Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.
Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.
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发表时间：
1998-12
期刊：
影响因子：
11.2
通讯作者：
Mengping Liu;Deliang Cao;R. Russell;R. E. Handschumacher;G. Pizzorno
中科院分区：
文献类型：
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作者：
Mengping Liu;Deliang Cao;R. Russell;R. E. Handschumacher;G. Pizzorno
尿苷磷酸化酶（UPase）催化尿苷可逆磷酸解为尿嘧啶。我们纯化的酶从小鼠结肠26肿瘤使用两步程序通过5-氨基-苄基acyclouridine亲和层析。在兔中产生的抗纯化蛋白的抗体在正常人组织和肿瘤提取物的Western印迹中显示单一条带。用于筛选人肝表达文库的多克隆抗体允许分离含有人UPase的整个开放阅读框的1.2-kb克隆。已使用pMal-C2载体在大肠杆菌中将UPase cDNA表达为融合蛋白。动力学分析表明，重组UPase优先使用尿苷，5-氟尿嘧啶，尿嘧啶作为底物，虽然较低的活性水平，观察到2-脱氧尿苷和胸苷。检测了人肿瘤和邻近正常组织的临床样品的磷酸分解活性和对5-苄基酰基尿苷（BAU）的敏感性，BAU是目前在I-II期临床试验中的酶的有效抑制剂。正常组织中的活性似乎较低，但对BAU非常敏感（在10 μ M时约90%抑制）。肿瘤的活性通常是邻近正常组织的2-3倍。然而，在乳腺癌标本和头颈部鳞状细胞癌中，尿苷裂解仅被10或100 μ M BAU部分抑制（40-60%）。BAU不敏感的活性需要与正常酶相似的磷酸盐和pH条件，并且新的磷酸解活性不依赖于胸苷磷酸化酶。在选定的肿瘤中的BAU不敏感的磷酸化活性，加上BAU对正常人体组织中的“经典”尿苷磷酸化酶的有效抑制活性，为BAU与5-氟尿嘧啶联合治疗乳腺和头颈部肿瘤提供了理论基础。
Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.