TUMOR SUPPRESSOR RB FAMILY/PRB2 IN MEDULLOBLASTOMA

项目#2：髓母细胞瘤中的肿瘤抑制因子Rb家族/pRb 2。 人类嗜神经病毒JCV广泛存在于人类中，并且是中枢神经系统（CNS）致命性脱髓鞘疾病进行性多灶性白质脑病（PML）的确定病原体。与其他乳多空病毒一样，JCV基因组由环状双链DNA组成，其通过病毒细胞类型特异性调控区分为早期和晚期编码序列。病毒早期基因T-抗原在协调病毒裂解周期中具有重要的调节功能，并具有与几种重要的细胞蛋白相互作用的能力，包括肿瘤抑制蛋白pRb，其指向 这种病毒的致癌潜力为了支持这一观点，早期的研究表明，JCV具有在几种动物模型中诱导神经源性肿瘤的能力。最近，与Khalili博士（领导项目#1）合作，创造了一种转基因动物，该动物从小脑的外部颗粒层产生肿瘤，模仿人类髓母细胞瘤。表达T-抗原的转基因表现出与pRb和p53相互作用的能力，并且可能去调节导致肿瘤演变的细胞周期途径。然而，这两种肿瘤抑制因子，特别是pRb的反式激活的潜在机制尚不清楚。为了更好地了解T抗原诱导的髓母细胞瘤发生的分子机制，我们从小鼠肿瘤中建立了细胞系。有趣的是，与肿瘤组织相似，并非所有细胞都表达T抗原，这导致推测随着肿瘤细胞的发展，可能不再需要T抗原的表达，并且T抗原的表达变得沉默。T-抗原表达的丧失伴随着pRb 2/p130的低磷酸化形式（活性形式）的缺乏，这表明在肿瘤发生的早期阶段，T-抗原的表达及其与pRb 2/p130的功能相关性降低。 使该蛋白失活，而在肿瘤发展的后期，由于pRb 2/p130磷酸化水平的增加和/或其蛋白水解降解的增强，pRb 2/p130的活性形式可能会丢失。对小鼠髓母细胞瘤的观察与我们对人髓母细胞瘤的早期观察雅阁，即在表达T抗原的JCV相关髓母细胞瘤中检测到活性pRb 2/p130，但在缺乏JCV及其早期蛋白的肿瘤细胞中未检测到。由于pRb 2/p130的生物学功能是在几个阶段决定的，最值得注意的是通过其与p27 Kip 1和E2 F家族的伙伴关系，在这项研究计划中，我们将利用髓母细胞瘤的小鼠模型以及一组特征良好的人类髓母细胞瘤来破译参与pRb 2/pRb 1失调的分子事件。P130在体外细胞周期的各个阶段和实验动物小脑中肿瘤形成过程中的表达。结果应提供有用的信息，治疗干预。

Project #2: Tumor Suppressor Rb Family/pRb2 in Medulloblastoma. The human neurotropic virus, JCV, is widespread in the human population and is the established etiologic agent of the fatal demyelinating disease of the central nervous system (CNS), Progressive Multifocal Leukoencephalopathy (PML). Like other papovaviruses, the JCV genome consists of circular double-stranded DNA that is separated into early and late coding sequences by the viral cell type-specific regulatory region. The viral early gene, T-antigen has important regulatory functions in orchestrating the viral lytic cycle and possesses the ability to interact with several important cellular proteins including the tumor suppressor protein, pRb pointing to the oncogenic potential of this virus. In support of this notion, earlier studies have revealed that JCV has the ability to induce neural origin tumors in several animal models. More recently, in collaboration with Dr. Khalili (Leader Project #1), a transgenic animal which developed tumor from external granular layer of cerebellun_ mimicking human medulloblastoma was created. The transgene which express T-antigen exhibited the ability to interact with pRb as well as p53 and that may de-regulate cell cycle pathway leading to evolution of tumor However, the underlying mechanism of transactivation of these two tumor suppressors, particularly pRb remains unknown. To better understand the molecular events involved in the genesis of T-antigen inducec medulloblastomas, we have developed cell lines from mouse tumors. Interestingly, similar to tumor tissue, not all cells expressed T-antigen, leading to speculation that as tumor cells develop, expression of T-antigen may no longer be needed and expression of T-antigen becomes silent. Loss of T-antigen expression which is concomitant with the lack of the hypophosphorylated form (active form) of pRb2/pl30 suggests that at the early stage of tumorigenesis, expression of T-antigen and its association with pRb2/pl30 functionally inactivates this protein, while at the latter stage of tumor development, the active form of pRb2/pl30 may be lost due to an increase in the level of phosphorylation of pRb2/pl30 and/or enhancement in its proteolytic degradation. The observation on murine medulloblastoma tumors is in accord with our earlier observation on human medulloblastoma that demonstrated detection of active pRb2/p 130 in JCV-associated medulloblastomas expressing T-antigen, but not in tumor cells lacking JCV and its early protein. As the biological function ot pRb2/pl30 is dictated at several stage, most notably through its partnership with p27 Kip1 and the E2F family, in this research proposal we will utilize the mouse model of medulloblastoma as well as a collection of well-characterized human medulloblastomas to decipher the molecular events involved in dysregulation o1 pRb2/pl30 during various stages of the cell cycle in vitro and during the course of tumor formation in the cerebellum of experimental animals. The outcome should provide useful information for therapeutic intervention.