrocytes were removed by washing cells 3 times lyophilized to dryness, resuspended in 200 ¿J with 10 to 15 volumes of a Ca2'-Mg2 -free phosphate- and heated to 90°for 10 min. After 4 extractions with 10 buffered NaCI solution (8.0 g NaCI, 0.2 g KCI, 2.16 g NaJ-tPO,-7H2O, and 0.2 g KH,PO, per liter of H,0, pH 7.4) following centrifugation at 75 x g for 5 min at room temperature. For in vitro experiments, washed cells (4 x 106 cells/ml) were incubated at 37°in the absence or presence of 6-TG in Fischer's medium supplemented with 5 mM potassium phosphate (pH 7.0) and 10% horse serum. At various times thereafter, [3H]glucosamine or [3H]fucose (0.5 /uCi/ml) was added, and the incorporation of radioac tivity into total cellular glycoprotein was measured by addi tion of 10 ml of ice-cold 10% TCA to 0.25 ml of either untreated or treated cells. Pellets were collected by centrif ugation at 4°and washed consecutively with 10 ml of icecold 5% TCA (twice), 5 ml of chloroform:methanol:ether (2:2:1. v/v), and 5 ml of methanol. The pellet was hydrolyzed with 1 ml of 1 M NaOH at 80°for 30 min and then neutralized with 5 M HCI. Radioactivity was determined with a Packard Tri-Carb liquid scintillation spectrometer (Packard Instru ments Co., Downers Grove, III.). The incorporation of radio activity after exposure of cells to 6-TG and radioactive tracer in vivo was determined in a similar manner. Washed cells were resuspended in 10 volumes of Ca2'-Mg2 -free phosphate-buffered NaCI solution (1.64 x 107 cells/ml), and 1-ml samples were added to 10 ml of ice-cold 10% TCA. The resulting precipitate was collected by centrifuga tion and washed twice with 10 ml of ice-cold 5% TCA. Less than 15 and 27% of the acid-precipitable radioactive fucose and glucosamine products, respectively, were removable with chloroform:methanol:ether (2:2:1, v/v). The pellet was hydrolyzed and neutralized, and its radioactivity was deter mined as described above. The intracellular concentrations of [3H]fucose and GDP-[3H]fucose were measured by the method of Yurchenco and Atkinson (27). Briefly, washed radiolabeled cells were extracted twice with 5 to 6 volumes of 60% ethanol in a boiling water bath for 5 min each. The com bined extracts were centrifuged at 1,000 x g for 5 min, and the supernatant solution was recentrifuged at 33,000 x g for 20 min. The supernatant fraction was evaporated to dryness under vacuum at 30°,redissolved in 1 ml of water, and centrifuged at 33,000 x g for 20 min to remove ethanolsoluble but water-insoluble material. Descending Whatman No. 3MM paper chromatography was carried out with 95% ethanol:1.0 M ammonium acetate (7:3, v/v) for 9 hr. Airdried paper strips were cut into 1-cm pieces and extracted twice with 60% ethanol in a boiling water bath. The extracts, which contained 90% of the radioactivity, were evaporated to dryness, and radioactivity therein was determined in Aquasol (New England Nuclear). The counting efficiency in all single-labeled experiments ranged between 15 and 30% for 3H and 31 and 35% for MC and radioactivity for doublelabeled experiments was 17% for 3H and 27% for I4C, with 40% 14Cto 3H spillover. In those experiments in which the specific activity of the GDP-fucose was determined, GDP-fucose was separated from other ethanol-soluble, water-insoluble materials with volumes of ether, the solution was chromatographed on Whatman No. 3MM paper for 6 hr with n-butyl alcohol:pyridine:water (6:4:3, v/v) as solvent (5). After sep aration, L-fucose was eluted from the paper, and its concen tration was measured according to the method of Diseñe and Shettles (6). RESULTS The rate of [3H]fucose incorporation into glycoprotein was measured following exposure of Sarcoma 180 cells to 60 MM 6-TG for either 