CELL CYCLE TRANSCRIPTION REGULATION IN MEDULLOBLASTOMAS

项目#1：髓母细胞瘤的细胞周期/转录调控 通过使用人类亲神经性乳多空病毒（JCV）的早期基因组，我们创造了转基因动物，这些动物发展出与人类髓母细胞瘤非常相似的小脑原始神经外胚层肿瘤。遗传和生物化学研究的结果表明，虽然在某些细胞中T抗原表达并与p53相关，但存在一群T抗原产生水平极低（如果有的话）的肿瘤细胞。来源于肿瘤组织的细胞系允许进一步表征T抗原阳性和T抗原阴性细胞，并且已经鉴定了外显子4缺失的p53的新突变体的存在。这一观察结果与以下假设一致，即在疾病的早期阶段，原始神经外胚层细胞中JCV T抗原的表达及其与p53的相关性可以在功能上抑制这种肿瘤抑制蛋白。由于p53在细胞周期的G1和G2阶段控制细胞增殖，并在基因组稳定性中起重要作用，p53的失活可导致细胞周期正常事件的失调，从而导致基因组不稳定。T抗原阳性与阴性细胞致瘤性的评价 揭示了与T-抗原阴性细胞不同，T-抗原阳性细胞在裸鼠中具有高度致瘤性，这表明T-抗原通过T-抗原影响的另外的途径可能涉及该事件。对与神经发生和肿瘤发生有关的Wnt信号通路的检查显示，β-连环蛋白及其核外观在T抗原阳性细胞中稳定，但在T抗原阴性细胞中不稳定。稳定的β-连环蛋白，在与LEF转录因子结合后，可以进入细胞核，并通过刺激细胞周期蛋白D1和cmye，解除细胞周期的调节，诱导细胞快速增殖。这些观察使我们假设T抗原介导的髓母细胞瘤的演变是通过不同的途径，如p53失活和Wnt失调。在本研究项目中，我们提出：i）创建并利用诱导型动物模型系统，该系统允许在发育期间在小鼠脑中条件性表达JCV T抗原，并将T抗原 ii）检测JCV T抗原和突变型p53的表达能力，（外显子4缺失）在不存在野生型p53 B的情况下诱导成神经管细胞瘤中的作用b）在p53无效背景下产生JCV T抗原和p53突变转基因动物; iii）研究T-抗原转基因小鼠中的WNI信号传导途径，所述T-抗原转基因小鼠被编程用于在各个阶段期间发展PNNI，通过评估β-连环蛋白的细胞质和细胞核水平以及细胞核中LEF与连环蛋白的结合水平，以及肿瘤形成过程中LEF应答基因（包括c-myc和细胞周期蛋白D）的活性，鉴于最近的数据指出，JCV与人类髓母细胞瘤和p53突变体参与这些肿瘤的潜力，JCV T抗原转基因动物的研究应该产生重要的信息，在儿童髓母细胞瘤的发展机制。

Project #1: Cell Cycle/Transcription Regulation in Medulloblastoma By using the early genome of the human neurotropic papovavirus, JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors with extraordinary similarity to human medulloblastoma. Results from genetic and biochemical studies have revealed that while in some cells T-antigen is expressed and is found in association with p53, there exists a population of tumor cells with extremely low, if any, levels of T-antigen production. Cell lines derived from tumor tissue have allowed further characterization of T-antigen positive and T-antigen negative cells and have identified the presence of a novel mutant of p53 with a deletion in exon 4. This observation is consistent with a hypothesis in which, at the early stage of the disease, expression of JCV T-antigen in primitive neuroectodermal cells and its association with p53 can functionally inactivate this tumor suppressor protein. As p53 controls cell proliferation at the G1 and G2 stages of the cell cycle, and plays an important role in genomic stability, inactivation of p53 can cause deregulation of normal events of the cell cycle resulting in genomic instability. Evaluation of tumorigenecity of T-antigen positive and T-antigen negative cells revealed that unlike T-antigen negative cells, T-antigen positive cells are highly tumorigenic in nude mice suggesting that an additional pathway affected by T-antigen by T-antigen may be involved in this event. Examination of a Wnt signalling pathway which is implicated in neurogenesis and oncogenesis revealed stabilization of beta-catenin and its nuclear appearance in T-antigen positive, but not in T-antigen negative cells. The stabilized beta-catenin, upon association with the LEF transcription factor, can enter nuclei and by stimulating cyclin D 1 and cmye, deregulate the cell cycle and induce rapid cell proliferation. These observations led us to hypothesize that the evolution of medulloblastomas by T-antigen is mediated through distinct pathways such as inactivation of p53 and deregulation of Wnt. In this research project we propose to: i) Create and utilize an inducible animal model system that permits conditional expression of the JCV T-antigen in mouse brain during development and correlate T-antigen expression with histological markers and biochemical parameters; ii) Examine the ability of JCV T-antigen and mutant p53 (with a deletion in exon 4) in the induction of medulloblastoma in the absence of wild-type p53 b) creating JCV T-antigen and p53 mutant transgenic animals on the p53 null background; iii) Study the WNI signaling pathway in T-antigen transgenie mice which is programmed for developing PNETs during various stage, of brain development by evaluating the cytoplasmic and nuclear levels of beta-catenin and the level of LEF association with catenin in nuclei, and the activity of LEF-responsive genes including c-myc and cyclin D during the course of tumor formation. In light of recent data pointing to the association of JCV with human medulloblastomas and the potential for the participation of p53 mutant in these tumors, the study of JCV T-antigen transgenic animals should yield important information regarding the mechanism involved in the development of pediatric medulloblastoma.