[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

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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 marrow cellularity, an aspirate show-

Table 1 Comparison of fucosyltransferase level versus degradar/Ve reactions in plasmas product GDP-fucose degrada DonorC. status"NormalNormalAMI, ferase6250400130450160020004508009002300550950Fucosidase'263227642448714945393946%degradation''121216151213161715171718%tion'785835764567 M.K. K.S. R.B. untreatedAMI, C.Ph. untreatedAMI, C.P.C.I. remissionAMI, remissionNHL, M.J. untreatedNHL, D.J. untreatedNHL, H.D. drugNHL,remission, on T.J. drugNHL.remission, on M.V. drugNHL,remission, off A.Disease remission, off drugFucosyltrans " AML, acute myelogenous lymphoma; NHL, non-Hodgkin's lymphoma. * Units [cpm/50 n\ plasma during 60-min incubations representing incorporation of radioactivity into high- molecular-weight acceptors (see Chart 1)]. '' Measured with p-nitrophenyl fucose as substrate; expressed as Amólesfucose formed per ml plasma per hr. '' Degradation of substrate (GDP-fucose) during 60-min incubations measured via paper Chromatographie analysis of reaction mixtures. Omission of plasma results in approximately 13 to 15% degradation. ' Degradation of high-molecular-weight product (fucosidase activity) by plasma enzymes; expressed as percentage of total high-molecular-weight product initially provided.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. Plasma Fucosyltransferase ing no malignant cells, normal marrow elements with tri- agents, the plasma level of this enzyme was substantially linear maturation, and an increase in normal precursor elevated above control values. Since drug maintenance cells. therapy in leukemia patients could not be discontinued, we The therapy used in acute leukemia was a combination measured enzyme activity in the plasma of lymphoma pa of Adriamycin, vincristine, prednisone, and 1-j8-D-arabino- tients in unmaintained remissions, 6 months after the last furanosylcytosine. Lymphoma patients received Adriamy drug administration. The level of plasma fucosyltransferase cin, vincristine, cyclophosphamide, and prednisone. Pa in such patients was in the normal range. tients sampled during unmaintained remissions had not These data indicate that the plasma level of this fucosyl received any antitumor drug for at least 6 months before transferase provides a measure of increased normal bone the plasma was sampled. marrow regeneration. Whether this specifically correlates Our data showed essentially normal levels of enzyme with normal erythroid, myeloid, megakaryocytic, or mono- activity in plasmas obtained from patients with untreated cytic hyperplasia is not known. The assay described here leukemia or lymphoma and from patients with lymphomas may prove useful in defining normal marrow recovery and in unmaintained remissions. Substantially elevated levels in timing the cyclic combination chemotherapy in patients of enzyme activity were found in the plasma of patients on with acute leukemia and solid tumors. drug therapy. When repeated samples were taken on 3 The nature of the product formed during the enzyme successive days, these values did not vary by more than reaction studied here is unknown. Schachter (19) has de ±10%.These results are summarized in Chart 1. In a group scribed transfer of fucose from a GDP-fucose donor onto of 9 patients with acute myelogenous leukemia, who were an internal /v-acetylglucosamine residue of a glycopeptide not responding to drug therapy, the mean value of plasma acceptor containing a terminal /v-acetylglucosamine resi fucosyltransferase was 300 enzyme units, with a range of due. The limited studies described here on product struc 150 to 495 (data not shown). ture are compatible with the supposition that a similar biosynthesis occurs in our incubation mixtures. Appropri ate studies with an endo-0-A/-acetylglucosaminidase should DISCUSSION clarify this point. In previous studies, elevated levels of plasma glycosyl- transferases were associated with neoplasia (2-6, 9, 11- ACKNOWLEDGMENTS 14), perhaps as a result of shedding of cell membrane components containing membrane-bound enzymes into the We thank Joanne Blahnik and Cheryl Murphy for their technical assistance and Terri Buvia for manuscript preparation. plasma. In this study we have described evidence that indicates an elevated level of glycosyltransferase associated with regeneration of normal marrow population in patients REFERENCES with lymphoma or leukemia after myelosuppressive drug 1 Alhadeff, J. A.. Miller. A. L.. Wenaas. H., Vedvick, T.. and 0 Brien, J. S. therapy. This elevation of enzyme activity coincided with Human Liver <>-L-Fucosidase.Purification. Characterization, and Immu- nochemical Studies. J. Biol. Chem , 250 7106-7113. 1975. morphological evidence of increased bone marrow regen 2 Bernacki, R.. and Kim, U. Concomitant Elevations in Serum Sialyltrans- eration. The level of GDP-fucose:/v-acetylglucosaminide ferase Activity and Sialic Acid Content in Rats with Metastasizing Mammary Tumors. Science, 795. 577-580, 1977. fucosyltransferase activity in plasma was not elevated in 3 Bhattacharya, M., Chatterjee. S. K.. and Barlow, J. J. Uridine 5'-Diphos- patients with untreated disease, suggesting that the enzyme phate-Galactose:Glycoprotein Galactosyltransferase Activity in the Ovar was not derived from a neoplastic cell population or was ian Cancer Patient. Cancer Res., 36: 2096-2100, 1976. not a result of any paraneoplastic syndrome associated 4. Bosmann, H. B., Hagopian, A., and Eylar, E. H. Glycoprotein Biosyn with acute myelogenous leukemia or non-Hodgkin's lym thesis: The Characterization of Two Glycoprotein: Fucosyl Transferases in HeLa Cells. Arch. Biochem Biophys.. 128: 470-481, 1968 phoma. Nonresponding patients did not show elevated 5. Bosmann. H. B., and Hilf. R. Elevations in Serum Glycoprotein:N- Acetylneuraminic Acid Transferases in Rats Bearing Mammary Tumors levels of plasma enzyme activity, ruling out a direct drug Federation European Biochem. Soc Letters, 44: 313-316. 1974. effect on enzyme activity. During drug-maintained remis 6 Bosmann, H. B., Spataro, A. C., and Myers, M W. Serum and Host sions involving periodic administration of myelosuppressive Liver Activities of Glycosidases and Sialyltransferases in Animals Bear ing Transplantable Tumors. Res. Commun. Chem. Pathol. Pharmacol.. 12: 499-511, 1975. 7 Chester, M A.. Yates. A. D., and Watkins. W M. Phenyl 0-D-Galactopy- ranoside as an Acceptor Substrate for the Blood-Group H Gene-Associ 2000 ated Guanosine Diphosphate L-Fucose: /3-o-Galactosyl <»-2-L-Fucosyl- transferase. European J Biochem., 69 583-592. 1976 8. Chou, T. H.. Murphy C , and Kessel. D. Selective Inhibition of a Plasma Fucosyltransferase by N-Ethylmaleimide Biochem. Biophys. Res Com mun., 74: 1001-1006, 1977. IOOO 9. Henderson, M.. and Kessel, D. Alterations in Plasma Sialyltransferase Levels in Patients with Neoplastic Disease Cancer. 39. 1129-1134, 1977. 10 Jabbal I., and Schachter, H. Pork Liver Guanosine Diphosphate-L- ii- Fucose Glycoprotein Fucosyltransferases J Biol Chem., 246. 5154- ABC D^ E F 5161, 1971. 11. Kessel, D.. and Allen, J Elevated Plasma Sialyltransferase in the Cancer Chart 1. Comparison of GDP-fucose:N-acetylglucosaminide fucosyl Patient. Cancer Res.. 35 670-672. 1975. transferase levels in plasma samples. A, disease-free controls; B. patients 12. Kessel. D., and Chou, T H Fucosyltransferase Levels in Plasma and with untreated acute myelogenous leukemia; C, acute myelogenous leuke Tissues of the Cancer Patient Federation Proc., 35: 1442. 1976. mia in drug-maintained remission; D, untreated non-Hodgkin s lymphoma; 13. Khilanani, P.. Chou, T-H., Lomen, P. L., and Kessel. D Variation of E, lymphomas in drug-maintained remission; F, lymphomas in unmaintained Levels of Plasma Guanosine Disphosphate L-Fucose:#-D-Galactosyl <>- remission. Data are in terms of cpm radioactivity incorporated into glycopro- 2-L-Fucosyltransferase in Acute Adult Leukemia Cancer Res.. 37: 2557- tein during 60-min incubations of 50-/jl plasma samples. 2559, 1977.

