Le X F, McWatters A, Wiener J, Wu J Y, Mills G B, Bast R C
Department of Clinical Investigation, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Clin Cancer Res. 2000 Jan;6(1):260-70.
Previous reports have shown that certain anti-HER2 antibodies and heregulin can inhibit clonogenic growth of breast and ovarian cancers that overexpress HER2. Anti-HER2 antibodies bind to HER2 directly, whereas heregulin does not bind to HER2 alone, but rather interacts with HER2 through the formation of heterodimers with HER3 or HER4. The purpose of the present study was to elucidate the mechanisms by which anti-HER2 antibody and heregulin inhibit tumor growth. The anti-HER2 monoclonal antibody (mAb) ID5 was found to block G1-S progression of the cell cycle, whereas heregulin inhibited passage through G2-M. Compatible with the effects on the cell cycle, treatment with mAb ID5 decreased levels of cyclin-dependent kinase (CDK) 2, cyclin E, and CDK6 proteins and reduced cyclin E-CDK2-associated kinase activity; mAb HD5-treated cells had increased p27Kip1 expression and an increased association of p27Kip1 with CDK2. In contrast, treatment with heregulin increased protein levels of CDK2, CDK6, CDC2, and cyclin B1. More Retinoblastoma protein was found in the hypophosphorylated state in the cells treated with mAb ID5, whereas more retinoblastoma protein was in the hyperphosphorylated state in heregulin-treated cells. Heregulin was able to induce cell differentiation as assessed by Oil Red O staining and apoptosis as assessed by sub-G1 peak on flow cytometry and the presence of DNA fragmentation in ApopTag histochemistry staining. Neither differentiation nor apoptosis was observed in the cells treated with mAb ID5. We conclude that anti-HER-2 mAb ID5 and heregulin exert growth inhibition through different mechanisms. In mammary cells overexpressing HER2, anti-HER2 mAb ID5 induces G1 arrest, whereas heregulin induces G2-M arrest, cell differentiation, and apoptosis.
先前的报道表明，某些抗HER2抗体和表皮生长因子（HRG）可抑制过表达HER2的乳腺癌和卵巢癌的克隆生长。抗HER2抗体直接与HER2结合，而HRG不能单独与HER2结合，而是通过与HER3或HER4形成异二聚体与HER2相互作用。本研究的目的是阐明抗HER2抗体和HRG抑制肿瘤生长的机制。发现抗HER2单克隆抗体（mAb）ID5可阻断细胞周期的G1-S期进程，而HRG可抑制细胞通过G2-M期。与对细胞周期的影响一致，用mAb ID5处理可降低细胞周期蛋白依赖性激酶（CDK）2、细胞周期蛋白E和CDK6蛋白的水平，并降低细胞周期蛋白E-CDK2相关激酶活性；经mAb HD5处理的细胞p27Kip1表达增加，且p27Kip1与CDK2的结合增加。相反，用HRG处理可增加CDK2、CDK6、细胞周期蛋白依赖性激酶2（CDC2）和细胞周期蛋白B1的蛋白水平。在用mAb ID5处理的细胞中，发现更多的视网膜母细胞瘤蛋白处于低磷酸化状态，而在用HRG处理的细胞中，更多的视网膜母细胞瘤蛋白处于高磷酸化状态。通过油红O染色评估，HRG能够诱导细胞分化；通过流式细胞术检测亚G1峰以及在凋亡检测试剂盒（ApopTag）组织化学染色中检测DNA片段化来评估，HRG能够诱导细胞凋亡。在用mAb ID5处理的细胞中未观察到分化或凋亡现象。我们得出结论，抗HER-2 mAb ID5和HRG通过不同机制发挥生长抑制作用。在过表达HER2的乳腺细胞中，抗HER2 mAb ID5诱导G1期停滞，而HRG诱导G2-M期停滞、细胞分化和凋亡。
