Mechanisms of Induction and Suppression of Viral and Tum

病毒和肿瘤的诱导和抑制机制

• 批准号: 6761464
• 负责人: DONALD BLAIR
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761464
• 关键词: bone morphogenetic proteins; cell line; fibroblasts; gene induction /repression; genetic mapping; green fluorescent proteins; human tissue; mitogen activated protein kinase; neoplasm /cancer genetics; neoplastic growth; neoplastic transformation; oncogenes; tissue /cell culture; transfection; tumor suppressor genes

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. While many primary cells expressed the gene, most transformed cells in culture fail to express DRM/Gremlin, and its expression could be suppressed in fibroblasts by oncogene-mediated activation of the MAPK pathway. We had hypothesized that this loss of expression might be important for tumor initiation or progression, and we have continued to focus our work on the effects of Drm on transformed cells. When retroviral or plasmid clones expressing DRM/Gremlin are introduced into tumor-derived cells that express little or no Drm/Gremlin, several cell lines exhibit a reduction in their transformed phenotype. We used an ecdysone-inducible Drm/Gremlin construct to show that 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 following Drm induction. Using p21Cip1/Waf1 promoter driven luciferase reporters and Real-time PCR, we demonstrated that growth inhibition correlated with the transcriptional induction of p21Cip1/Waf1 and an increase in the level of phosphoryalted p42/44 Mitogen Activated Protein Kinase (MAPK). The induction was independent of p53 and did not require activation of either the p38 or the p42/44 MAP kinases. Our data suggests that the BMP antagonist Drm/Gremlin can act to affect tumor cell growth through its affects on the level of p21Cip1/Waf1 and phoshorylated p42/44 MAPK. In an attempt to identify novel targets for Drm/Gremlin, we also conducted yeast-two hybrid analysis using a kidney library, in collaboration with members of Dr. Alan Perantoni's laboratory. These screens identified several novel potential interacting proteins. In particular, we confirmed that Slit 1, the soluble ligand of the Robo receptor, could interact with Drm. The Slit/Robo pathway has been shown to be involved in kidney development , axon guidance, and lymphocyte chemotaxis, and we are investigating the possible biological significance of Drm-Slit interactions in these processes.

Drm（Down-regulated by mos）/Gremlin是一种骨形态发生蛋白（BMP）拮抗剂，最初由我们实验室鉴定，在体内以组织特异性方式表达。虽然许多原代细胞表达该基因，但培养物中的大多数转化细胞不能表达DRM/Gremlin，并且其表达可以通过癌基因介导的MAPK途径激活在成纤维细胞中被抑制。我们假设这种表达的缺失可能对肿瘤的发生或进展很重要，我们继续将工作重点放在Drm对转化细胞的影响上。 当将表达DRM/Gremlin的逆转录病毒或质粒克隆引入表达很少或不表达Drm/Gremlin的肿瘤衍生细胞中时，几种细胞系表现出其转化表型的减少。我们使用蜕皮激素诱导的Drm/Gremlin构建体来显示p53阴性的原始神经外胚层来源的Daoy和骨肉瘤来源的Saos 2细胞系在培养中表现出较慢的生长，并且在Drm诱导后体内形成肿瘤的能力降低。利用p21 Cip 1/Waf 1启动子驱动的荧光素酶报告基因和实时荧光定量PCR技术，我们证明了生长抑制与p21 Cip 1/Waf 1的转录诱导和磷酸化p42/44丝裂原活化蛋白激酶（MAPK）水平的增加有关。这种诱导作用不依赖于p53，也不需要激活p38或p42/44 MAP激酶。我们的数据表明，BMP拮抗剂Drm/Gremlin可以通过影响p21 Cip 1/Waf 1和磷酸化p42/44 MAPK的水平来影响肿瘤细胞的生长。 为了确定Drm/Gremlin的新靶点，我们还与Alan Perantoni博士实验室的成员合作，使用肾脏文库进行了酵母双杂交分析。这些筛选鉴定了几种新的潜在相互作用蛋白。特别地，我们证实了Robo受体的可溶性配体Slit 1可以与Drm相互作用。Slit/Robo通路已被证明参与肾脏发育，轴突导向和淋巴细胞趋化性，我们正在研究Drm-Slit相互作用在这些过程中可能的生物学意义。