Kumar R, Shepard H M, Mendelsohn J
Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Mol Cell Biol. 1991 Feb;11(2):979-86. doi: 10.1128/mcb.11.2.979-986.1991.
Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.
单克隆抗体（MAb）4D5用于分析SK - BR - 3细胞中p185HER2（c - erbB - 2/HER2的基因产物）的磷酸化情况。在持续存在4D5的情况下培养，可使p185HER2磷酸化的体内稳态水平以剂量依赖方式降低80%，这表明单克隆抗体4D5可能干扰了p185HER2磷酸化的激活过程。观察到的单克隆抗体介导的p185HER2磷酸化减少不能完全用下调来解释。当细胞在无血清条件下培养时，p185HER2磷酸化的稳态水平降低了56%，添加4D5进一步将磷酸化抑制至稳态水平的20%。在这些培养物中持续暴露于浓度不断增加的新生小牛血清时，p185HER2的酪氨酸特异性磷酸化呈线性增加，在10%新生小牛血清时增加了5.4倍。新生小牛血清存在时p185HER2的磷酸化并非归因于表皮生长因子或转化生长因子 - α对表皮生长因子受体的刺激。将这些观察结果扩展到另外两种乳腺癌细胞系MDA - MB - 453和BT - 474，也证明了血清诱导p185HER2磷酸化的显著能力。在无血清条件下抗体介导的磷酸化部分抑制的证明表明，乳腺癌细胞也可能产生和分泌一种或多种可能激活p185HER2的因子。我们观察到具有生长抑制作用的单克隆抗体4D5能够降低新生小牛血清和细胞衍生因子对p185HER2的磷酸化作用，这表明存在一种生长因子，它利用p185HER2的磷酸化作为信号转导途径来调节细胞增殖。
