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Guanosine Diphosphate-T-Fucose Plasma:N-Acetylglucosaminide Fucosyltransferase As in Index of Bone Marrow Hyperplasia After Chemotherapy1

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Guanosine Diphosphate-T-Fucose Plasma:N-Acetylglucosaminide Fucosyltransferase As in Index of Bone Marrow Hyperplasia After Chemotherapy1 [CANCER RESEARCH 38, 181-184, January 1978] Guanosine Diphosphate-t-Fucose Plasma:N-Acetylglucosaminide Fucosyltransferase as in Index of Bone Marrow Hyperplasia after Chemotherapy1 Prem Khilanani, Ta-Hsu Chou, and David Kessel2 Department of Oncology ¡T-H.C., D. K.¡and Section of Hematology ¡P.K.], Wayne State University School of Medicine, and Michigan Cancer Foundation [T-H. C., D. K.], Detroit, Michigan 48201 ABSTRACT also measured plasma levels of another fucosyltransferase, which utilizes sugar acceptors with terminal /V-acetylglu- We have measured the plasma level of a fucosyltrans- cosamine residues. This enzyme has been described before ferase in patients with acute myelogenous leukemia and non-Hodgkin's lymphoma at various stages of the disease (4, 10, 16, 19), but its biological role is unknown. We found an elevated level of GDP-fucose:/V-acetylglucosaminide fu and in normal controls. This enzyme transfers the sugar cosyltransferase activity in patients during drug-maintained fucose from a guanosine diphosphate-i-fucose donor to remissions. The plasma level of this enzyme apparently high-molecular-weight acceptors with a terminal W-acetyl- reflects regeneration of a normal marrow population after glucosamine residue. The enzyme levels of fucosyltrans- myelosuppression. ferase in individuals free from disease and in patients with untreated leukemia or lymphoma were comparable. MATERIALS AND METHODS A substantial increase in plasma enzyme level was mea sured during drug-induced remissions, three weeks after Plasma Samples. Blood was collected from normal do drug therapy. The enzyme level fell to the normal range nors and patients with neoplastic disease, with EDTA as during unmaintamed remissions in patients with lympho- the anticoagulant. Erythrocytes were removed by centrifu- mas; comparable information for the leukemia is not gation at 1,000 x g for 10 min; platelets were removed by a available since all remissions were drug maintained. second centrifugation at 10,000 x g for 10 min. All opera These data, together with microscopic examination of tions were carried out at 4°.The final plasma preparation marrow samples, indicate that the level of this fucosyl- was stored at -70°(1 to 5 days) before assay procedures. transferase is correlated with regeneration of a normal Enzyme Assay. We had previously determined that the marrow population after chemotherapy. The enzyme as GDP-fucose:/V-acetylglucosaminide fucosyltransferase was say may prove useful in defining normal bone marrow not inhibited by the presence of 3 mM N-ethylmaleimide recovery and in timing cyclic combination chemotherapy (6); this reagent was therefore added to all incubation in patients with neoplastic disease. mixtures. The assay mixture, in a total volume of 150 ¿¿I, contained 50 ¿ilplasma, 50 mM cacodylate buffer [pH 7.0; mM ATP, 3 mM A/-ethylmaleimide, 10 mM MgCL, 10 mM INTRODUCTION ethylene glycol bis(/3-aminoethyl ether)-A/,/V-tetraacetic The glycosyltransferases are a series of enzymes that acid (which chelates serum calcium, an enzyme inhibitor)], 1 fíMGDP-[14C]fucose, and 0.5 ml fetuin from which the add specific sugar residues to protein or glycoprotein acceptors (15). Elevated levels of sialyltransferase (2, 5, 6, terminal sialic acid and galactose moieties had been re 9, 11, 15), galactosyltransferase (3), and fucosyltransferase moved (18). Each mixture contained 50,000 cpm radioac (12, 13) have been detected in plasma of tumor-bearing tivity. After incubation at 37°for60 min, a 120-^1 portion of animals and in patients with neoplastic disease. The expla the mixture was washed through a 0.5- x 1-cm column of nation for this phenomenon is unknown; "shedding" of Dowex 1 (OH) with 1 ml of water. The product appears in membrane-bound enzyme into the plasma has been sug the eluate, whereas radioactive GDP-fucose, fucose, and gested as a relevant factor (2). other fucose derivatives are retained or their elution is re In a study of patients with acute myelogenous leukemia tarded by the resin (8). The column eluate is taken up in an (12) and non-Hodgkin's lymphoma (14), we found that the appropriate solvent for determination of radioactivity by level of plasma GDP-fucose3:galactoside fucosyltransferase liquid scintillation counting. We confirmed by Chromato was correlated with the extent of the disease. This enzyme graphie analysis (16) that no low-molecular-weight material catalyzes transfer of the sugar fucose onto glycoprotein appeared in this column eluate. Results are reported in acceptors with terminal galactose residues and is presum terms of cpm radioactivity per 50 /nl plasma, representing incorporation of [14C]fucose into glycoprotein. ably specified by the H gene (17) responsible for ABO blood group activity. During the course of this study, we Substrate (GDP-fucose) degradation was monitored in selected samples by high-voltage electrophoresis on paper, as described in Ref. 16, and by a Chromatographie proce 1 This work was supported in part by Grant CA 7177 from the National dure (7). «-Fucosidasewas measured with p-nitrophenyl-a- Cancer Institute. NIH. fucoside (1). Product degradation was measured as follows. 