Batra S K, Castelino-Prabhu S, Wikstrand C J, Zhu X, Humphrey P A, Friedman H S, Bigner D D
Duke University Medical Center, Durham, North Carolina 27710, USA.
Cell Growth Differ. 1995 Oct;6(10):1251-9.
The type III deletion-mutant gene for the epidermal growth factor receptor (EGFRvIII) is frequently expressed in glioblastomas and in breast and non-small cell lung carcinomas. To understand its contribution to the malignant phenotype in humans, we transfected NR6 cells with the mammalian vector pH beta APr-1-neo containing cDNA for either EGFRvIII or wild-type EGFR. Western blot analyses showed that NR6 transfected with wild-type EGFR (NR6W) contained a normal-sized protein (170 kilodaltons); cells transfected with EGFRvIII (NR6M) contained a truncated protein (145 kilodaltons). NR6W cells demonstrated a saturation binding curve with 125I-labeled EGF (affinity, 1.8 x 10(8); r2 = 0.96). NR6M cells, however, showed a low but consistent level of 125I-labeled EGF binding (affinity, 4 x 10(7); r2 = 0.99) compared with NR6, which lacked binding. The population doubling time was shorter for NR6M (0.64 days) than for NR6W (1.1 days) and NR6V (2.27 days). Soft agar focus formation assay by NR6M was 4- to 5-fold higher than that by NR6W. In nude mice, NR6M (1 x 10(7) cells), without exogenous ligand, formed tumors within 12 days; no tumors were observed over 90 days in mice receiving identical doses of NR6W, NR6V, or NR6 cells. EGF stimulated autophosphorylation of receptor in NR6W (4- to 9-fold) but caused only slight (1.8- to 1.9-fold) to no enhancement in NR6M. Further, there was no difference in constitutive tyrosine kinase activity between NR6M and NR6W. Our results clearly indicate that EGFRvIII functions as an oncoprotein, but its intrinsic tyrosine kinase activity may not be responsible for its biological function.
表皮生长因子受体（EGFRvIII）的III型缺失突变基因在胶质母细胞瘤、乳腺癌和非小细胞肺癌中频繁表达。为了解其对人类恶性表型的作用，我们用含有EGFRvIII或野生型EGFR cDNA的哺乳动物载体pH beta APr-1-neo转染NR6细胞。蛋白质印迹分析表明，用野生型EGFR转染的NR6（NR6W）含有正常大小的蛋白质（170千道尔顿）；用EGFRvIII转染的细胞（NR6M）含有截短的蛋白质（145千道尔顿）。NR6W细胞对125I标记的表皮生长因子（EGF）表现出饱和结合曲线（亲和力，1.8×10⁸；r² = 0.96）。然而，与缺乏结合能力的NR6相比，NR6M细胞对125I标记的EGF的结合水平较低但较为稳定（亲和力，4×10⁷；r² = 0.99）。NR6M的群体倍增时间（0.64天）比NR6W（1.1天）和NR6V（2.27天）短。NR6M的软琼脂集落形成试验比NR6W高4至5倍。在裸鼠中，NR6M（1×10⁷个细胞）在没有外源性配体的情况下，12天内形成肿瘤；接受相同剂量的NR6W、NR6V或NR6细胞的小鼠在90天内未观察到肿瘤。EGF刺激NR6W中受体的自磷酸化（4至9倍），但在NR6M中仅引起轻微（1.8至1.9倍）或无增强。此外，NR6M和NR6W之间的组成型酪氨酸激酶活性没有差异。我们的结果清楚地表明，EGFRvIII作为一种癌蛋白发挥作用，但其内在的酪氨酸激酶活性可能与其生物学功能无关。
