The WEHI231 and CH33 immature B-cell lines have served extensively as models for the study of B-cell tolerance through clonal deletion via apoptosis. The investigator has recently shown that treatment of these lines with antisera against surface IgM or TGF-beta-1 leads to a reduction in activity of NF-kappaB/Rel transcription factors, causing a drop in c-myc expression which induces apoptosis. Rescue from apoptosis by CD40 ligation leads to maintenance of NF-kappaB/Rel and c-Myc expression.
In Specific Aim 1, the regulation of expression and role in apoptosis of the NF-kappaB/Rel inhibitor I-kappaB-beta following anti-IgM and CD40 ligation will be assessed. Also the control of transcriptional induction of I-kappaB-alpha by TGF-beta-1 and of its sustained reduction by CD40 ligation will be characterized.
In Specific Aim 2, the functional role of the drop in c-Myc levels in the induction of apoptosis will be pursued. Myc has been shown to either induce transcription of E-box driven promoters via interaction with its partner MAX, or to repress transcription of genes driven by Inr elements. The investigator has identified an alternate transcript of Max that generates protein functioning as a dominant negative Max (dMax) in B-cells, which inhibits c-Myc transactivation. The investigator proposes that expression of dMax serves to reduce the level of positive transactivation by c-Myc in immature B-cells, such that a drop in c-Myc levels results in de-repression of gene transactivation. Therefore, dMax will be characterized: 1) in different functional B-cell stages; 2) as a negative regulator of c-Myc-promoted transactivation and apoptosis in myeloid cells; and 3) with respect to c-Myc de-repression of Inr-mediated transcription. Finally, since expression of Mad1, the Max binding partner, was induced following the drop in c-Myc expression and activated apoptosis in WEHI 231 cells following microinjection, the regulation of Mad1 expression and its role in apoptosis will be characterized. Together, these studies should provide important insights into the role of the c-Myc oncogene and NF-kappaB/Rel in the control of apoptosis of immature B-cells and the induction of tolerance via clonal deletion.
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