Immortalized embryonic mouse fibroblasts lacking the RelA subunit of transcription factor NF-κB have a malignantly transformed phenotype

Abstract

The RelA transcription factor is part of dimeric complexes, most commonly either p50-RelA (NF-kB) heterodimers or RelA homodimers, that control a variety of cellular processes. Immortalized embryonic ®broblasts established from rela knockout mice have previously been shown to be more sensitive to apoptosis induced by tumor necrosis factor (TNF) than are control ®broblasts. In this report, we show that one line of rela7/7 ®broblasts has additional phenotypes that distinguish them from control mouse ®broblasts. As compared to normal 3T3 cells, RelAde®cient ®broblasts have a spindled morphology, are less adherent to culture dishes, grow to a higher saturation density, and can form colonies in soft agar. These properties are consistent with a weakly transformed phenotype for rela7/7 cells. Furthermore, RelA-de®cient ®broblasts can form tumors in immunode®cient mice, but these tumors regress, probably because of the sensitivity of these cells to TNF. The ability of rela7/7 ®broblasts to form colonies in soft agar can be reverted by re-expression of wild-type mouse RelA, but not by expression of RelA mutants that cannot form homodimers. There is no clear correlation between the absence of RelA and the levels of expression of other Rel/NF-kB family members or adhesion proteins (ICAM-1 and VCAM-1) whose genes have upstream kB sites. Taken together, these results suggest that RelA has tumor suppressing activity under some circumstances and that RelA complexes are involved in the control of a variety of cellular properties associated with oncogenesis.

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

“…Cell lines 1 and 4 have a spindled morphology, which is characteristic of transformed cells (see also Gapuzan et al, 2002). In contrast, RelA-deficient cell lines 2 and 3 have a flat morphology, which is similar to normal 3T3 cells.…”

“…We have previously characterized one immortalized mouse fibroblast cell line derived from RelA knockout embryos (cell line 1, herein) that is weakly transformed (Gapuzan et al, 2002). In this study, three additional RelA-deficient cell lines have been characterized.…”

“…Ras transformation also increased the expression of c-Rel and p50 in wild-type 3T3 cells (Ras/wt-1 and Ras/wt-2), but not in p50-and RelA-deficient cells, which already have elevated levels of c-Rel (Figure 6c; see also Gapuzan et al, 2002). Both c-Rel and p50 are encoded by NF-kB response genes (Loop and Pahl, 2003).…”

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

“…Virus stocks for the expression of NEMO proteins were prepared by cotransfecting 15 μg of a pBABE-puro-NEMO plasmid with 15 μg of the pKAT helper plasmid using 60 μg of PEI into a 60-mm dish of BOSC23 packaging cells as described [12]. Virus stocks were then used to infect NEMO-deficient fibroblasts, and selection was performed using 2.5 μg/ml puromycin (Sigma) for two days [12].…”

“…Virus stocks were then used to infect NEMO-deficient fibroblasts, and selection was performed using 2.5 μg/ml puromycin (Sigma) for two days [12].…”