NM23 PROTEINS AND MECHANISMS OF PDGF A CHAIN SILENCING

NM23 蛋白和 PDGF A 链沉默机制

• 批准号: 6377515
• 负责人: David M Kaetzel
• 金额: $ 20.68万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377515
• 关键词: DNA binding protein; DNA footprinting; cell line; gel mobility shift assay; genetic regulation; genetic regulatory element; genetic transcription; immunoglobulin genes; metastasis; neoplastic cell; platelet derived growth factor; southern blotting; transfection; tumor suppressor proteins

This proposal is focused on the molecular mechanisms underlying transcriptional silencing of the platelet-derived growth factor (PDGF) A-chain gene. Polypeptide growth factors (GFs) such as PDGF, EGF and TGFalpha, among many others, are important positive growth effectors in malignant cells, while others such as TGFbeta have been implicated as inhibitory regulators. However, very little is known about the mechanisms through which GF genes are repressed, nor about the mechanisms through which control of GF gene transcription is subverted in cancer. We have identified multiple silencer elements in the 5'-flanking region of the A-chain gene, one of which was localized to a 33 bp sequence and denoted the 5'-S1 nuclease-hypersensitive (5'SHS) silencer. Two 5'SHS-binding factors were identified as NM23-H1 and NM23-H2 (H1 and H2), proteins implicated previously in suppression of metastasis in breast cancer and melanoma. We have also shown that H1 and H2 expression is required for A-chain silencing, and that DNA-binding is required for their silencing and growth-suppressing activities. We have also observed binding of a number of other protein species (p97, p87, p70, p44/48) to the 5'SHS silencer element. These findings appear to place this project in an excellent position to study molecular mechanisms of transcriptional silencing in a GF gene model. The linkage between NM23s and silencer function also provides a foundation for study of mechanisms through which NM23s mediate transcriptional repression and thereby suppress tumor progression. The proposed studies will test the hypothesis that binding of NM23 and other 5'SHS-binding proteins is critical to function of the structurally related 5'SHS and intSHS silencers of the A-chain gene. In addition, we will determine the extent to which DNA-binding and structure-modifying activities of H1 and H2 are required for silencing and growth-suppression. DNA structure will be assessed by nuclease- and chemical-hypersensitivity assays, while DNA-protein interactions will be characterized by electrophoretic mobility shift assay (EMSA), Southwestern blot and DNA footprinting. Silencer function will be determined by transient transfection analysis in a panel of tumor cell lines that exhibit a range of A-chain silencer activities and expression levels of H1 and H2. Together, the proposed studies should provide important new insights into the mechanisms through which precise control of GF gene transcription is compromised in cancer.

该提案的重点是血小板源性生长因子 (PDGF) A 链基因转录沉默的分子机制。 PDGF、EGF 和 TGFα 等多肽生长因子 (GF) 是恶性细胞中重要的正生长效应因子，而 TGFbeta 等其他因子则被认为是抑制性调节因子。 然而，人们对 GF 基因被抑制的机制以及癌症中 GF 基因转录控制被破坏的机制知之甚少。 我们在 A 链基因的 5' 侧翼区域鉴定了多个沉默元件，其中一个位于 33 bp 序列，称为 5'-S1 核酸酶超敏 (5'SHS) 沉默元件。 两个 5'SHS 结合因子被鉴定为 NM23-H1 和 NM23-H2（H1 和 H2），这两种蛋白质先前与抑制乳腺癌和黑色素瘤的转移有关。 我们还表明，A 链沉默需要 H1 和 H2 表达，并且它们的沉默和生长抑制活性需要 DNA 结合。 我们还观察到许多其他蛋白质种类（p97、p87、p70、p44/48）与 5'SHS 沉默元件的结合。 这些发现似乎使该项目处于研究 GF 基因模型中转录沉默的分子机制的绝佳位置。 NM23 和沉默子功能之间的联系也为研究 NM23 介导转录抑制从而抑制肿瘤进展的机制提供了基础。拟议的研究将检验以下假设：NM23 和其他 5'SHS 结合蛋白的结合对于 A 链基因结构相关的 5'SHS 和 intSHS 沉默子的功能至关重要。 此外，我们将确定 H1 和 H2 的 DNA 结合和结构修饰活性在多大程度上需要沉默和生长抑制。 DNA 结构将通过核酸酶和化学超敏测定进行评估，而 DNA-蛋白质相互作用将通过电泳迁移率变动测定 (EMSA)、Southwestern 印迹和 DNA 足迹进行表征。 沉默子功能将通过一组肿瘤细胞系中的瞬时转染分析来确定，这些肿瘤细胞系表现出一系列 A 链沉默子活性以及 H1 和 H2 的表达水平。 总之，拟议的研究应该为癌症中 GF 基因转录的精确控制受到损害的机制提供重要的新见解。