Kucera G L, Capizzi R L
Comprehensive Cancer Center, Wake Forest University, Winston-Salem, North Carolina 27157.
Cancer Res. 1992 Jul 15;52(14):3886-91.
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.
为了确定在培养细胞以及白血病患者来源的细胞中，1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C）处理后生成1-β-D-阿拉伯呋喃糖基胞嘧啶二磷酸（ara-CDP）-胆碱的途径，我们探究了磷脂酰胆碱生物合成的CDP-胆碱途径中涉及的酶促步骤。在所用条件下，三磷酸ara-C不是CTP：磷酸胆碱胞苷转移酶活性的底物，而CTP和dCTP分别用于形成CDP-胆碱和dCDP-胆碱。当同时存在时，三磷酸ara-C和CTP在磷酸胆碱存在下抑制CTP向CDP-胆碱的酶促转化，其Ki为6 mM。由于CTP：磷酸胆碱胞苷转移酶似乎与ara-CDP-胆碱水平升高无关，我们接下来研究了磷脂酰胆碱合成途径中另一种可形成ara-CDP-胆碱的酶，即CDP-胆碱：1,2-二酰基甘油胆碱磷酸转移酶。从L5178Y小鼠白血病细胞分离的微粒体中存在的CDP-胆碱：1,2-二酰基甘油胆碱磷酸转移酶活性表现出其正常催化活性的逆转，利用CMP和1-β-D-阿拉伯呋喃糖基胞嘧啶单磷酸（ara-CMP）以及磷脂酰胆碱产生CDP-胆碱或ara-CDP-胆碱，外加二酰基甘油。CMP的Vmax和Km值分别为0.78±0.04 nmol/分钟/毫克和340±20 microM，而ara-CMP的Vmax和Km分别为0.22±0.06 nmol/分钟/毫克和1410±540 microM。在所使用的无细胞测定条件下，ara-CMP的Ki值为3 mM。这些结果表明，ara-CDP-胆碱很可能源于利用ara-CMP的CDP-胆碱：1,2-二酰基甘油胆碱磷酸转移酶的逆转，而不是由CTP：磷酸胆碱胞苷转移酶催化三磷酸ara-C加磷酸胆碱生成ara-CDP-胆碱。据推测，这种机制可能部分解释了高剂量ara-C治疗时观察到的快速细胞裂解现象。
