TUMOR SUPPRESSOR RB FAMILY/PRB2 IN MEDULLOBLASTOMA

项目2：髓母细胞瘤中的肿瘤抑制基因Rb家族/pRb2。 人类嗜神经性病毒JCV在人群中广泛存在，是中枢神经系统(CNS)致命脱髓鞘疾病-进行性多灶性白质脑病(PML)的病原体。与其他乳头病毒一样，JCV基因组由环状双链DNA组成，通过病毒细胞类型特定的调节区将其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，并具有与包括肿瘤抑制蛋白在内的几种重要细胞蛋白相互作用的能力，pRB指出 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发神经源性肿瘤的能力。最近，在与Khalili博士(Leader Project#1)的合作下，创造了一个从小脑外部颗粒层发展成肿瘤的转基因动物--模仿人类髓母细胞瘤。表达T抗原的转基因具有与pRb和pR53相互作用的能力，并可能下调细胞周期途径，从而导致肿瘤的发生发展，但这两种抑癌基因，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，与肿瘤组织类似，并非所有细胞都表达T抗原，这导致人们猜测，随着肿瘤细胞的发展，可能不再需要T抗原的表达，T抗原的表达变得沉默。伴随着pRb2/PL30低磷酸化形式(活性形式)的T抗原表达的缺失表明，在肿瘤发生的早期，T抗原的表达及其与pRb2/PL30功能上的相关性 在肿瘤发展的后期，由于pRb2/PL30磷酸化水平的增加和/或其蛋白降解的增强，pRb2/PL30的活性形式可能会丢失。对小鼠髓母细胞瘤的观察与我们早期对人髓母细胞瘤的观察一致，即在表达T抗原的JCV相关性髓母细胞瘤中检测到活性的pRb2/p130，而在缺乏JCV及其早期蛋白的肿瘤细胞中检测到活性的pRb2/p130。由于pRb2/pl30的生物学功能是在几个阶段决定的，最明显的是通过它与p27Kip1和E2F家族的伙伴关系，在本研究方案中，我们将利用髓母细胞瘤的小鼠模型以及一组具有良好特征的人类髓母细胞瘤来破译体外细胞周期的不同阶段和实验动物小脑肿瘤形成过程中涉及的pRb2/pl30失调的分子事件。结果应该为治疗干预提供有用的信息。

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.