2 or 6 hr (Chart 1). A linear rate of fucose incorporation into acid-precipitable material oc curred for up to 1 hr in untreated Sarcoma 180 cells that had been preincubated for 2 or 6 hr. 6-TG markedly de creased the rate of utilization of [3H]fucose for cellular glycoprotein synthesis under both experimental conditions. The rate of [3H]glucosamine incorporation into the glyco protein of both untreated and 6-TG-treated Sarcoma 180 cells under similar conditions is shown in Chart 2. Un treated Sarcoma 180 cells incorporated radioactive gluco samine into acid-insoluble material at essentially a linear rate for at least 1 hr. At both concentrations of 6-TG (6 and 60 /¿M),an enhanced incorporation of [3H]glucosamine was observed after the 2-hr preincubation, but it decreased after 6 hr of drug exposure. However, this reduction in [3H]glucosamine utilization observed after 6 hr of exposure to 60 /UM 6-TG was significantly less than that seen with [3H]fucose. For more detailed examination of the differential effects of 6-TG on the incorporation of these precursors into glycoproteins, cells were incubated with a range of concen trations of 6-TG (0.6 to 100 ¡¿M) for 2, 4, or 6 hr (Chart 3). B 30 60 90 120 30 60 90 120 TIME OF INCUBATION (min) Chart 1. Incorporation of [3Hjfucose into glycoproteins of Sarcoma 180 cells treated with 6-TG in vitro. Sarcoma 180 cells isolated from ascites fluid were preexposed to 0.9% NaCI solution or 6-TG (60 MM)for 2 hr (A) or 6 hr (B). After drug treatment, [3H]fucose (0.5 ¿iCi/ml)was added, and cells were incubated at 37°for various periods of time. Radioactivity present in TCAprecipitable material from 10* cells was determined. Each value represents the mean of results obtained from duplicate samples of 2 separate experi ments. • , control; G, 6-TG. DECEMBER 1977 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 4251 J. S. Lazo et al. the time of exposure to the 6-purinethione was increased to 4 or 6 hr, the incorporation of glucosamine into acidinsoluble material was gradually decreased as a function of both concentration and time, with a maximum inhibition of 35% occurring after 6 hr of exposure to 100 /J.M6-TG. The in vivo incorporation of [3H]fucose into acid-precipitable material of Sarcoma 180 ascites cells was essentially linear for at least 30 min after the i.p. injection of 0.07 ¿¿g of fucose per mouse (Chart 4A). Similarly, [14C]glucosamine B m -i LJ O m 8 Z o. o 20 40 60 20 40 60 TIME OF INCUBATION (min) Chart 2. Incorporation of [3H]glucosamine into glycoproteins of Sarcoma 180 cells treated with 6-TG in vitro. Sarcoma 180 cells, preexposed to 0.9% NaCI solution or 6-TG (6 or 60 /¿M) for 2 hr (A) or 6 hr (B), were incubated with [3H]glucosamine (0.5 Ã-Ã-Ci/ml) at 37°for various periods of time. Radio activity present in TCA-precipitable material from 106cells was determined. Each value represents the mean of results obtained from 2 to 3 separate experiments. •,control; D, 6-TG, 6 /¿M; A, 6-TG, 60 ¡J.M. 2hr 120 incorporation into macromolecules was linear for 30 min (data not shown). Cells from mice treated with 6-TG (20 mg/kg) 2 hr before exposure to 6-TG had significantly less radioactivity in both acid-soluble and acid-precipitable frac tions at 30 and 60 min (Chart 4). The reduction in [3H]fucose incorporation into glycoprotein was time dependent; it occurred as early as 1 hr after exposure to 6-TG and reached a maximum of 65% inhibition at 2 hr (Chart 5). Inhibition of [3H]fucose incorporation by 6-TG persisted for up to 6 hr after the administration of the purine antimetab olite. By 24 hr after the drug, however, the utilization of fucose for glycoprotein synthesis was enhanced. No signif icant decrease in the rate of [14C]glucosamine