Immortalized fibroblasts from NF-κB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor α after transformation by v-Ras

Activation of the NF-kappa B pathway can either promote or block apoptosis and oncogenesis in different cell types and circumstances. In this report, we show that independently derived immortalized mouse embryonic fibroblast cell lines prepared from RelA knockout mice have different phenotypes, based on their sensitivity to tumor necrosis factor alpha (TNF alpha)-induced apoptosis, morphology, ability to form colonies in soft agar, and the presence of distinct kappa B site-binding complexes. In addition, these RelA-deficient cell lines appear to have distinct alterations in the p53 pathway, which correlate with the normal vs transformed status of individual cell lines. We have also infected mouse embryonic fibroblasts lacking RelA, c-Rel or p50 with a retrovirus for the expression of v-Ha-Ras to determine whether individual NF-kappa B family members are required for Ras-mediated transformation. All three NF-kappa B-deficient cell types could be transformed by v-Ha-Ras. However, v-Ras-infected RelA-deficient cells formed colonies in soft agar at an approximately fourfold reduced efficiency compared to v-Ras-transformed control mouse 3T3 and p50-deficient cells. Ras transformation did not alter the sensitivity of RelA-deficient cells to TNF alpha-induced apoptosis, and Ras transformation did not affect the general resistance of 3T3, c-Rel-deficient, and p50-deficient cells to TNF alpha-induced apoptosis. However, TNF alpha specifically and dose-dependently decreased the ability of v-Ras-transformed RelA-deficient cells to form colonies in soft agar. These results suggest that RelA is a potential protein target for human tumors driven by oncogenic Ras mutations, but caution that inhibition of RelA may promote tumorigenesis in some circumstances.