The use of Rel/NF-kappaB mutants and knockout cell lines to study the role of individual Rel/NF-kappaB transcription factors in oncogenesis

Rel/NF-κB transcription factors regulate the expression of many genes and constitutive Rel/NF-κB activity is associated with oncogenesis by enabling increased cell growth, metastasis, and survival. This thesis further investigates the role of Rel/NF-κB in cellular transformation and describes Rel/NF-κB mutants with altered DNA-binding and dimerization properties.; It is demonstrated that one immortalized embryonic fibroblast cell line derived from rela knockout mice has a transformed phenotype. These RelA-deficient fibroblasts have a spindled morphology, are less adherent to culture dishes, grow to a high saturation density, form colonies in soft agar, and form tumors in scid mice that regress, possibly because of their increased sensitivity to tumor necrosis factor (TNF). Re-expression of RelA in these cells abolishes their ability to form colonies in soft agar; however, RelA mutants that are unable to form homodimers or activate transcription fail to suppress the ability of these cells to form colonies in soft agar.; To determine whether individual Rel/NF-κB subunits are required for Ras transformation, RelA-deficient, c-Rel-deficient, or p50-deficient fibroblasts were infected with a v-Ha-Ras retroviral vector. v-Ras transformed wild-type 3T3 cells and all three knockout cell lines; however, RelA-deficient cells were transformed at a reduced efficiency. Ras-transformation did not alter the increased sensitivity of RelA-deficient cells to TNF-induced apoptosis. TNF selectively suppressed the ability of Ras-transformed RelA-deficient cells, but not Ras-transformed 3T3 cells, to grow in soft agar. These results suggest that RelA is a potential adjuvant therapeutic target for Ras-induced tumors.; Conditional mutants have been useful for studying complex pathways. Conversion of arginine-266 to histidine caused mouse c-Rel to be temperature-sensitive for binding DNA in vitro and for transcription activation in vivo, and the analogous mutation abolished the ability of RelA homodimers to bind DNA in vitro. Mutagenesis of nearby residues was also used to create partially-defective c-Rel and RelA mutants that could form heterodimers but not homodimers.; The results presented in this thesis may be useful for the development of anticancer therapies that target the Rel/NF-κB pathway and may aid in the development of strategies to identify the role of individual Rel/NF-κB proteins in the regulation of cellular processes.