Cell Cycle Control and Tumor Suppressors

细胞周期控制和肿瘤抑制剂

• 批准号: 6950166
• 负责人: CURTIS 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/6950166
• 关键词: apoptosis; cell cycle; cell growth regulation; chromosome inversion; cytogenetics; fluorescent in situ hybridization; gel mobility shift assay; gene expression; gene interaction; genetic library; genetic mapping; human genetic material tag; human tissue; molecular cloning; neoplasm /cancer genetics; p53 gene /protein; tissue /cell culture; transcription factor; tumor suppressor genes

When p33ING1 (ING1) was shown to physically and functionally interact with p53, we hypothesized that p33ING1 was the proband member of a family of genes encoding p53 co-transcription factors, and thus, initiated a search for ING1-related genes. By using methodologies already established in the LHC, we have identified four related genes (p47ING3, p29ING4 and p28ING5; ING2-5). We are systematically investigating each of these members of the ING gene family. For example, we have previously reported that unlike ING1b, an alternatively spliced form of ING1, ING2, is induced by the DNA-damaging agents, etoposide or neocarzinostatin. ING1b or ING2 negatively regulate cell growth and survival in a p53-dependent manner through the induction of G1-S cell cycle checkpoints and apoptosis. ING2 strongly enhances the transcriptional transactivation activity of p53. Furthermore, ING2 expression increases the acetylation of p53 at lysine-382. These results have been recently confirmed and extended by others, i.e., ING2 is a nuclear phosphoinositide receptor. Taken together, ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through the activation of p53 by enhancing its acetylation. Our recent data from SiRNA knockdown of ING2 also indicates that ING2 can contribute to the induction of the scenescent phenotype. In collaboration with Karl Riabowol, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity, but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) upregulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity on promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein. We have discovered that ING3-5 also participate in the p53 response pathway to cellular stress. Our preliminary data indicate that different members of the ING family physically and functionally interact with multiprotein complexes of either histone deacetylases, e.g., SIR2, or histone acetyltransferase, e.g., p300, and coregulate p53-mediated transcription by chromatin remodeling.

当p33 ING 1（ING 1）被证明与p53的物理和功能相互作用，我们假设，p33 ING 1是一个家族的基因编码p53共转录因子的先证者成员，因此，开始寻找ING 1相关基因。通过使用LHC中已经建立的方法，我们已经确定了四个相关基因（p47 ING 3，p29 ING 4和p28 ING 5; ING 2 -5）。我们正在系统地研究ING基因家族的每一个成员。例如，我们以前曾报道过，与ING 1b不同，ING 1的一种选择性剪接形式ING 2是由DNA损伤剂依托泊苷或新制癌素诱导的。ING 1b或ING 2通过诱导G1-S细胞周期检查点和凋亡以p53依赖的方式负调节细胞生长和存活。ING 2强烈增强p53的转录反式激活活性。此外，ING 2表达增加p53在赖氨酸-382处的乙酰化。这些结果最近得到了其他人的证实和扩展，即，ING 2是一种核磷酸肌醇受体。总之，ING 2是一种DNA损伤诱导基因，通过增强其乙酰化激活p53来负调控细胞增殖。我们最近从ING 2的SiRNA敲低的数据也表明，ING 2可以有助于诱导衰老表型。 与Karl Riabowol合作，我们发现ING家族成员的一个子集强烈抑制人甲胎蛋白（AFP）启动子活性，但在相同细胞类型的平行实验中刺激p21（WAF 1）启动子，类似于p53的作用。p47（ING 1a）亚型也抑制AFP启动子活性，但与其他ING亚型相反，它抑制p21（WAF 1）启动子。p47（ING 3）上调p21（WAF 1）启动子活性，但对AFP启动子无影响。ING 1b和ING 2也抑制了Hep 3B p53-null细胞系中的AFP启动子，并且p53共表达增强了这种转录抑制。ING对AFP基因转录的抑制强烈依赖于与肝细胞核因子1（HNF 1）转录因子结合的AT基序。事实上，电泳迁移率变动分析证实，HNF 1结合AT-基序，但我们发现，令人惊讶的是，ING 1复合物结合这些AT-基序是缺乏HNF 1蛋白。ING 1和p53都能够抑制AFP转录并引起p21诱导; p53蛋白的负调节因子hSIR 2对AFP启动子表现出相反的作用，并且像HDAC 1一样，抑制p21启动子活性。此外，我们发现p33（ING 1b）与hSIR 2物理相互作用，逆转其诱导AFP启动子的能力，并诱导p53残基在Lys（373）和/或Lys（382）处的乙酰化。这些发现提供了新的证据表明，p33（ING 1b）抑制AFP转录至少有两种机制，其中之一包括p53。第一种是通过与AT基序结合并排除HNF 1结合，同时可能靶向启动子区域上的HAT活性，第二种是通过结合和抑制hSIR 2使p53蛋白脱乙酰化的能力来增加活性乙酰化p53的水平。 我们已经发现ING 3 -5也参与了p53对细胞应激的反应途径。我们的初步数据表明，ING家族的不同成员在物理上和功能上与组蛋白脱乙酰酶的多蛋白复合物相互作用，例如，SIR 2或组蛋白乙酰转移酶，例如，p300，并通过染色质重塑共调节p53介导的转录。