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Effects of Pyrimidine Antagonists on Sialic Acid Regeneration in HL-60 Cells¹

Division of Oncology, Department of Medicine, Columbia University, New York, New York [A. A. H., R. N. T., S. G., G. C.], and Department of Medicine, Toronto Western Hospital, University of Toronto, Toronto, Canada [M. A. B.]

Because alterations in cell membrane sialoglycoconjugates can affect the behavior of neoplastic cells, we investigated the effects of in vitro treatment with antimetabolites used in cancer therapy on the expression of membrane sialic acid in cultured HL-60 leukemic cells. In these studies, cells were incubated with Vibrio cholerae neuraminidase to remove surface sialic acid. Reappearance of membrane sialic acid during drug treatment was followed (a) by measuring changes in radioactive surface labeling of viable cells with sodium metaperiodate-sodium[³H]-borohydride, (b) by measuring the decline in accessible surface galactosyl receptor sites which occurred coincident with membrane sialic acid replacement, and (c) by measuring the incorporation of [³H]glucosamine into membrane-associated neuraminidase-labile sialic acid.

We were especially interested in learning whether drugs that affect intracellular pools of cytidine triphosphate (CTP), an important nucleotide intermediate in sialylation reactions, could inhibit regeneration of membrane sialic acid. 3-Deazauridine, a competitive inhibitor of CTP synthetase, depleted CTP pools and curtailed surface membrane resialylation with little or no effect on synthesis of de novo sialic acid from precursor sugars. The addition of cytidine restored CTP pools and sialic acid regeneration.

Acivicin, a glutamine antagonist, also depleted CTP pools and curtailed surface membrane resialylation. In addition, it retarded de novo synthesis of sialic acid. The addition of cytidine restored intracellular CTP pools and sialic acid regeneration. However, both cytidine and guanosine were required to restore sialic acid synthesis from precursor sugars.

1-ß-D-Arabinofuranosylcytosine, a competitive inhibitor of sialic acid synthetase and of sialyltransferase, inhibited both de novo sialic acid synthesis and membrane resialylation. Only the latter effect was reversed by the addition of exogenous cytidine.

Hydroxyurea, an agent shown previously to inhibit glycoconjugate production in hamster fibroblasts, curtailed membrane resialylation and de novo synthesis of sialic acid without depleting CTP pools.

Doxorubicin, at levels that caused marked arrest of cell proliferation, had no effect on sialic acid synthesis or expression on the membrane surface.

These data suggest that antimetabolites, apart from their cytotoxic effects or effects on cellular growth, may directly inhibit the expression of membrane sialic acid. Acivicin and 3-deazauridine may act by depleting intracellular CTP pools, 1-ß-D-arabinofuranosylcytosine by competing with cytidine derivatives and curtailing de novo synthesis of sialic acid, and hydroxyurea by inhibiting de novo synthesis of sialic acid. The idea that inhibition of membrane sialylation by chemotherapeutic drugs may alter cell surface properties of tumor cells and contribute to the antineoplastic effect should be further explored.

¹ Supported by the National Cancer Institute, Grants CA-31761, CA-31762, and CA-35601, the Medical Research Council of Canada, the National Cancer Institute of Canada, and the William J. Matheson Foundation.

² Fellow of the Leukemia Society of America and Junior Clinical Faculty Fellow of the American Cancer Society.

³ Scholar of the Leukemia Society of America.

Received 6/15/84. Revised 3/27/85. Accepted 4/ 3/85.

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