Gallagher W M, Cairney M, Schott B, Roninson I B, Brown R
CRC Department of Medical Oncology, CRC Beatson Laboratories, Glasgow, UK.
Oncogene. 1997 Jan 16;14(2):185-93. doi: 10.1038/sj.onc.1200813.
Loss of p53 function is associated with the acquisition of cisplatin resistance in the human ovarian adenocarcinoma A2780 cell line. Selection for cisplatin resistance of A2780 cells was used to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human or murine TP53 cDNA. Six GSEs were identified, encoding either dominant negative mutant peptides or antisense RNA molecules which corresponded to various regions within the TP53 gene. Both types of GSE induced cisplatin resistance when introduced individually into A2780 cells. Expression of antisense GSEs led to decreased intracellular levels of p53 protein. One sense GSE induced loss of p53-mediated activities such as DNA damage induced cell cycle arrest and apoptosis. A synthetic peptide, representing the predicted amino acid sequence of this GSE, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence specific DNA binding activity of p53 protein in vitro. Overall, these results directly indicate that inactivation of p53 function confers cisplatin resistance in these human ovarian tumour cells. We have identified short structural domains of p53 which are capable of independent functional interactions and highlighted the efficacy of this approach to discriminate biologically active GSEs from a random fragment library.
p53功能缺失与人类卵巢腺癌A2780细胞系顺铂耐药性的获得相关。利用对A2780细胞进行顺铂耐药性筛选，从表达人或鼠TP53 cDNA随机片段的逆转录病毒文库中分离遗传抑制元件（GSEs）。鉴定出6种GSEs，它们编码显性负性突变肽或与TP53基因内不同区域相对应的反义RNA分子。当分别导入A2780细胞时，这两种类型的GSE均诱导顺铂耐药性。反义GSEs的表达导致细胞内p53蛋白水平降低。一种正义GSE诱导p53介导的活性丧失，如DNA损伤诱导的细胞周期停滞和凋亡。一种代表该GSE预测氨基酸序列的合成肽，导入A2780细胞时赋予顺铂耐药性，并在体外抑制p53蛋白的序列特异性DNA结合活性。总体而言，这些结果直接表明p53功能失活赋予这些人卵巢肿瘤细胞顺铂耐药性。我们已经鉴定出p53的短结构域，它们能够独立进行功能相互作用，并突出了这种从随机片段文库中区分生物活性GSEs方法的有效性。
