Maity A, Pore N, Lee J, Solomon D, O'Rourke D M
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19004, USA.
Cancer Res. 2000 Oct 15;60(20):5879-86.
Glioblastomas are highly vascular malignant brain tumors that often overexpress vascular endothelial growth factor (VEGF). They also frequently overexpress epidermal growth factor receptor (EGFR) and contain regions of hypoxia, both conditions that can induce VEGF. We examined VEGF regulation in U87 MG human glioblastoma cells and in U87/T691 cells, a clonal derivative that contains a truncated erbB2/Neu receptor that interferes with EGFR signaling through the formation of nonfunctional heterodimeric receptor complexes. U87/T691 cells contained approximately one-half as much VEGF mRNA as did U87 MG cells under normoxic conditions (21% oxygen). Pharmacological inhibition of EGFR, Ras, or PI(3) kinase, but not MAP kinase, led to a significant decrease in VEGF mRNA levels in U87 MG cells. VEGF promoter activity in transient transfections was decreased by either pharmacological or genetic inhibition of EGFR, Ras, or phosphatidylinositol 3'-kinase [PI(3) kinase]. However, inhibition of PI(3) kinase or EGFR did not completely abolish induction of VEGF mRNA by hypoxia (0.2% oxygen). Likewise, VEGF mRNA expression was induced 3-fold by hypoxia in EGFR-inhibited U87/T691 cells, comparable with the fold induction seen in parental U87 MG cells, although the absolute level of message under hypoxia was higher in U87 MG cells. In transient transfections, a luciferase reporter construct containing a 1.2-kb fragment of the VEGF promoter, lacking the known hypoxic-responsive element (HRE), showed up-regulation after EGF stimulation to the same degree as the full-length, 1.5-kb VEGF promoter construct retaining the HRE. Furthermore, activity of the HRE-deleted, 1.2-kb promoter luciferase reporter was down-regulated by PI(3) kinase inhibition. Therefore, in glioblastoma cells, transcriptional regulation of the VEGF promoter by EGFR appears to involve Ras/PI(3) kinase and to be distinct from signals induced by hypoxia.
胶质母细胞瘤是高度血管化的恶性脑肿瘤，常过度表达血管内皮生长因子（VEGF）。它们还经常过度表达表皮生长因子受体（EGFR），并含有缺氧区域，这两种情况均可诱导VEGF。我们研究了U87 MG人胶质母细胞瘤细胞和U87/T691细胞中的VEGF调节，U87/T691细胞是一种克隆衍生物，含有截短的erbB2/Neu受体，该受体通过形成无功能的异二聚体受体复合物干扰EGFR信号传导。在常氧条件（21%氧气）下，U87/T691细胞中的VEGF mRNA含量约为U87 MG细胞的一半。对EGFR、Ras或PI(3)激酶（而非MAP激酶）的药理学抑制导致U87 MG细胞中VEGF mRNA水平显著降低。在瞬时转染中，通过对EGFR、Ras或磷脂酰肌醇3'-激酶[PI(3)激酶]的药理学或基因抑制，VEGF启动子活性降低。然而，抑制PI(3)激酶或EGFR并不能完全消除缺氧（0.2%氧气）对VEGF mRNA的诱导。同样，在EGFR抑制的U87/T691细胞中，缺氧可使VEGF mRNA表达增加3倍，与亲代U87 MG细胞中的诱导倍数相当，尽管缺氧条件下U87 MG细胞中的mRNA绝对水平更高。在瞬时转染中，一个含有1.2 kb VEGF启动子片段、缺乏已知缺氧反应元件（HRE）的荧光素酶报告构建体，在EGF刺激后上调程度与保留HRE的全长1.5 kb VEGF启动子构建体相同。此外，PI(3)激酶抑制可下调缺失HRE的1.2 kb启动子荧光素酶报告基因的活性。因此，在胶质母细胞瘤细胞中，EGFR对VEGF启动子的转录调节似乎涉及Ras/PI(3)激酶，且与缺氧诱导的信号不同。
