This study will focus on the identification and characterization of nuclear matrix proteins (NMPs) of the rat osteosarcoma (ROS) cell. Our working hypothesis is that NMPs unique to the osteosarcoma cell are related to loss of growth and control and deregulation of differentiation related genes. We have identified several NMPs in the ROS cell line 17/2.8 that are not observed in normal rat osteoblast cells at any stage of differentiation. We will further purify and characterize these proteins and other NMPS, at least 10 fold more abundant in the osteosarcoma cells compared to diploid cells, by a combination of two-dimensional gel electrophoresis, reparative gel electrophoresis, HPLC and other appropriate techniques. Peptide sequences will be determined directly from the purified proteins. Utilizing peptide sequence data for each NMP, we will clone and sequence cDNAs for each protein. These molecular probes will also be used to study expression and regulation the NMPs in several osteosarcoma cell lines. Monoclonal antibodies (produced from either pure protein or from a fusion protein) will be used to characterized the biologically important properties of the expression of each NMP including the nuclear localization, cell cycle alterations and post-transnational modifications that each protein undergoes in the osteosarcoma cell. From an analysis of the amino acid sequence and the biological properties described above, we will begin to address the question of protein function using transfection constructs and antisense down regulation protocols where preliminary evidence indicates that these approaches will permit a meaningful functional analysis. These studies will identify NMPs that contribute to alterations in nuclear architecture and deregulation of growth/differentiation relationships in osteosarcoma cells.
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