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.
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