1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase
Kucera, G.L.; Capizzi, R.L.
Cancer Research 52(14): 3886-3891
1992
ISSN/ISBN: 0008-5472 PMID: 1377599 Document Number: 402707
In an effort to identify the pathway leading to the formation of 1-.beta.-D-arabinofuranosylcytosine-diphosphatase (ara-CDP)-choline from 1-.beta.-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 Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-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 choline-phosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase acivity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-.beta.-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 .+-. 0.04 nmol/min/mg and 340 .+-. 20 .mu.M, respectively, whereas the Vmax and Km for ara-CMP were 0.22 .+-. 0.06 nmol/min/mg and 1410 .+-. 540 .mu.M, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay condtions 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 cytidylyl transferase. It is speculated that this mechanism may explain, inpart, the rapid cellular lysis observed with high dose ara-C therapy.