Pati Debananda, Haddad Bassem R, Haegele Albert, Thompson Henry, Kittrell Frances S, Shepard Anne, Montagna Cristina, Zhang Nenggang, Ge Gouqing, Otta Subhendu Kumar, McCarthy Maureen, Ullrich Robert L, Medina Daniel
Department of Pediatrics, Hematology-Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA.
Cancer Res. 2004 Aug 15;64(16):5608-16. doi: 10.1158/0008-5472.CAN-03-0629.
The absence of p53 function increases risk for spontaneous tumorigenesis in the mammary gland. Hormonal stimulation enhances tumor risk in p53-null mammary epithelial cells as well as the incidence of aneuploidy. Aneuploidy appears in normal p53-null mammary epithelial cells within 5 weeks of hormone stimulation. Experiments reported herein assessed a possible mechanism of hormone-induced aneuploidy. Hormones increased DNA synthesis equally between wild-type (WT) and p53-null mammary epithelial cells. There were two distinct responses in p53-null cells to hormone exposure. First, Western blot analysis demonstrated that the levels of two proteins involved in regulating sister chromatid separation and the spindle checkpoint, Mad2 and separase (ESPL1) were increased in null compared with WT cells. In contrast, the levels of securin and Rad21 proteins were not increased in hormone-stimulated p53-null compared with WT cells. ESPL1 RNA was also increased in p53-null mouse mammary cells in vivo by 18 h of hormone stimulation and in human breast MCF7 cells in monolayer culture by 8 h of hormone stimulation. Furthermore, both promoters contained p53 and steroid hormone response elements. Mad2 protein was increased as a consequence of the absence of p53 function. The increase in Mad2 protein was observed also at the cellular level by immunohistochemistry. Second, hormones increased gene amplication in the distal arm of chromosome 2, as shown by comparative genomic hybridization. These results support the hypothesis that hormone stimulation acts to increase aneuploidy by several mechanisms. First, by increasing mitogenesis in the absence of the p53 checkpoint in G2, hormones allow the accumulation of cells that have experienced chromosome missegregation. Second, the absolute rate of chromosome missegregation may be increased by alterations in the levels of two proteins, separase and Mad2, which are important for maintaining chromosomal segregation and the normal spindle checkpoint during mitosis.
p53功能的缺失会增加乳腺自发肿瘤发生的风险。激素刺激会增加p53基因缺失的乳腺上皮细胞的肿瘤风险以及非整倍体的发生率。在激素刺激的5周内，非整倍体出现在正常的p53基因缺失的乳腺上皮细胞中。本文报道的实验评估了激素诱导非整倍体的一种可能机制。激素在野生型（WT）和p53基因缺失的乳腺上皮细胞之间同等程度地增加DNA合成。p53基因缺失的细胞对激素暴露有两种不同的反应。首先，蛋白质印迹分析表明，与WT细胞相比，在基因缺失的细胞中，参与调节姐妹染色单体分离和纺锤体检查点的两种蛋白质Mad2和分离酶（ESPL1）的水平增加。相比之下，与WT细胞相比，在激素刺激的p53基因缺失的细胞中，securin和Rad21蛋白的水平没有增加。在体内，经18小时激素刺激后，p53基因缺失的小鼠乳腺细胞中的ESPL1 RNA也增加；在单层培养的人乳腺MCF7细胞中，经8小时激素刺激后，ESPL1 RNA同样增加。此外，两个启动子都含有p53和类固醇激素反应元件。Mad2蛋白的增加是p53功能缺失的结果。通过免疫组织化学在细胞水平上也观察到了Mad2蛋白的增加。其次，如比较基因组杂交所示，激素增加了2号染色体长臂上的基因扩增。这些结果支持了这样一种假说，即激素刺激通过多种机制增加非整倍体。首先，在G2期缺乏p53检查点的情况下，激素通过增加有丝分裂，使经历了染色体错分离的细胞积累。其次，两种蛋白质（分离酶和Mad2）水平的改变可能会增加染色体错分离的绝对速率，这两种蛋白质对于在有丝分裂期间维持染色体分离和正常的纺锤体检查点很重要。