incorporation into glycoprotein was detected; although, in a manner similar to [3H]fucose, [14C]glucosamine utilization for the formation of glycoproteins was increased at 24 hr. In both the acid-precipitable and acid-soluble fractions, the inhibi tion after a 2-hr exposure of Sarcoma 180 to 6-TG reached 100 4hr O ¡E 80 Z o u o 6hr 60 40 o IO O *v Z CL io- io" io-1 -4 IO O [e-TGMOLARITY] Chart 3. Effects of various concentrations of 6-TG on [3H]fucose and [3H]glucosamine incorporation into glycoproteins of Sarcoma 180 cells in vitro. Sarcoma 180 cells were exposed to various concentrations of 6-TG (0.6 to 100 /J.M)for either 2, 4, or 6 hr. [3H]Glucosamine (0.5 ¿iCi/ml)or [3H]fucose (0.5 /iCi/ml) was added after 6-TG treatment and incubated for 1 hr. Determination of the incorporation of fucose and glucosamine into glycoproteins was performed as described in Chart 1. Each value represents the mean of results obtained from 2 separate experiments. [3H]Fucose incorporation: O, 2 hr; D, 4 hr; A, 6 hr. [3H]Glucosamine incorporation; •, 2 hr; •4 hr; A. 6 hr. Fifty % inhibition of [3H]fucose utilization for the synthesis of glycoproteins occurred at 8, 5, and 4 /¿M6-TG after respectively, 2, 4, and 6 hr, of incubation with this agent. The incorporation of [3H]glucosamine into glycoprotein was enhanced at all concentrations of 6-TG tested after 2 hr of preincubation with the purine antimetabolite but, as 4252 IS 30 60 MINUTES AFTER [3h] FUCOSE Chart 4. The effect of 6-TG on the incorporation of [3H]fucose into Sarcoma 180 cells in vivo. Sarcoma 180-bearing mice were given i.p. injections of 0.9% NaCI solution or 6-TG (20 mg/kg; 0.3 to 0.4 ml) 2 hr prior to injection of 5 fiCi [3H]fucose per mouse. At various times thereafter, cells were collected and washed 3 times. The radioactivity associated with the TCA-insoluble (A) and TCA-soluble (B) material was calculated as the average of separate determinations from 2 to 3 mice. Bars, S.E. or range of values; •,0.9% NaCI solution; D, 6-TG. CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 6-TG Effects on Fucose Incorporation a maximum at a 10-mg/kg dose of drug (Chart 6). Control cells incorporated 25 pg of [3H]fucose per 106 cells into glycoproteins after injection of 8.8 /¿Ci/permouse, and cells from animals treated with 10 mg 6-TG per kg incorpo rated only 4.5 pg of [3H]fucose per 106 cells into these macromolecules. The [3H]fucose utilization was measured in a subline of Sarcoma 180 resistant to 6-TG (S180/TG), in which the mechanism of resistance involves an increased catabolism of the active nucleotide form(s) of the antimetabolite by elevated levels of alkaline phosphatase (28). Table 1 shows that the decrease in [3H]fucose incorporation into glycoproteins of Sarcoma 180 by 6-TG did not occur to the same degree in S180/TG cells. Thus, a 77% reduction in the rate of [3H]fucose utilization for glycoproteins of Sarcoma 180 cells was observed 2 hr after 6-TG (10 mg/ kg), whereas only a 38% reduction was detected in S180/ TG cells under the same conditions. Similarly, a 45% decrease in acid-soluble radioactivity from [3H]fucose was produced by the antimetabolite in Sarcoma 180 cells, while no decrease was caused by this treatment in S180/TG cells. These data support the concept that the inhibition of glycoprotein synthesis by 6-TG requires 6-TG nucleotide and may be part of the mechanism of cytotoxicity of this drug. Since Nelson ef al. (20) have shown that GTP levels are decreased in neoplastic cells treated with 6-TG, it was important to determine whether the mechanism by which 6-TG reduced [3H]fucose incorporation into glycoprotein was due to an inability to activate fucose to the level of 30 J5 ISO 20 IO 123 o F ico o o o o CL er o o LJ cc 