NM23 PROTEINS AND MECHANISMS OF PDGF A CHAIN SILENCING

本研究的重点是研究血小板衍生生长因子(PDGF)A链基因转录沉默的分子机制。多肽生长因子(GFS)如PDGF、EGF和TGFα等是恶性肿瘤细胞中重要的正向生长效应因子，而其他如TGFβ等则被认为是抑制因子。然而，关于GF基因被抑制的机制，以及在癌症中GF基因转录控制被颠覆的机制，人们知之甚少。我们已经在A链基因5‘侧翼区发现了多个沉默元件，其中一个位于33bp的序列中，被称为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)，这两个蛋白以前与抑制乳腺癌和黑色素瘤的转移有关。我们还证明了A链沉默需要H1和H2的表达，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物种(p97、p87、p70、p44/48)与5‘SHS沉默元件结合。这些发现似乎将这个项目置于一个很好的位置，来研究在GF基因模型中转录沉默的分子机制。NM23和沉默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。这些研究将验证NM23和其他5‘SHS结合蛋白的结合对A链基因的结构相关的5’SHS和intSHS消音器的功能至关重要的假设。此外，我们将确定H1和H2的DNA结合和结构修改活性对于沉默和生长抑制所需的程度。DNA结构将通过核酸酶和化学超敏分析来评估，而DNA与蛋白质的相互作用将通过凝胶迁移率改变分析(EMSA)、西南印迹和DNA足迹来表征。沉默功能将通过对一组肿瘤细胞株的瞬时转染分析来确定，这些肿瘤细胞株表现出一系列A链沉默分子活性和H1和H2的表达水平。总之，拟议的研究应该为癌症中精确控制GF基因转录受损的机制提供重要的新见解。

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.