MUTATIONAL AND FUNCTIONAL ANALYSIS OF THE P53 TUMOR SUPPRESSOR GENE

P53的羧基末端可以与受损的DNA结合，重新退火 互补的单链核酸并导致 P53蛋白的四聚化。因为我们的数据表明 羧基末端P53也参与了细胞凋亡，我们正在使用 遵循免费的策略来定义这个领域的“死亡领域” P53区域：(A)微量注射C-末端合成的P53 多肽(AA 319-393、361-393、319-360)转化为正常的Li-Fraumeni (041；P53-/-)或P53突变(错义或P53缺失)人肿瘤细胞 DAPI染色检测细胞凋亡率 (B)穿透性偶联或聚乙二醇化标记合成p53 上面列出的多肽，上面列出的细胞类型和 DAPI、TUNEL和FACS对凋亡细胞的评分，即亚G1DNA 含量分析；(C)CMV驱动的表达载体的显微注射 含有人类癌症来源的P53错义和移码突变 位于P53的羧基末端；(D)微量注射 巨细胞病毒驱动的含人工合成p53突变体的表达载体 推测的磷酸化位点(366ser、376ser和378ser)或缺失 突变株(Aa1-353、1-356、1-363和1-370) P53。我们的初步数据将一个重要的区域缩小到 成为AA363到AA370。因为莱文和沃克(P53研讨会，Dundee， 1996)表明删除了位于 P53的AA62到AA91抑制其凋亡活性，而P53 转录交易不受影响，我们正在显微注射 正常人成纤维细胞和乳腺组织中aa62~aa91多肽的表达 上皮细胞。如果假定的细胞蛋白(S)与 P53的SH3区在其凋亡途径中位于p53的上游，然后 多肽可阻断DNA损伤诱导的细胞凋亡。如果推定的 P53结合蛋白(S)是P53下游的显微注射 多肽可能启动细胞凋亡。

The carboxy-terminus of p53 can bind to damaged DNA, reanneal complementary single strands of nucleic acids and lead to tetramerization of the p53 protein. Because our data indicate that the carboxy-terminus p53 is also involved in apoptosis, we are using the following complimentary strategies to define a "death domain" in this region of p53: (a) microinjection of C-terminal p53 synthetic polypeptides (aa 319-393, 361-393, 319-360) into normal, Li-Fraumeni (041; p53-/-) or p53 mutant (missense or p53 null) human tumor cells and determining the frequency of apoptotic cells by DAPI staining and TUNEL assays; (b) penetratin coupling or pegylated tagged synthetic p53 polypeptides listed above, exposure of the cell types listed above and scoring apoptotic cells by DAPI, TUNEL and FACS, i.e., subG1 DNA content assays; (c) microinjection of CMV-driven expression vectors containing human cancer-derived p53 missense and frameshift mutants located in the carboxy-terminus of p53; (d) microinjection of CMV-driven expression vectors containing synthetic p53 mutants of putative phosphorylation sites (366ser, 376ser and 378ser) or deletion mutants (aa1-353, 1-356, 1-363 and 1-370) in the carboxy-terminus of p53. Our preliminary data have narrowed down one important region to be aa363 to aa370. Because Levine and Walker (p53 Workshop, Dundee, 1996) have shown that deletion of the five PXXP, SH3 motif, located in aa62 to aa91 of p53 inhibits its apoptotic activity whereas p53 transcription transaction is not affected, we are microinjecting the aa62 to aa91 polypeptide in normal human fibroblastic and mammary epithelial cells. If the putative cellular protein(s) binding to the SH3 region of p53 is upstream of p53 in its apoptotic pathway, then the polypeptide may block DNA damage induced apoptosis. If the putative p53 binding protein(s) is downstream of p53, the microinjected polypeptide might initiate apoptosis.