NM23 PROTEINS AND MECHANISMS OF PDGF A CHAIN SILENCING

本研究的重点是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TGFalpha等，是恶性细胞中重要的正生长效应因子，而其他多肽生长因子，如TGFbeta，则被认为是抑制调节因子。然而，人们对GF基因被抑制的机制知之甚少，也不知道在癌症中GF基因转录的控制被破坏的机制。我们在a链基因的5‘侧区发现了多个沉默元件，其中一个定位在一个33 bp的序列上，表示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)，这两个蛋白先前参与抑制乳腺癌和黑色素瘤的转移。我们还证明了H1和H2的表达是a链沉默所必需的，而它们的沉默和生长抑制活性则需要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。这些发现似乎为研究GF基因模型中转录沉默的分子机制奠定了良好的基础。nm23与沉默者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。本研究将验证NM23与其他5’shs结合蛋白的结合对a链基因结构相关的5’shs和intSHS沉默子的功能至关重要的假设。此外，我们将确定沉默和生长抑制需要H1和H2的dna结合和结构修饰活性的程度。DNA结构将通过核酸酶和化学超敏试验来评估，而DNA-蛋白质相互作用将通过电泳迁移转移试验（EMSA）、西南印迹和DNA足迹来表征。沉默者的功能将通过对一组肿瘤细胞系的瞬时转染分析来确定，这些细胞系表现出一系列a链沉默者活性和H1和H2的表达水平。总之，提出的研究应该提供重要的新见解，通过精确控制GF基因转录在癌症中受损的机制。

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.