MECHANISMS OF INDUCTION AND SUPPRESSION OF VIRAL AND TUMOR-DERIVED ONCOGENE-DEPEN

该项目是正在进行的通过MAP激酶信号通路作用的癌基因转化机制研究的延续。在过去的一年里，我们专注于DRM (Down- Regulated by Mos)/Gremlin基因的研究，这是一个我们最初发现的基因，其表达在多种癌基因转化的细胞中下调。我们推测，这种表达缺失可能对特定肿瘤的发生或发展很重要。我们目前的工作主要集中在表征DRM的合成和性质，并分析其可能的功能。我们已经证明，体外合成的DRM可以在微粒体膜系统中被切割和糖基化，产生加工后的蛋白质形式，其流动性与培养中产生DRM的细胞中产生的形式相同。预测的n链糖基化位点的突变在体内和体外都阻断了糖基化，但不影响内质网/高尔基体的加工或蛋白质运输。我们对表达内源性DRM或通过转染表达构建体编码的外源性DRM的细胞进行了分析，结果显示，虽然大部分蛋白质与细胞相关，但一小部分以可溶性形式释放到培养基中。细胞相关和可溶性形式均能干扰BMP-4信号，诱导培养的C2C12细胞成骨分化。直接免疫沉淀和蛋白交联研究也表明，DRM以相对稳定的复合物结合BMP，提示DRM/Gremlin作为BMP拮抗剂的可能机制。突变的非糖基化DRM在两种实验中都具有功能，表明BMP结合不需要糖基化。目前研究的重点是可溶性DRM和细胞相关DRM的作用，以及DRM改变胶质瘤和胶质母细胞瘤细胞系生长特性的能力。- mos，癌基因，逆转录病毒，肿瘤抑制因子，骨形态发生蛋白，MAPK， -人体组织，液体，细胞等。

This project is a continuation of ongoing studies of the mechanism of transformation by oncogenes that act through the MAP kinase signaling pathway. During the past year we have focused our studies on DRM (Down- Regulated by Mos)/Gremlin, a gene which we initially identified and whose expression was down-regulated in cell transformed by a variety of oncogenes. We have theorized that this loss of expression may be important for the initiation or progression of specific tumors. Our current efforts have focused on characterizing the synthesis and properties of DRM and analyzing its possible function. We have demonstrated that in-vitro synthesized DRM can be cleaved and glycosylated in a microsomal membrane system, generating processed forms of the protein with mobilities identical to the forms generated in DRM producing cells in culture. Mutation of the predicted N-linked glycosylation site blocked glycosylation both in vivo and in vitro but did not affect processing or protein trafficking through the ER/Golgi. Our analysis of cells expressing either endogenous DRM, or exogenous DRM encoded by transfected expression constructs, revealed that while the bulk of the protein was cell associated, a fraction was released into the media in a soluble form. Both cell associated and soluble forms were capable of interfering with BMP-4 signaling which induced osteogenic differentiation of C2C12 cells in culture. Direct immunoprecipitation and protein crosslinking studies also demonstrated that DRM bound BMP in a relatively stable complex, suggesting a possible mechanism of DRM/Gremlin function as a BMP antagonist. Mutant non-glycosylated DRM was functional in both assays, indicating that glycosylation was not required for BMP binding. Ongoing work is focusing on the role of soluble and cell associated DRM and the ability of DRM to alter the growth properties of gliomas and glioblastoma cell lines. - mos, Oncogene, retrovirus, Tumor Suppressor, Bone Morphogenetic Proteins, MAPK, - Human Tissues, Fluids, Cells, etc.