雙微染色體基因(mdm2)已知是抑癌基因p53產物的下游轉錄調控的基因之一。當mdm2的基因被轉錄活化時,即代表是具有活性的p53抑癌功能所轉錄調控。本研究主要將已知三個具有p53突變的肺癌患者組織,其p53基因自組織cDNA以聚合？增幅(T-Y234N及T-L194R)或以定點突變(V143A)方式選殖入pCDNA-3表現載體中及另一對照組自p53基因的起始密碼區至終止密碼區全長為1182bp的(p53S)基因,另外構築mdm2 promoter(1076bp)以接合於螢光報告(Luc)基因載體上。二者共同轉染至p53缺失的肺癌(H1299)細胞株中。以西方吸漬法證實自肺癌組織所增幅的p53基因只有T-Y234N無表現,其餘皆有p53蛋白表現。但是以螢光活性分析其促進基因轉錄表現量則非常低,表示此三種p53突變皆無法轉錄活化mdm2基因。證實肺癌患者p53的蛋白功能喪失。 MDM2 is an oncoprotein that inhibits p53 tumour suppressor protein. MDM2 gene is one of the downstream target genes for transcriptional activation by the product of p53 tumor suppressor gene. Transactivation of MDM2 gene expression is represented by the presence of a functional p53 protein. To understand biological function of the mt p53 for tumorigenesis. In the study, we construct three of p53 mutants from lung carcinomas tissues (T-Y234N and T-L194R) or site directed mutagenesis (V143A). Mdm2 promoter (1076bp) was ligated into pGL-3 luciferase reporter gene. Another recombinant plasmid containing only the coding regions of 1182bp for p53 (p53S) has been constructed. We study their transactivation properties of p53 mutants by co-transfecting them with a reported plasmid caring the promoter of MDM2 gene into the p53 null non small cell lung carcinoma cell line (NSCLC) H1299. We observed these mutant p53 protein expression by Western blot. We determine the transactivation activity by observing the luciferase activity. We find that all three mutants of p53 proteins (T-Y234N, V143A and T-L194R) loss transactivation activity.
