CELL CYCLE TRANSCRIPTION REGULATION IN MEDULLOBLASTOMAS

项目1：髓母细胞瘤的细胞周期/转录调控 利用人类嗜神经性乳头状瘤病毒JCV的早期基因组，我们创造了转基因动物，这些动物患有与人类髓母细胞瘤极其相似的小脑原始神经外胚层肿瘤。遗传和生化研究的结果表明，虽然在一些细胞中表达T抗原，并发现与P53相关，但仍有一批肿瘤细胞产生极低水平的T抗原。来自肿瘤组织的细胞系进一步鉴定了T抗原阳性和T抗原阴性的细胞，并发现了一个新的p53突变，外显子4缺失。这一观察结果与一种假说是一致的，即在疾病的早期，原始神经外胚层细胞中JCVT抗原的表达及其与P53的关系可以在功能上灭活这种肿瘤抑制蛋白。由于P53在细胞周期的G1和G2期控制细胞的增殖，并在基因组稳定中发挥重要作用，P53的失活可以导致细胞周期正常事件的解除调控，从而导致基因组不稳定。T抗原阳性和T抗原阴性细胞的致瘤性评价 与T抗原阴性细胞不同的是，T抗原阳性细胞在裸鼠体内的致瘤性很高，提示T抗原通过T抗原影响的另一条途径可能参与了这一事件。对与神经发生和肿瘤发生有关的Wnt信号通路的检测发现，在T抗原阳性的细胞中，β-连环蛋白及其核出现稳定，但在T抗原阴性的细胞中不出现。稳定的β-连环蛋白与Lef转录因子结合后，可以进入细胞核，通过刺激细胞周期蛋白D1和cmye，解除对细胞周期的调控，诱导细胞快速增殖。这些观察结果使我们假设，T抗原导致的髓母细胞瘤的进化是通过不同的途径来介导的，例如P53的失活和Wnt的去调节。在这个研究项目中，我们建议：i)建立和利用一个可诱导的动物模型系统，该系统允许JCVT抗原在发育过程中在小鼠脑中有条件地表达，并与T抗原相关 这些研究包括：i)利用组织学标志物和生化参数检测JCVT抗原和突变型p53(外显子4缺失)在诱导髓母细胞瘤中的作用；ii)检测JCVT抗原和突变型p53(外显子4缺失)在没有野生型p53的情况下诱导髓母细胞瘤的能力；b)在p53缺失的背景下产生JCVT抗原和p53突变型转基因动物；iii)通过检测T抗原转基因小鼠在脑发育的不同阶段中β-catenin的胞质和核水平，以及Lef应答基因，包括c-myc和Cyclin D的活性，研究WNI信号通路。鉴于最近的数据表明JCV与人类髓母细胞瘤有关，以及P53突变体参与这些肿瘤的可能性，JCV T抗原转基因动物的研究应该能为儿童髓母细胞瘤的发生机制提供重要的信息。

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.