Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.
Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.
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DOI：
10.1038/bjc.1995.392
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发表时间：
1995-09
影响因子：
8.8
通讯作者：
Harris, A L
中科院分区：
文献类型：
--
作者：
Patterson, A V;Zhang, H;Moghaddam, A;Bicknell, R;Talbot, D C;Stratford, I J;Harris, A L
血小板衍生内皮细胞生长因子(PD-ECGF)与人胸苷磷酸化酶(DThdPase)相同。将dThdPase基因导入人乳腺癌细胞系MCF-7，表达了45 kDa的蛋白，并用抗dThdPase抗体进行了检测。细胞裂解产物具有较高的dThdPase活性，细胞对前药5‘-脱氧-5-氟尿苷(5’-DFUR)的敏感性在体外增加了165倍。对5-氟尿嘧啶(5-FU)和5-氟-2‘-脱氧尿苷(5-FUDR)的敏感性不变。重组dThdPase可直接催化5‘-DFUR的磷酸裂解生成5-FU。外源性胸腺嘧啶核苷(DThd)逆转了5-FudR对亲本细胞的毒性(1微米dThd使IC50值增加1000倍)，但dThd的拯救在表达dThdPase的克隆4(1微米dThd使IC50值增加3倍)中被大量调节。我们观察到，当小部分表达dThdPase的细胞与亲本MCF-7细胞混合时，会产生实质性的“旁观者”杀伤效应。乳腺肿瘤的dThdPase活性平均是正常乳腺的27倍。野生型MCF-7的表达水平与肿瘤表达的低端相似。因此，在某些肿瘤中，对5‘-DFUR治疗的耐药性可能是由于dThdPase活性低所致，而在较宽的肿瘤范围内或更高的范围内提高dThdPase水平的转基因应该会显著增强对前药的敏感性。这些结果证实了dThdPase是5‘-DFUR代谢活化的主要途径，旁观者效应表明dThdPase可能是一种适合于基因治疗导向的酶/前药激活治疗的酶。
Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.