Mechanisms of Induction and Suppression of Viral and Tum

我们一直在研究通过丝裂原活化蛋白激酶(MAPK)信号通路发挥作用的癌基因的转化机制。DRM(Down-Regular by Mos)/Gremlin是本实验室首次发现的骨形态发生蛋白(BMP)拮抗剂，在体内以组织特异性方式表达。除原代成纤维细胞和少数细胞株外，大多数转化细胞不表达DRM/Gremlin，我们观察到癌基因转化可通过MAPK途径抑制其表达。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。我们最近的工作集中在分析DRM在转化细胞中的功能和作用机制。当逆转录病毒和表达DRM/Gremlin的质粒克隆被导入到表达很少或不表达DRM/Gremlin的转化细胞中时，一些细胞的生长和转化相关表型特征发生了变化。为了更仔细地研究DRM/Gremlin表达对肿瘤细胞系的影响，我们构建了蜕皮激素诱导的DRM/Gremlin表达载体，并用诱导剂Ponasterone A处理后产生了过表达DRM/Gremlin的细胞系。当DRM/Gremlin过表达时，P53阴性的原始神经外胚层来源的DAOY和骨肉瘤来源的Saos2细胞株在培养中生长缓慢，体内成瘤能力降低。相比之下，几个表达P53的细胞系要么显示出生长和肿瘤形成能力的增加，要么与对照细胞没有变化。在DAOY和SAOS2中，DRM/Gremlin的过度表达与p21Cip1/Waf1的过度表达有关，p21Cip1/Waf1是一种已知的细胞周期蛋白依赖的激酶抑制物，参与了G1/S细胞周期的转变。其他生长相关蛋白的水平，包括ERK1/ERK2，没有受到影响。初步证据表明，在DRM/Gremlin诱导后，这些细胞中p21启动子的活性降低。相反，HT1080(纤维肉瘤)细胞表现出p21Cip1/Waf1水平降低，增殖率和成瘤率增加。这些数据表明，BMP拮抗剂DRM/Gremlin可以以新的方式影响肿瘤细胞的生长和功能。

We have been studying the transformation mechanisms of oncogenes that act through the Mitogen Activated Protein Kinase (MAPK) signaling pathway. Drm (Down-regulated by mos)/Gremlin is a Bone Morphogenetic Protein (BMP) antagonist, initially identified by our lab, which is expressed in a tissue-specific fashion in vivo. With the exception of primary fibroblasts and a few cell lines, most transformed cells in culture fail to express DRM/Gremlin, and we observed that its expression could be suppressed by oncogene transformation, via the MAPK pathway. We hypothesized this loss of expression may be important for the initiation or progression of specific tumors. Our recent work has focused on analyzing Drm's function and mechanism of action in transformed cells. When retroviral and plasmid clones expressing DRM/Gremlin were introduced into transformed cells that expressed little or no Drm/Gremlin, some showed alterations in growth and transformation-associated phenotypic characteristics. To more carefully examine the effect of Drm/Gremlin expression on tumor cell lines, we constructed an ecdysone-inducible Drm/Gremlin construct and generated cell lines that overexpressed Drm/Gremlin following treatment with the inducer Ponasterone A. In parallel we generated cell lines which constitutively overexpress the protein. The p53-negative, primitive neuroectodermal-derived Daoy and the osteosarcoma-derived Saos2 cell lines exhibited slower growth in culture and reduced tumor-forming ability in vivo when Drm/Gremlin was overexpressed. In contrast several p53-expressing cell lines show either increased growth and tumor forming ability, or no change from control cells. In Daoy and Saos2, overexpression of Drm/Gremlin correlated with overexpression of p21Cip1/Waf1, a known inhibitor of the cyclin-dependent kinases involved in progression through the G1/S cell cycle transition. Levels of other growth associated proteins, including ERK1/ERK2, were not affected. Preliminary evidence suggests that p21 promoter activity is decreased following Drm/Gremlin induction in these cells. In contrast, HT1080 (fibrosarcoma) cells exhibit a reduction in p21Cip1/Waf1 levels and increased rates of proliferation and tumoriginicity. These data suggest that the BMP antagonist Drm/Gremlin can act in novel ways to affect tumor cell growth and function.