Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.
Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.
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发表时间：
2001-06
期刊：
影响因子：
11.2
通讯作者：
C. Jing;C. Beesley;C. Foster;Hai-Lan Chen;P. Rudland;D. West;H. Fujii;Paul H. Smith;Y. Ke
中科院分区：
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作者：
C. Jing;C. Beesley;C. Foster;Hai-Lan Chen;P. Rudland;D. West;H. Fujii;Paul H. Smith;Y. Ke
人皮肤脂肪酸结合蛋白 (C-FABP) 基因在非转移性大鼠 Rama 37 细胞中过表达时能够诱导转移表型。然而，其如何诱导转移的机制尚不清楚。 Northern 和槽印迹分析表明，与仅转染质粒 DNA 产生的非转移对照转染子 pSV-R37 细胞相比，C-FABP 转染细胞 (pSV-CFABP-R37) 及其转移亚系（例如 Met-1）中内源性血管内皮生长因子 (VEGF) 基因的表达增加了 3.8-5.2 倍。恶性 C-FABP 表达细胞也分泌较高水平的 VEGF 免疫反应蛋白。逆转录-PCR 在非转移对照转染 pSV-R37 细胞和恶性转移 Met-1 细胞中检测到两种 VEGF 转录亚型：VEGF(164) 和 VEGF(188)。鸡绒毛尿囊膜测定表明，对照pSV-R37细胞的条件培养基仅具有非常弱的血管生成活性，而来自转移性C-FABP转染子及其亚系的条件培养基具有强烈的血管生成活性，并且可以被VEGF抗体抑制。将表达载体中的 VEGF(164) cDNA 转染至非转移性 Rama 37 细胞中，产生表达高水平 VEGF 的细胞克隆 (R37-VEGF-2)。将 R37-VEGF-2 细胞接种到同系 Wistar Furth 大鼠中，在大量（Fisher 精确检验，P < 0.01）动物（31 只动物中的 18 只）中产生转移，而对照、仅载体转染的 R37-PSV 细胞未产生转移（30 只动物中的 0 只）。免疫细胞化学方法表明，与对照转染子产生的肿瘤相比，PSV-VEGF-2 细胞产生的原发性肿瘤中 VEGF 呈强阳性染色，且微血管密度增加。因子 VIII 的免疫细胞化学染色检测到，与对照 pSV-R37 细胞产生的原发性肿瘤相比，PSV-VEGF-2 细胞产生的原发性肿瘤的微血管密度增加了 3.5 倍。因此，我们认为，在该大鼠 Rama 37 模型系统中，原始转染子中 C-FABP 基因的过度表达通过上调 VEGF 基因的表达来诱导转移，因此 VEGF 可能在这种特定的转移级联中发挥关键作用。
Human cutaneous fatty acid-binding protein (C-FABP) gene is capable of inducing the metastatic phenotype when overexpressed in nonmetastatic rat Rama 37 cells. However, the mechanism of how it induces metastasis is not clear. Northern and slot blot analyses revealed that expression of the endogenous vascular endothelial growth factor (VEGF) gene was increased by 3.8-5.2-fold in the C-FABP-transfected cells (pSV-CFABP-R37) and in their metastatic sublines (e.g., Met-1) when compared with that in the nonmetastatic control transfectant pSV-R37 cells generated by transfection of only plasmid DNA. Higher levels of VEGF immunoreactive protein were also secreted from the malignant C-FABP-expressing cells. Reverse transcription-PCR detected two VEGF transcript isoforms, VEGF(164) and VEGF(188), in both the nonmetastatic control transfectant pSV-R37 cells and the malignant metastatic Met-1 cells. Chick chorioallantoic membrane assays showed that the conditioned medium of the control pSV-R37 cells possessed only very weak angiogenic activity, whereas conditioned media from the metastatic C-FABP transfectants and their sublines were strongly angiogenic and could be inhibited by antibodies to VEGF. Transfection of VEGF(164) cDNA in an expression vector into nonmetastatic Rama 37 cells produced a cell clone (R37-VEGF-2) that expressed high levels of VEGF. Inoculation of R37-VEGF-2 cells into syngeneic Wistar Furth rats produced metastases in a significant number (Fisher's exact test, P < 0.01) of animals (18 of 31 animals), whereas the control, vector alone-transfected R37-PSV cells produced no metastases (0 of 30 animals). Immunocytochemical methods demonstrated a strong positive staining for VEGF and an increased microvessel density in the primary tumors produced from PSV-VEGF-2 cells in comparison with tumors produced from control transfectants. Immunocytochemical staining for factor VIII detected a 3.5-fold increase in microvessel density of the primary tumors produced by PSV-VEGF-2 cells when compared with that of the primary tumors developed from the control pSV-R37 cells. Therefore, we suggest that overexpression of the C-FABP gene in the original transfectants induces metastasis through up-regulation of expression of the VEGF gene in this rat Rama 37 model system, and thus VEGF may play a crucial role in this particular metastatic cascade.