1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.
In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (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 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-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 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki 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.
Kucera
G L
GL
Comprehensive Cancer Center, Wake Forest University, Winston-Salem, North Carolina 27157.
Capizzi
R L
RL
D000818:Animals; D019916:Choline-Phosphate Cytidylyltransferase; D003561:Cytarabine / Q000031:analogs & derivatives* / Q000378:metabolism*; D003566:Cytidine Diphosphate Choline / Q000031:analogs & derivatives* / Q000378:metabolism; D002798:Diacylglycerol Cholinephosphotransferase / Q000378:metabolism*; D007940:Leukemia L5178 / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism*; D001084:Vidarabine Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D003561:Cytarabine; D001084:Vidarabine Phosphate; C000101:ara-ATP; D003566:Cytidine Diphosphate Choline; C016241:1 beta-D-arabinofuranosylcytosine diphosphate choline; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D002798:Diacylglycerol Cholinephosphotransferase