Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates
Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates
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DOI：
10.1016/j.biocel.2003.08.014
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发表时间：
2004-03-01
影响因子：
4
通讯作者：
Christopherson, RI
中科院分区：
文献类型：
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作者：
Kamal, MA;Christopherson, RI
氨基磷酸核糖基转移酶(APRT)催化嘌呤核苷酸从头合成的第一步，即5-磷酸核糖基-1-焦磷酸(PRPP)转化为5-磷酸核糖胺(PRA)。APRT是开发抗癌药物抑制剂的有效靶点。我们建立了一种从细胞中提取的PRPP的薄层色谱分析方法。在偶联酶的作用下，过量的[2-C-14]甲酸(OA)的PRPP被定量地转化为[2-C-14]OMP，然后通过PPI的水解得到[2-C-14]UMP。反应产物在聚乙二亚胺-纤维素(PEI-C)色谱图上分离。在培养中生长的人CCRF-CEM白血病细胞已经暴露于一些抗叶酸，并确定了它们对细胞内PRPP水平的影响。在CCRF-CEM细胞中测得PRPP的稳态水平为102+/-11微米。在培养物中加入抗叶酸后，细胞中PRPP的积累表明了APRT的抑制程度。在人CCRF-CEM白血病细胞中，洛美曲索(LTX)、2，4-diamino-6-(3，4，5-trimethoxybenzyl)-5，6，7，8-tetrahydro-quinazoline(PY899)、甲氨蝶呤(MTX)、N-alpha(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine(PT523)、吡替曲辛(PTX)、美托品、2，4-diamino-6-(3，4，5-trimethoxyanilino)-methylpyrido[3，2-d]pyrimidine(PY873)和多靶点抗叶酸，N-[4[2-(2-amino-3，4-dihydro-4-oxo-7H-pyrrolo[2，3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic酸(NITA)直接或间接地抑制APRT，表现为PRPP积累的时程，最大值为3-12倍。这些数据表明，LTX对APRT的抑制作用最强。(C)2003爱思唯尔有限公司。保留所有权利。
Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-C-14]orotate (OA) is quantitatively converted to [2-C-14]OMP and then [2-C-14]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose(PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102 +/- 11 muM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N-alpha(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (NITA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT. (C) 2003 Elsevier Ltd. All rights reserved.