The Nuclear Factor kappa B (NF-κB) family of transcription factors regulates genes involved in a diverse range of cellular processes including cell growth, inflammatory response, differentiation and apoptosis. Members of the family are regulated by at least two distinct pathways. In the well-studied classical pathway, numerous stimuli lead to ubiquitin-dependent degradation of the IkBα regulatory protein allowing the functionally active p50/p65 complex to accumulate in the nucleus and stimulate transcription. The non-canonical NF-κB pathway is regulated by the p100 NF-κB2 protein which encodes an IκB-like activity within its C-terminus. p100 is processed to p52 NF-κB2 in response to cellular stimulation through a subset of TNFR family members. Mutations, commonly truncations in the IκB-like domain of the NFKB2 gene, have been found in B-cell non-Hodgkin’s lymphomas, chronic lymphocytic leukemia, multiple myelomas, cutaneous T-cell lymphomas (CTCL), breast and colon cancers. These mutations lead to stimulation-independent processing of NFKB2 to the active p52. To identify potential target genes that might play a role in malignancies associated with activation of the non-canonical NFKB pathway, we cloned the truncated NFKB2 gene (p80) from the HUT 78 human CTCL cell line downstream of a Tetracycline-repressible promoter. This construct was stably integrated into the Jurkat T-lymphocyte cell line and RNA was harvested under conditions of elevated and repressed p80 expression. Whole genome expression analysis using Affymetrix microarrays revealed several genes correlated with p80 expression. One of these genes, BIRC3 (API2, cIAP2, AIP1), is a member of the Inhibitor of Apoptosis family of proteins that has been previously linked to activation by the classical NFKB pathway. We confirmed the microarray results by performing RT-qPCR reactions and show elevated levels of BIRC3 mRNA in cells expressing high levels of p80. Further, in transient transfection assays in Jurkat cells with the BIRC3 promoter cloned upstream of the Luciferase reporter gene, we have shown that plasmids expressing p80, and the processed p52 are capable of stimulating the BIRC3 promoter whereas activation by the wild type p100 was diminished. BIRC3 promoter activity was further stimulated by cotransfection of p80 (or p52) with RELB or p65, indicating possible binding partners in NFKB dimers. Additional work is ongoing to elucidate the similarities and differences in classical and non-canonical NFKB activation of the BIRC3 gene.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA