Diermeier Simone, Horváth Gábor, Knuechel-Clarke Ruth, Hofstaedter Ferdinand, Szöllosi János, Brockhoff Gero
Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
Exp Cell Res. 2005 Apr 1;304(2):604-19. doi: 10.1016/j.yexcr.2004.12.008. Epub 2005 Jan 21.
Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors.
BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation.
EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent.
The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.
生长因子和赫赛汀对肿瘤细胞的增殖具有特异性和差异性调节作用。然而，其在erbB受体水平的作用机制尚未完全明确。我们评估了赫赛汀在有或无相关生长因子存在的情况下，调节erbB受体在细胞表面水平的激活及相互作用，进而可能抑制HER2/neu（c-erbB2）过表达乳腺癌细胞增殖的能力。
用表皮生长因子（EGF）、这里菌素以及不同组合的赫赛汀处理BT474和SK-BR-3乳腺癌细胞系。基于BrdU标记，通过流式细胞术评估细胞增殖动力学。采用荧光共振能量转移、酶联免疫吸附测定以及磷酸化位点特异性蛋白质免疫印迹法研究erbB受体的相互作用和激活情况。
EGF诱导的EGFR/EGFR和EGFR/c-erbB2相互作用与BT474细胞的增殖刺激相关。在SK-BR-3细胞中，同源和异源二聚化均明显较弱，且EGF处理会进一步减少异源相互作用，从而抑制细胞增殖。这里菌素在两种细胞系中均广泛刺激细胞增殖。与SK-BR-3细胞相比，赫赛汀能更有效地使BT474细胞进入静止期，从而阻断细胞周期进程。在SK-BR-3细胞中，赫赛汀处理导致Y877和Y1248位点的c-erbB2磷酸化，EGF诱导Y877和Y1112位点磷酸化。在SK-BR-3细胞中由EGF激活的Y1112磷酸化位点，在BT474细胞中则不出现。此外，赫赛汀对BT474和SK-BR-3细胞增殖的抑制能力在很大程度上取决于生长因子的有无。
赫赛汀对c-erbB2过表达乳腺癌细胞的生长抑制作用受到EGFR共表达以及由此产生的EGFR/c-erbB2同源和异源相互作用，以及生长因子存在与否的显著调节。单纯的c-erbB2过表达不足以预测生长因子和抗体对细胞增殖的影响。基于erbB受体靶向的治疗方法的优化和细化需要考虑EGFR共表达以及erbB受体相关生长因子的潜在存在情况。
