Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.
Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.
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发表时间：
2002-04
期刊：
影响因子：
11.2
通讯作者：
D. Cao;R. Russell;Dekai Zhang;J. Leffert;G. Pizzorno
中科院分区：
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作者：
D. Cao;R. Russell;Dekai Zhang;J. Leffert;G. Pizzorno
我们已经报道了尿苷磷酸化酶（UPase）在许多实体瘤中的升高，以及在乳腺癌组织中存在变异的磷酸化活性（M。Liu等人，癌症研究所，58：5418-5424，1998）。为了更好地理解这些发现的生物学和药理学意义，我们通过特异性基因打靶技术建立了UPase基因敲除的胚胎干细胞模型。在这个细胞模型中，我们确定了UPase作为一种重要的合成代谢酶在5-氟尿嘧啶（5-FU）激活和嘧啶补救途径调节中的关键作用。人们早就知道，UPase调节尿苷的血浆浓度;然而，很少有人知道UPase在5-FU及其衍生物的活化和代谢中的作用，主要是因为缺乏适当的模型系统。实验数据表明，鼠ES细胞中UPase活性的破坏导致5-FU IC（50）增加10倍，其掺入核酸中减少2-3倍，而其他嘧啶核苷类似物如5-氟尿苷、2 '-脱氧-5-氟尿苷、和1-β-D-阿拉伯呋喃糖基胞嘧啶。苯甲酰吖啶能特异性地抑制WT ES细胞对5-FU的敏感性。我们的数据还显示了UPase对5-FU前药5 '-脱氧-5-氟尿苷（5' DFUR）的细胞毒性的影响。与野生型细胞相比，在敲除细胞中IC（50）增加了几乎16倍，证明了UPase在催化5 'DFUR转化为5-FU中的作用。这些发现还阐明了卡培他滨的肿瘤特异性选择性，卡培他滨是批准用于治疗转移性乳腺癌和结直肠癌的口服氟嘧啶前药。敲除细胞不仅表现出5-FU掺入核酸的减少，而且对嘧啶补救途径的依赖性增加。UPase敲除细胞对嘧啶从头合成的依赖性降低反映在对膦酰乙酰-L-天冬氨酸（嘧啶途径的特异性抑制剂）的明显抗性中，与WT相比，UPase无效细胞的IC（50）升高5倍。总之，我们已经成功地产生了UPase基因敲除细胞模型，该模型对5-FU、5 'DFUR和膦酰乙酰-L-天冬氨酸的敏感性降低，尽管它在正常组织培养条件下不影响基本的细胞生理学。考虑到UPase在5-FU代谢中的作用以及与配对正常组织相比该蛋白在癌细胞中的表达升高，应进行额外的研究以确定UPase在5-FU和卡培他滨的肿瘤选择性激活中的临床作用。
We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.