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.
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