75 B 100 eo 50 60 25 i 10 40 4 6 24 HOURS AFTER 6-TG Chart 5. Incorporation of [3H]fucose and [14C]glucosamine into Sarcoma 180 cells in vivo at various times after 6-TG. Sarcoma 180-bearing mice were given i.p. injections of 0.9% NaCI solution or 6-TG (20 mg/kg; 0.3 to 0.4 ml). At various times thereafter, either 5 /¿Ci [3H]fucose or 7.5 ¿iCi ("Cjglucosamine were injected ¡.p.into each mouse. Cells were collected 30 min later, and radioactivity in the TCA-precipitable material was measured as previously described. Points, average ±S.E. of separate determinations from 6 to 9 mice. The range of control ["Cjglucosamine incorporation (•) was 3.6 to 6.3 dpm/103 cells. The range of control [3H]fucose incorporation (O) was 2.4 to 3.7 dpm/103 cells. 5 6-TG IO 15 DOSE (mg/kg) 20 Chart 6. The effect of various dose levels of 6-TG on [3H]fucose incorpo ration into acid-soluble and acid-insoluble material of Sarcoma 180 cells. Sarcoma 180-bearing mice were given i.p. injections of various doses of 6TG 2 hr prior to being given injections of 8.8 (¿Ci of [3H]fucose. Radioactivity was determined as described in Chart 1. Points, average of triplicate determinations from a single mouse. A, acid-precipitable radioactivity; B. acid-soluble radioactivity. Table 1 The effects of 6-TG on [3H]fucose incorporation into glycoproteins of Sarcoma 180 and S 180/TG cells Mice were given i.p. injections of 0.9% NaCI solutions with or without 6-TG (10 mg/kg) and 2 hr later were given i.p. injections of [3H]fucose (5 ¿¿Ci/mouse).Cells were collected 30 min later, and radioactivity present in acid-precipitable material was determined. Triplicate determinations were made of samples from each animal. pg ¡3H]fucose equivalents incorpo pg [3H]fucose equivalents incorpo rated/106 Sarcoma 180/TG cells" rated/106 Sarcoma 180 cells" Treatment0.9% NaCI solution 6-TGAcid insoluble10.7 ±1.7(7)" soluble47.0 ±2.7 (7) 21.0 ±0.6 (4)Acid 2.4 ±0.4 (4)Acid " Mean ±S.E. * Numbers in parentheses, number of mice per group. DECEMBER insoluble13.7 soluble43.5 ±1.6 (7) ±10.4 (5) 8.5 ±0.9 (7)Acid 40.8 ± 3.4 (6) 1977 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 4253 J. S. Lazo et al. GDP-fucose. For this reason the intracellular content of [3H]fucose and GDP-[3H]fucose was measured by the method of Yurchenco and Atkinson (27). The ethanol-soluble radioactivity extracted from whole cells was identical with that found in TCA-soluble fractions (49.8 ±9.3 versus 47.0 ±2.7 pg of [3H]fucose equivalents per 106cells). GDP[3H]fucose represented 93.5% of the radioactivity found in control cell extracts (Chart 7). The only other 3H-labeled species extracted from cells was [3H]fucose, which repre sented 4.9% of the recovered radioactivity. Cells obtained from mice treated with 20 mg of 6-TG per kg contained only 9.1 pg of [3H]fucose equivalents per 106 cells in the ethanolic extracts, while untreated cells had 49.8 pg of [3H]fucose equivalents per 106 cells (Table 2). Only 75.4% of the radioactivity recovered from 6-TG-treated cells was found to be present in GDP-fucose, while over 20.6% of the radioactivity from 6-TG-treated cells migrated as free fucose. The specific activity of the GDP-fucose pool was measured in cells from mice treated with 0.9% NaCI solution or with an identical solution containing 6-TG (20 mg/kg). Table 3 Specific activity and intracellular content of GDP-fucose in Sarcoma 180 cells labeled with [3H]fucose after treatment with 6TG (20 mg/kg) The specific activity of the GDP-fucose pool was measured in 2 experiments in which cells from 2 mice were exposed in vivo for 2 hr to 0.9% NaCI solution with or without 6-TG (20 mg/kg). The intracellular content of GDP-fucose was calculated from the spe cific activity of GDP-fucose and