MUTATIONAL AND FUNCTIONAL ANALYSIS OF THE P53 TUMOR SUPPRESSOR GENE

P53 肿瘤抑制基因的突变和功能分析

• 批准号: 6100879
• 负责人: C C HARRIS
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100879
• 关键词: DNA binding protein; DNA damage; apoptosis; carcinogenesis; flow cytometry; frameshift mutation; gene deletion mutation; gene mutation; helicase; human genetic material tag; human tissue; microinjections; molecular oncology; neoplasm /cancer genetics; protein sequence; protein structure function; stainings; tumor suppressor genes

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 "apoptotic 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. We also have found that somatic mutations in the C-terminus of p53 from human cancers lose apoptotic activity and retain transcriptional activity. We have shown that p53 binds to DNA helicases, e.g., XPD, XPB, and Rad54, and are currently investigating p53 modulation of BLM and WRN helicase activities.

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“凋亡区域” P53的这个区域：(A)显微注射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)人肿瘤细胞和 DAPI染色和TUNEL法检测细胞凋亡率 分析；(B)穿透性偶联或聚乙二醇化标记的合成p53 上面列出的多肽，上面列出的细胞类型和 用DAPI、TUNEL和FACS对凋亡细胞进行评分，即亚G1DNA含量 (C)显微注射CMV驱动的表达载体，包括 人类癌症来源的P53错义和移码突变位于 P53的羧基末端；(D)显微注射巨细胞病毒驱动的表达 含有假定磷酸化突变体的合成p53的载体 位点(366ser、376ser和378ser)或缺失突变体(aa1-353、1-356、 1-363和1-370)。我们的初步数据 已经将一个重要区域缩小到AA363到AA370。因为 Levine和Walker(P53研讨会，Dundee，1996)已经证明了这种缺失 在PXXP的5个基序中，SH3基序位于P53的Aa62至Aa91之间 细胞凋亡活性，而P53转录活性不是 受影响，我们正在正常情况下显微注射aa62到aa91多肽。 人成纤维细胞和乳腺上皮细胞。如果推定的 结合P53 SH3区的细胞蛋白(S)位于P53的上游 在其凋亡途径中，多肽可能会阻止DNA损伤 诱导细胞凋亡。如果假定的P53结合蛋白(S)在下游 在P53中，显微注射的多肽可能启动细胞凋亡。我们也 已发现人类P53 C末端的体细胞突变 癌症失去了凋亡活性，并保留了转录活性。我们 已经证明P53与DNA解旋酶结合，例如XPD、XPB和Rad54， 目前正在研究P53对BLM和WRN解旋酶的调控 活动。