: Early thymocyte development (prior to the CD4+CD8 + stage) is critical for the generation of T cells. Genetic alterations that cause several human immunodeficiencies have been mapped in genes that play a key role in these early stages of thymocyte development. Several important processes occur during the differentiation of immatureCD25?CD44 - thymocytes into CD25-CD44- thymocytes, such as V(D)J recombination, expression of the T cell receptor (TcR) and expression of the pre-TcR complex. In addition to pre-TcR-mediated signals, inactivation of p53 appears to be required for differentiation of CD25+CD44 thymocytes. We have demonstrated that activation of p38 MAP kinase arrests cell cycle progression and differentiation ofCD25+CD44- thymocytes. Moreover, we have also shown that p38 MAP kinase induces an accumulation of p53 in these thymocytes. It has been shown that p38 MAP kinase activates p53 in response to DNA damage to induce a cell cycle checkpoint for repair of mutations. We propose that the activation of p38 MAP kinase by IL-7 in CD25+CD44 -thymocytes phosphorylates and activates p53 to promote a G2/M checkpoint, a/lowing the repair of deleterious mutations that could be caused by V(D)J rearrangement. Later, the expression of pre-TcR on CD25+CD44 - thymocytes triggers signals that inactivate p38 MAP kinase and, thereby, p53 to allow cell cycle progression and further differentiation. To test this hypothesis we propose to demonstrate that:- p38 MAP kinase activates p53 at the CD25 +CD44- stage to induce a cell cycle checkpoint. We will examine: a) p53 phosphorylation and expression of 14-3-3 sigma (a p53 target at the G2/M checkpoint) in thymocytes arrested at theCD25+CD44 - stage by activation of p38 MP kinase in vivo, and b) the effect of p53 inactivation in cell cycle progression and differentiation of these cells.- pre-TcR signals inactivate p38 MAP kinase and p53 to end the cell cycle checkpoint and allow differentiation of CD25+CD44 - thymocytes. We will examine p38 MAP kinase and p53 activity in CD25+CD44 - thymocytes in the presence or absence of pre-TcR or pre-TcR-signals.- IL-7 activates p38 MAP kinase and, thereby, p53 in CD25+CD44 thymocytes to induce a cell cycle checkpoint.- pre-TcR provides survival signals during the cell cycle checkpoint of CD25+CD44 - thymocytes to prevent death of these cells while DNA repairs takes place.
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