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1-ß-D-Arabinofuranosylcytosine-diphosphate-choline Is Formed by the Reversal of Cholinephosphotransferase and not via Cytidylyltransferase¹

Comprehensive Cancer Center of Wake Forest University, Winston-Salem, North Carolina 27157

In an effort to identify the pathway leading to the formation of 1-ß-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-ß-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a K_i of 6 mM.

Since CTP:phosphocholine cytidylytransferase did not appear to be responsible for the increased levels of area-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-ß-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The V_max and K_m values for CMP were 0.78 ± 0.04 nmol/min/mg and 340 ± 20 µM, respectively, whereas the V_max and K_m for ara-CMP were 0.22 ± 0.06 nmol/min/mg and 1410 ± 540 µM, respectively. A K_i value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy.

¹ This work was supported by grants from the American Cancer Society (CH471N), the Cancer Services, Inc., the Gaston County Cancer Society, Inc., and the Comprehensive Cancer Center of Wake Forest University Core Grant NIH NCI CA 12197.

² To whom requests for reprints should be addressed.

³ Present address: U.S. Bioscience, One Tower Bridge, 100 Front Street, P.O. Box 851, West Conshohocken, PA 19428.

Received 2/ 3/92. Accepted 5/ 4/92.

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