1 To whom requests for reprints should be addressed, at Department of Oncology. 4160 John R Street. Detroit. Mich. 48201. With a plasma sample with high fucosyltransferase activity, 3 The abbreviation used is: GDP-fucose. guanosine diphosphate-L-fucose. the reaction mixture was scaled up by a factor of 10. After JANUARY 1978 181 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. P. Khilanani et al. incubation for 60 min at 37°.the reaction mixture was min at 37°.Under these conditions, approximately 3 to 10% eluted through a 1- x 5-cm column of Dowex 1 (OH) and of the total radioactivity was solubilized (Table 1). This lyophilized. We determined via a Chromatographie proce latter experiment provides a more stringent test of fucosi dure (16, 17) that this product was free from radioactive dase levels in plasma samples that did the study done with fucose and GDP-fucose. We then incubated portions of p-nitrophenyl-«-L-fucopyranoside; no correlation between this product (each containing 10,000 cpm radioactivity) such activity and disease status was found. with 50-/nl samples of plasmas, ethylene glycol bis(/y-ami- The product formed in the presence of /V-ethylmaleimide noethyl etherJ-N./V'-tetraacetic acid, MgCU, and cacodylate was subjected to mild acid and alkaline hydrolysis. Acid buffer (pH 7.0) under usual experimental conditions. Solu- hydrolysis yielded only radioactive L-fucose. as measured bilization of radioactivity into a form not precipitated by 1% by a Chromatographie procedure (14). Alkaline hydrolysis phosphotungstic acid in 0.5 M HCI was taken to represent of the product afforded a 60 to 80% yield of free fucose, the action of plasma fucosidases on the product. This suggesting that a fucose (1—»2)galactoselinkage was not product was also partly characterized by alkaline and acid involved. hydrolysis (16). We measured the level of the GDP-fucose:/V-acetylglu- cosaminide fucosyltransferase in the plasma of 54 individ RESULTS uals, including 14 normal donors, 7 patients with acute leukemia in drug-induced complete remission, 9 patients As provided by New England Nuclear, Boston, Mass., the with acute leukemia not responding to drug therapy, 4 preparation of radioactive GDP-fucose contained 97% GDP- patients with untreated non-Hodgkin's lymphoma, 6 pa fucose and 0.55% fucose by Chromatographie analysis, tients with non-Hodgkin's lymphoma in drug-maintained with the solvent systems described in Ref. 7. Incubation of remission, and 7 such patients in unmaintained remissions. radioactive GDP-fucose for 60 min at 37°in the assay Patients with untreated leukemias were chosen from a system used here caused degradation of the nucleotide- group with at least >50% marrow myeloblasts. Those with sugar, resulting in 15% conversion to fucose. When se untreated lymphoma were characterized by lymphadenop- lected plasma samples were added to the incubation mix athy and/or hepatosplenomegaly and/or positive lymphan- ture, although no exogenous acceptor was added, the giogram with diagnosis confirmed by lymph node biopsy. extent of GDP-fucose degradation was not altered. We Complete remission in leukemia was defined as a patient conclude that the plasma samples examined here, under whose marrow contained <5% myeloblasts (or <10% mye these conditions, did not cause any detectable substrate loblasts + promyeloblasts) with qualitatively and quantita degradation. tively normal granulopoiesis, erythropoiesis, and thrombo- The measurement of «-fucosidase activity, with p-nitro- poiesis. Complete remission in non-Hodgkin's lymphoma phenyl-a-L-fucopyranoside as substrate, showed variable was equated with no evidence of lymphadenopathy or levels of enzyme activity in selected plasma samples. How hepatosplenomegaly, a negative lymphangiogram, and ever, we found no correlation between such activity and negative liver and bone marrow biopsies. disease status (Table 1). Since some fucosidases are inac In patients with drug-induced complete remission, we tive with low-molecular substrates, we incubated the radio measured regeneration of marrow components by needle active product of fucosyltransferase activity, with desialated biopsy and marrow aspiration. Normal marrow hyperplasia fetuin as substrate, with selected plasma samples for 60 resulted in increased
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