Merlo G R, Venesio T, Taverna D, Marte B M, Callahan R, Hynes N E
Friedrich Miescher Institute, Basel, Switzerland.
Oncogene. 1994 Feb;9(2):443-53.
p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.
p53突变在人类乳腺癌中很常见。为了了解p53在乳腺癌突变积累背景下的作用，人们寻找了一种未转化的乳腺细胞模型。HC11细胞是永生化的、未转化的啮齿动物乳腺上皮细胞，在用催乳激素刺激后能合成乳蛋白。用PAb421抗体在HC11蛋白提取物中很容易检测到p53蛋白。在HC11细胞的p53 cDNA中鉴定出两个突变：密码子138处的错义突变，用色氨酸取代半胱氨酸，以及外显子5中密码子123至130的微缺失。后者是由内含子4/外显子5连接处剪接受体位点的内含子突变引起的。这些突变影响不同的p53等位基因，未发现野生型等位基因。野生型p53通过逆转录病毒载体在Cd(++)诱导型启动子的控制下被导入HC11细胞。在硫酸镉存在的情况下，观察到显著的生长抑制。一个温度敏感的突变型p53基因也被转染到HC11细胞中。当p53处于野生型构象时，这导致在32摄氏度时细胞生长受到明显抑制，而当突变型构象占主导时，在37摄氏度时未观察到影响。野生型p53介导的单层生长抑制不涉及程序性细胞死亡的诱导，也不激活分化特异性乳蛋白的从头合成。我们得出结论，p53基因中的突变可能在其永生化过程中起作用。HC11细胞为评估乳腺肿瘤发生中其他突变的协同作用提供了一个模型。
