Biotin supplementation increases expression of the cytochrome P4501B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks
Biotin supplementation increases expression of the cytochrome P4501B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks
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DOI：
10.1093/jn/134.9.2222
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发表时间：
2004-09-01
影响因子：
4.2
通讯作者：
Zempleni, J
中科院分区：
文献类型：
--
作者：
Rodriguez-Melendez, R;Griffin, JB;Zempleni, J
DNA微阵列研究提供的证据表明，生物素补充增加了人类淋巴细胞中编码细胞色素P-450 1B 1（CYP 1B 1）的mRNA的丰度。CYP 1B 1羟基化前致癌物，产生亲电诱变剂。在这里，我们试图确定增加CYP 1B 1在生物素补充的人T（Jurkat）细胞中的表达的信号通路，并确定CYP 1B 1基因的激活是否与单链DNA断裂的发生率增加有关。Jurkat细胞在生物素缺乏（0.025 nmol/L）和生物素补充（10 nmol/L）的培养基中培养。与缺乏生物素的细胞相比，添加生物素的细胞中CYP 1B 1报告基因构建体的转录活性高24%（P < 0.01）。同样地，补充生物素的细胞中CYP 1B 1 mRNA的丰度比缺乏生物素的细胞高72%（P < 0.05）。电泳迁移率变动分析表明，在CYP 1B 1基因的调控区Sp1位点发挥重要作用，生物素的转录激活。与生物素缺乏的细胞相比，添加生物素的细胞中CYP 1B 1蛋白的丰度和CYP 1B 1活性分别增加了124%和35%（P < 0.05）。与生物素缺乏细胞相比，补充生物素的细胞中CYP 1B 1表达增加与单链DNA断裂发生率增加相关; CYP 1B 1的合成抑制剂可防止链断裂，表明生物素的作用对CYP 1B 1具有特异性。这些研究提供的证据表明，具有Sp1位点亲和力的转录因子介导补充生物素的T细胞中CYP 1B 1基因的转录激活，增加单链DNA断裂的发生。
DNA microarray studies provided evidence that biotin supplementation increases the abundance of mRNA encoding cytochrome P-450 1B1 (CYP1B1) in human lymphocytes. CYP1B1 hydroxylates procarcinogens, generating electrophilic mutagens. Here, we sought to identify the signaling pathways that increase the expression of CYP1B1 in biotin-supplemented human T (Jurkat) cells and to determine whether activation of the CYP1B1 gene is associated with increased occurrence of single-stranded DNA breaks. Jurkat cells were cultured in biotindeficient (0.025 nmol/Q and biotin-supplemented (10 nmol/L) media. The transcriptional activity of a CYP1B1 reporter gene construct was 24% greater in biotin-supplemented compared with biotin-deficient cells (P < 0.01). Similarly, the abundance of CYP1B1 mRNA was 72% greater in biotin-supplemented than in biotin-deficient cells (P < 0.05). Electrophoretic mobility shift assays suggested that Sp1 sites in the regulatory region of the CYP1B1 gene play important roles in transcriptional activation by biotin. The abundance of CYP1B1 protein and activity of CYP1B1 were 124 and 35% greater, respectively, in biotin-supplemented compared with biotin-deficient cells (P < 0.05). The increased expression of CYP1B1 in biotin-supplemented cells was associated with an increase in the occurrence of single-stranded DNA breaks compared with biotin-deficient cells; synthetic inhibitors of CYP1B1 prevented strand breaks, suggesting that the effects of biotin were specific for CYP1B1. These studies provide evidence that transcription factors with an affinity for Sp1 sites mediate transcriptional activation of the CYP1B1 gene in biotin-supplemented T cells, increasing the occurrence of single-stranded DNA breaks.