the total radioactive content of this nucleotide sugar per cell as reported in Table 2. GDPfucose per 107 Treatment0.9% NaCI solution 6-TGnmoles cpm//j.g cells2660 GDP-fucose ± 70" 2650 ±810 13.6 2.0 " Mean ±range. No change in the specific activity of the GDP-fucose pool was detected, but an 85% decrease in the total intracellular quantity of GDP-fucose was observed following treatment with 6-TG (TableS). DISCUSSION Q. Ãœ S£2 - IO I5 MIGRATION 20 25 30 (cm) Chart 7. Chromatographie tracing of ethanol extracts from Sarcoma 180 cells after treatment with 6-TG. Sarcoma 180-bearing mice were given i.p. injections of 0.9% NaCI solution or 6-TG (20 mg/kg) 2 hr prior to being given injections of 5 /¿Ci of [3H]fucose. Cells were collected 30 min later and washed 3 times. Radiolabeled ethanol extracts from •,0.9% NaCI solution and D, 6-TG-treated cells were subjected to Whatman No. 3MM paper chromatography with 95% ethanoM M ammonium acetate (7:1, v/v) as solvent for 9 hr. Arrows, origin and solvent fronts. [3H]Fucose and GDP[14C]fucose migrated with RKvalues of 0.76 and 0.29, respectively. [3H]Fucose Sarcoma scribed in migrated in [14C]fucose determination Table 2 incorporation into ethanol extracts of Sarcoma 180 after treatment with 6-TG (20 mg/kg) 180-bearing mice were treated and assayed as de Chart 7. The radioactivity found in the peaks that a manner identical with that of [3H]fucose and GDPused as standards was calculated from the separate of cells from 3 to 4 mice. The [3H]fucose used in these experiments had 47.2 cpm/pg. Total pg [3H]fucose Treatment 0.9% NaCI solution 6-TG " Mean ±S.E. 4254 equivalents/ 106 cells" 49.8 ±9.3° 9.1 ±3.9 % total radioactivity GDP-[3H]fucose 93.5 ±1.6 75.4 ±8.7 | 'H]fucose 4.9 ±1.0 20.6 ±6.7 Radioactive fucose is a terminal carbohydrate of glycoproteins and has been extensively used to study the biosyn thesis of these molecules in eukaryotic cells (e.g., see Refs. 1, 3, and 15). L-Fucose is metabolically converted to /3-L-fucose-1-phosphate which is subsequently further anabolized by GDP-L-pyrophosphorylase to form GDP-L-fucose (14); GDP-fucose is then added to appropriate glycoprotein and glycolipid acceptors (3). In HeLa cells the radioactivity incorporated from fucose appears almost exclusively in plasma membranes in the form of fucosyl glycoproteins (1). However, the rate at which the radioactive fucose is incorporated into glycoprotein is not necessarily a reflec tion of the true rate of synthesis of these proteins, since the soluble precursor pool, primarily GDP-fucose, can be derived from 2 sources, i.e., through exogenous fucose (14) and endogenously synthesized GDP-mannose (8). In this investigation, after tumor-bearing mice were given injections of 0.07 /¿gof [3H]fucose, glycoprotein and the soluble precursor pool (almost completely GDP-fucose) contained radioactivity equivalent to 10.7 and 47.0 pg of [3H]fucose per 106 cells, respectively (Table 1). Since the intracellular pool size of GDP-fucose in Sarcoma 180 was found to be on the order of 13.6 nmoles (8.3 ¿¿g)/107 cells (Table 3), it is evident that only trace amounts of [3H]fucose are being incorporated into glycoproteins under the condi tions used. Sarcoma 180 cells, like HeLa cells (27), incor porate [3H]fucose almost exclusively into glycoprotein and contain predominantly GDP-fucose as the soluble radiolabeled fucose intermediate after a pulse of [3H]fucose (Chart 7; Table 2). Exposure of Sarcoma 180 cells to 6-TG both in vitro and in vivo caused an inhibition of [3H]fucose incorporation into glycoprotein without markedly reducing [14C]glucosamine incorporation into these macromolecules. This prefer ential decrease in exogenous fucose utilization occurs within 1 hr of drug treatment (Chart 4) and appears to be due, at least in part, to a decrease in intracellular GDPfucose content (Table 3). It is well established that 6-TG CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 6-7G Effects on Fucose Incorporation and the related antimetabolite 6-mercaptopurine inhibit the de novo biosynthesis of purine nucleotides (22, 23) and thereby cause a decrease in the intracellular pool of GTP (20). Such inhibition would be expected to lead to the suppression of purine nucleotide sugar biosynthesis by reducing the contents of both GDP-fucose and GDPmannose. The observed decrease in the content of GDPfucose in 6-TG-treated cells is consistent with such a concept. Moreover, since endogenously synthesized GDPmannose appears to be the major source of GDP-fucose (14), the failure to see a decrease in GDP-fucose specific activity after 6-TG treatment (Table 3) suggests that GDPmannose formation is also inhibited by the purine antime tabolite. Previous results from our laboratory indicate that at 6 hr, but not at 2 hr, after 6-TG treatment, a significant decrease occurred in the rate of cellular agglutination produced by the mannose-specific lectin, concanavalin A (11,12). This depression in the rate of agglutination appears to be due to a decrease in the degree of binding of concanavalin A to Sarcoma 180 cells (J. S. Lazo and A. C. Sartorelli, unpublished data). It has been demonstrated that surface components are associated with tumorigenicity, cell proliferation, and DNA replication (16, 21, 25). Moreover, it appears that at least some neoplastic tissues have significantly higher fucosyltransferase and fucosidase activities and higher GDP-fucose content than does normal tissue (2). Thus, it is conceivable that the alteration or inhibition of carbohydrate addition to surface membranes of tumor cells may disrupt the biosynthesis of cell surface receptors for external factors (e.g., serum factors) neces sary for cell proliferation (10). This suggestion is supported by the fact that inhibition of exogenous fucose incorpora tion is 1 of the earliest events inhibited by 6-TG and is sus tained for several hr after exposure of cells to this agent. Several other possible consequences of 6-TG inhibition of glycoprotein biosynthesis include osmotic imbalance and altered membrane transport, since these processes depend on membrane glycoprotein (13). Our results indicate that 6-TG has effects on surface glycoprotein biosynthesis and suggest that this phenomenon may be of relevance to the cytotoxicity produced by this agent. ACKNOWLEDGMENTS We wish to thank Charles W. Shansky for his technical assistance. REFERENCES 1. Atkinson. P. H. Synthesis and Assembly of HeLa Cell Plasma Membrane Glycoproteins and Proteins. J. Biol. Chem., 250. 2123-2134. 1975. 2. Bauer, C., Vischer, P., Grünholz,H., and Reutter, W. Glycosyltransferase and Glycosidases in Morris Hepatomas. Cancer Res., 37: ISISISIS. 1977. DECEMBER 3. Bekesi. J. G.. and Winzler, R J. The Metabolism of Plasma Glycopro teins J. Biol. Chem., 242. 3873-3879, 1967. 4. Brockman. R. W. Mechanisms of Resistance to Anticancer Agents. Advan. Cancer Res., 7: 129-234, 1963. 5. Crumpton, M. J. Identification of Amino Sugars. Biochem. J., 72: 479486, 1959. 6. Dische, Z.. and Shettles, L. B. 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Cancer Res.. 37. 1620-1626. 1971. 1977 Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research. 4255 Inhibition of l-Fucose Incorporation into Glycoprotein of Sarcoma 180 Ascites Cells by 6-Thioguanine John Stephen Lazo, Kou M. Hwang and Alan C. Sartorelli Cancer Res 1977;37:4250-4255. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/37/12/4250 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research.