Jeoung D I, Tang B, Sonenberg M
Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
J Biol Chem. 1995 Aug 4;270(31):18367-73. doi: 10.1074/jbc.270.31.18367.
Tumor necrosis factor-alpha (TNF-alpha) demonstrated antimitogenic activity in MCF-7 cells (estrogen receptor-positive human breast cancer cells) in a dose- and time-dependent manner (EC-50 of 2.5 ng/ml). This antimitogenic effect of TNF-alpha was accompanied by a decreased number of cells in S phase in a dose- and time-dependent manner. Based on growth arrest experiments using aphidicolin, it is apparent that TNF-alpha acted in early G1 phase. It did not show antimitogenic effects once cells reentered the S phase based on [3H]thymidine incorporation into DNA and cell cycle analysis. Specificity of TNF-alpha was established by using monoclonal anti-human TNF-alpha antibody. On the basis of Western immunoblot analysis of Rb, p53 and cell cycle inhibitory protein (Cip1) (p21) proteins, TNF-alpha decreased Rb protein expression in a dose- and time-dependent manner whereas it increased the expression level of tumor suppressor p53 protein. TNF-alpha also increased the expression level of Cip1 (p21) protein in a dose-dependent manner. This induction of Cip1 (p21) protein was preceded by the induction of p53 protein in MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also increased. Tumor suppressor Rb protein expression was increased during G1 to S phase progression. Cyclin D protein expression levels were not changed in response to TNF-alpha treatment, although serine/threonine kinase inhibitors such as H7 and the protein kinase C inhibitor staurosporine decreased cyclin D expression levels in MCF-7 cells. Based on experiments with staurosporine, it appears that TNF-alpha does not utilize a protein kinase C pathway in MCF-7 cells. Other cell cycle-related proteins such as Cdk2, Cdc2, and Cdk4 did not show any change in response to TNF-alpha. TNF-alpha did not affect complexes between cyclin D and Cdk2, Cdk4, and Rb proteins in MCF-7 cells. Taken together these results suggest that Rb, p53, and Cip1 (p21) proteins mediate TNF-alpha antimitogenic activity, and TNF-alpha induces growth arrest in the G1 phase in MCF-7 cells.
肿瘤坏死因子-α（TNF-α）在MCF-7细胞（雌激素受体阳性的人乳腺癌细胞）中呈剂量和时间依赖性地表现出抗有丝分裂活性（半数有效浓度为2.5 ng/ml）。TNF-α的这种抗有丝分裂作用伴随着S期细胞数量呈剂量和时间依赖性减少。基于使用阿非科林的生长停滞实验，很明显TNF-α作用于G1早期阶段。根据[3H]胸腺嘧啶掺入DNA和细胞周期分析，一旦细胞重新进入S期，TNF-α就不再显示抗有丝分裂作用。通过使用单克隆抗人TNF-α抗体确定了TNF-α的特异性。基于对Rb、p53和细胞周期抑制蛋白（Cip1）（p21）蛋白的Western免疫印迹分析，TNF-α呈剂量和时间依赖性地降低Rb蛋白表达，而增加肿瘤抑制蛋白p53的表达水平。TNF-α也呈剂量依赖性地增加Cip1（p21）蛋白的表达水平。在MCF-7细胞中，Cip1（p21）蛋白的这种诱导发生在p53蛋白诱导之前。与细胞周期蛋白D相关的Cip1（p21）蛋白也增加。在从G1期到S期的进程中，肿瘤抑制蛋白Rb的表达增加。尽管丝氨酸/苏氨酸激酶抑制剂如H7和蛋白激酶C抑制剂星形孢菌素可降低MCF-7细胞中细胞周期蛋白D的表达水平，但TNF-α处理后细胞周期蛋白D的表达水平并未改变。基于用星形孢菌素进行的实验，似乎TNF-α在MCF-7细胞中不利用蛋白激酶C途径。其他与细胞周期相关的蛋白如Cdk2、Cdc2和Cdk4对TNF-α无任何反应变化。TNF-α不影响MCF-7细胞中细胞周期蛋白D与Cdk2、Cdk4和Rb蛋白之间的复合物。综上所述，这些结果表明Rb、p53和Cip1（p21）蛋白介导TNF-α的抗有丝分裂活性，并且TNF-α诱导MCF-7细胞在G1期生长停滞。
