To study the nature of genes that are involved during apoptotic death of hepatocyte precursor cells, we identified a novel gene termed praja-1 (encoding a RING finger Protein involved in cell Apoptosis, Juxtaposition and Architecture). Inhibition of praja-1 results in dramatic apoptosis of hepatocytes utilizing embryonic liver explant cultures. PRAJA-1 is a unique protein with RING-H2, zinc finger domain and a proline rich C-terminal region that is down-regulated during apoptosis induced in 32Dcl3 cells that are strictly dependent on IL-3 for growth and apoptose when deprived of IL-3 from the medium. The experiments proposed are aimed at understanding the role of praja-1 in the apoptosis, differentiation and transformation of hepatocytes.
The aims are: 1. To carry out a detailed biochemical characterization of the protein encoded by praja-1 to determine its sub-cellular localization, post-translational modification patterns, and possible association with other signal transducing proteins via its RING-H2 domain and thus its mechanism of action. 2. To test (i) whether praja-1 expression is increased in v-abl, and bcr-abl-transformed 32Dcl3 cells. Transformation of 32Dcl3 cells with v-abl, and bcr-abl enables them to grow in the absence of IL-3, yet remain susceptible to apoptosis induced by xenobiotic agents. Therefore we will also test whether apoptotic stimuli produced by different xenobiotic agents such as calphostin C, methotrexate etoposide and mitomycin. C reduce the expression of praja-1 (ii) whether ectopic over-expression of praja-1 renders 32Dcl3 cells less sensitive to apoptotic death induced by the above xenobiotic agents. (iii) PRAJA-1 is also down-regulated when embryonic liver explants are induced to terminally differentiate into hepatocytes in the presence of Hepatocyte Growth Factor (HGF), Insulin, and IL-6. In this aim, we propose to study the effects of transgenic expression of praja on embryonic liver explant cultures, and hepatoma cell lines such as PLC/PRF/5 cells for cell growth, differentiation and apoptosis. 3. To study the functional and developmental role of praja-1 by generating mice lacking praja-1 expression by targeted mutagenesis. We have constructed a vector target praja-1 deletion by disrupting exon 2 and the RING-H2 domain. Electroporation into ES cells produced 148 clones. Further analysis by Southern blotting, using an 894-base-pair-pair (bp) 5' praja-1 probe has revealed five homologous recombinant events. These clones bearing the disrupted praja-1 gene will bee used to generate praja-1 +/- mice, and subsequent mating to generate praja-1-/- mice.
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