Phenotypic profiling of DPYD variations relevant to 5-fluorouracil sensitivity using real-time cellular analysis and in vitro measurement of enzyme activity.
Phenotypic profiling of DPYD variations relevant to 5-fluorouracil sensitivity using real-time cellular analysis and in vitro measurement of enzyme activity.
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DOI：
10.1158/0008-5472.can-12-3858
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发表时间：
2013-03-15
期刊：
影响因子：
11.2
通讯作者：
Diasio RB
中科院分区：
文献类型：
--
作者：
Offer SM;Wegner NJ;Fossum C;Wang K;Diasio RB
在其发展的45年中，嘧啶类似物5-氟尿嘧啶(5-FU)已成为许多癌症治疗中不可或缺的组成部分，尤其是用于结直肠癌的治疗。接受5-FU治疗的患者中有相当一部分会出现严重的不良反应，在极端情况下可能会导致死亡。二氢嘧啶脱氢酶(DPD，由DPYD编码)能迅速降解85%的5-FU，因此限制了可用于转化为活性代谢物的药物的数量。临床研究表明，DPYD的遗传变异增加了5-FU毒性的风险，但对于哪些变异是相关的预测因素，还没有明确的共识。在本研究中，我们在哺乳动物细胞中表达了DPYD变异体，并测定了表达蛋白相对于野生型的酶活性。同时测定了表达DPYD变异的细胞对5-FU的相对敏感性。DPYD*2A变异体(IVS14+1G>；A导致外显子14缺失)被证实是催化失活的。与野生型相比，S534N和C29R两个突变体的酶活性显著高于野生型。与表达野生型DPYD的细胞相比，表达S534N的细胞对5-FU介导的毒性具有更强的抵抗力。这些发现支持这样的假设，即选定的DPYD等位基因对严重的5-FU毒性具有保护作用，因此可能会降低5-FU作为抗肿瘤药物在携带者中的有效性。此外，这项研究还展示了一种方法，该方法可能有助于对药物相关途径中的其他遗传变异进行表型鉴定。
In the 45 years since its development, the pyrimidine analog 5-fluorouracil (5-FU) has become an integral component of many cancer treatments, most notably for the management of colorectal cancer. An appreciable fraction of patients who receive 5-FU suffer severe adverse toxicities, which in extreme cases may result in death. Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Clinical studies have suggested that genetic variations in DPYD increase the risk for 5-FU toxicity, however there is not a clear consensus as to which variations are relevant predictors. In the present study, DPYD variants were expressed in mammalian cells, and the enzymatic activity of expressed protein was determined relative to wild type. Relative sensitivity to 5-FU for cells expressing DPYD variations was also measured. The DPYD*2A variant (exon 14 deletion caused by IVS14+1G>A) was confirmed to be catalytically inactive. Compared to wild type, two variants, S534N and C29R, showed significantly higher enzymatic activity. Cells expressing S534N were more resistant to 5-FU mediated toxicity compared to cells expressing wild type DPYD. These findings support the hypothesis that selected DPYD alleles are protective against severe 5-FU toxicity, and, as a consequence, may decrease the effectiveness of 5-FU an anti tumor drug in carriers. Additionally, this study demonstrates a method that may be useful for phenotyping other genetic variations in pharmacologically relevant pathways.