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14. Khilanani, P., Chou, T. H., Ratanatharathorn, V., and Kessel, D. Evalua- tor, and Lewis Blood-Group Phenotypes. European J. Cancer, 30. 269- tion of Two Plasma Fucosyltransferases as Marker Enzymes in Non- 277, 1972. Hodgkin's Lymphoma. Cancer, in press. 18. Spiro, R. G. Periodate Oxidation of the Glycoprotein Fetuin. J. Biol. 15. Marshall, R. D. Glycoproteins. Ann. Rev. Biochem., 41: 673-702, 1972. Chem., 239: 567-573, 1964. 16. Munro, J. R., and Schachter, H. The Presence of Two GDP-L- 19. Wilson, J. R., Williams, D.. and Schachter. H. The Control of Glycopro- Fucose:Glycoprotein Fucosyltransferases in Human Serum. Arch. Bio- tein Synthesis: N-Acetylglucosamine Linkage to a Mannose Residue as chem. Biophys., 756: 534-542, 1973. a Signal for the Attachment of L-Fucose to the Asparagine-Linked N- 17. Schenkel-Brunner, H., Chester, M. A., and Watkins, W. M. 2-L-o-Fuco- Acetylglucosamine Residue of Glycopeptide from a Acid Glycoprotein. syltransferases in Human Serum from Donors of Different ABO, Secre- Biochem. Biophys. Res. Commun., 72: 909-916, 1976.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. Guanosine Diphosphate-l-Fucose Plasma:N -Acetylglucosaminide Fucosyltransferase as in Index of Bone Marrow Hyperplasia after Chemotherapy

Prem Khilanani, Ta-Hsu Chou and David Kessel

Cancer Res 1978;38:181-184.

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