Characterization of the mis-regulated REL/NF-kappaB pathway in the RC-K8 human lymphoma cell line and characterization of a conditionally active REL-estrogen receptor fusion protein.
The Rel or NF-kappaB family of transcription factors regulates adaptive and innate immune system functions in higher vertebrates. As part of their normal physiological functions, NF-kappaB proteins regulate cell proliferation and survival, and in many human and animal cancer cells the NF-kappaB pathway is mis-regulated. In this thesis, the mis-regulation of the NF-kappaB pathway is explored in cell culture models of both human and chicken lymphoma. The human diffuse large B-cell lymphoma-derived cell line RC-K8 contains multiple genetic alterations. It is shown that one of these genetic alterations leads to the deletion of 28 megabase pairs on chromosome 2 and to the fusion of sequences from the REL locus to sequences derived from a non-REL gene (NRG). Results show that the RC-K8 cells express both wild-type REL and mutant REL-NRG proteins. REL-NRG can bind to a kappaB-site DNA probe in vitro, but cannot activate transcription from a kappaB- site reporter plasmid in A293 cells. The RC-K8 cell line is shown to contain multiple nuclear NF-kappaB DNA-binding complexes due to mutations in the NF-kappaB inhibitor gene IKBA. These nuclear complexes consist of REL, REL-NRG, and p50, and their nuclear activity correlates with the upregulation of several NF-kappaB target genes. Inhibition of REL activity in RC-K8 cells by retroviral-mediated expression of IkappaBalpha inhibits cell culture proliferation. In other work, a conditionally active REL protein, RELDelta424-490-ER, was created by fusion of REL to the ligand-binding domain of the human estrogen receptor (ER). RELDelta424-490-ER has the unusual property of being active in the absence of estrogen and inactive in the presence of estrogen. Conditional inactivation, by estrogen, of this fusion protein inhibits both the transactivation function of RELDelta424-490-ER and the growth of RELDelta424-490-ER-transformed chicken spleen cells in soft agar. Furthermore, estrogen inhibits the growth of v-Rel- and RELDelta424-490-transformed cells that coexpress ERalpha in trans. These results indicate that sustained REL activity is required for the growth of some human and chicken lymphoma cell lines, thus validating REL as a possible therapeutic target. The ability of estrogen to inhibit the growth of Rel- transformed cells may prove to be a novel approach to treating REL-dependent/ERalpha-positive lymphomas.