Xia G, Kageyama Y, Hayashi T, Kawakami S, Yoshida M, Kihara K
Department of Urology and Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Cancer. 2001 Apr 15;91(8):1429-36. doi: 10.1002/1097-0142(20010415)91:8<1429::aid-cncr1149>3.0.co;2-v.
Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that expressed most abundantly in highly vascularized organs. The authors examined the effect of transfection of EPAS1 cDNA on the endogenous expression of vascular endothelial growth factor (VEGF) in the 293 Tet-Off cell line and the possible involvement of EPAS1 in the angiogenesis of renal cell carcinoma (RCC).
Complete cDNA of EPAS1 was cloned and transfected to cells from the 293 Tet-Off fetal kidney cell line, in which the expression of EPAS1 can be inhibited by doxycycline. The subsequent changes in expression pattern of VEGF and transferrin receptor (TfR), a target gene of hypoxia-inducible factor 1alpha (HIF-1alpha), were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. In addition, expression of EPAS1, HIF-1alpha, and VEGF were analyzed by semiquantitative RT-PCR in five RCC cell lines and in 13 RCC tissue samples. In situ hybridization was performed on 7 of the 13 RCC tissue samples.
Endogenous VEGF was increased significantly by the introduction of EPAS1 cDNA at both the mRNA level and the protein level. With the inhibition of EPAS1 by doxycycline treatment, the expression of VEGF was significantly decreased accordingly, whereas the expression of TfR was not affected. EPAS1 was detected in all of the RCC cell lines examined. In RCC tissue samples, EPAS1 mRNA and VEGF mRNA were increased significantly in tumor tissues compared with normal adjacent kidney tissues. In situ hybridization showed that EPAS1 and VEGF were coexpressed topographically in tumor tissues.
These results suggest that endogenous VEGF can be up-regulated transcriptionally by EPAS1, and EPAS1 may be involved in the angiogenesis of RCC.
内皮 PAS 结构域蛋白 1（EPAS1）是一种碱性螺旋-环-螺旋/PAS 结构域转录因子，在血管高度丰富的器官中表达最为丰富。作者研究了转染 EPAS1 cDNA 对 293 Tet-Off 细胞系中血管内皮生长因子（VEGF）内源性表达的影响，以及 EPAS1 可能参与肾细胞癌（RCC）血管生成的情况。
克隆 EPAS1 的完整 cDNA，并将其转染至 293 Tet-Off 胎儿肾细胞系的细胞中，在该细胞系中，强力霉素可抑制 EPAS1 的表达。通过半定量逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定法检测 VEGF 和转铁蛋白受体（TfR，缺氧诱导因子 1α（HIF-1α）的靶基因）表达模式的后续变化。此外，通过半定量 RT-PCR 分析了 5 种 RCC 细胞系和 13 个 RCC 组织样本中 EPAS1、HIF-1α和 VEGF 的表达。对 13 个 RCC 组织样本中的 7 个进行了原位杂交。
在 mRNA 水平和蛋白质水平上，引入 EPAS1 cDNA 均显著增加了内源性 VEGF。用强力霉素处理抑制 EPAS1 后，VEGF 的表达相应显著降低，而 TfR 的表达未受影响。在所检测到的所有 RCC 细胞系中均检测到 EPAS1。在 RCC 组织样本中，与相邻正常肾组织相比，肿瘤组织中 EPAS1 mRNA 和 VEGF mRNA 显著增加。原位杂交显示，EPAS1 和 VEGF 在肿瘤组织中呈拓扑共表达。
这些结果表明，EPAS1 可转录上调内源性 VEGF，且 EPAS1 可能参与 RCC 的血管生成。
