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.