The proposed studies utilize molecular probes to neurofilament (NF) proteins to characterize the full nature of NF protein alterations which occur during the transformation of the neuronal genotype in neoplastic conditions of the human nervous system. Biochemical methods will be combined with immunocytochemical applications of these probes. Comparisons will be made to determine the extent to which NF triplet proteins of transformed neurons are similar to or different from the NF protein subunits of immature, developing and mature neurons. Studies will be conducted to elucidate the mechanisms underlying NF abnormalities in transformed neurons using two neoplastic human cell lines derived from an embryonal carcinoma and a medulloblastoma. The replicating cells of the embryonal carcinoma cell line express keratin rather than NF proteins, but they can be induced to differentiate into non-dividing, neurons that express normal NF triplet proteins and no longer contain keratin proteins. In contrast, cells in the medulloblastoma cell line divide rapidly, are undifferentiated, and express all three NF subunits. However, the NF in these cells are abnormal. The molecular probes we will use to accomplish our objectives will be selected from our large and well-characterized library of monoclonal antibodies to human NF proteins. They recognize immunologically distinct, posttranslationally modified variants of each NF subunit that result from the differential phosphorylation of """"""""nascent"""""""" NF polypeptides. The complement our analysis of epigenetic mechanisms leading to NF abnormalities in transformed neurons, cDNA probes to human NF triplet protein mRNAs will be used to evaluate genetic mechanisms which may also account for these derangements. The accomplishment of these specific aims will: 1) Establish molecular criteria for the diagnostic and prognostic assessment of neuronal tumors; 2) Define the extent to which neoplastic NF proteins are deranged relative to those in normal mature and immature human neurons; 3) Elucidate mechanisms leading to abnormal NF expression in neoplastic neurons. This work has important implications for understanding the multi-step process of transformation and tumor progression, and it will lead to improvements in the management of patients with neuron derived malignancies.
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