A chicken c-Rel-estrogen receptor chimeric protein shows conditional nuclear localization, DNA binding, transformation and transcriptional activation

Abstract

In this report, we characterize the biological and biochemical properties of a conditional protein containing chicken c-Rel fused to the hormone-binding domain of the human estrogen receptor. This chimeric c-RelER protein causes estrogen-dependent, but otherwise c-Relspeci®c, transformation of avian ®broblasts in vitro. Our results demonstrate that c-RelER heterodimerizes with wild-type c-Rel and forms speci®c complexes with IkB-a. Estrogen causes translocation of c-RelER to the nucleus and stabilizes its binding to DNA. Hormone-activated cRelER induces transcription of at least four cellular genes that are constitutively active in wild-type c-Reltransformed ®broblasts. Two distinct cell populations were examined that di ered with respect to their growth phenotypes. The growth of ®broblasts with moderate expression levels of c-RelER was stimulated by estrogen. In contrast, the addition of estrogen to cells with high cRelER expression levels resulted in inhibition of cytokinesis and the arrest of growth. The carboxy terminal transactivation domain of c-Rel was required for the induction of these e ects since neither v-Rel nor c-Rel deletion mutants were able to induce similar changes. Taken together, our results demonstrate that high levels of c-Rel expression can a ect cell cycle control and/or cytokinesis. Furthermore, they also indicate that the biological properties of c-Rel in cell growth and di erentiation will potentially di er depending on the level of expression.

Multiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel

“…This suggests that v-Rel promotes toxicity by binding to DNA and a ecting gene expression (see below). Overexpression of chicken cRel can induce G 1 -S phase cell-cycle arrest in HeLa cells and CEF Zurovec et al, 1998), and this could also be the mechanism by which v-Rel is toxic. However, the cytotoxicity of v-Rel in mammalian cells has not been investigated in any comprehensive way, and it appears that at least one mammalian cell type, D17 dog osteosarcoma cells, can tolerate high-level expression of v-Rel (TD Gilmore, 1999 unpublished results).…”

Aberrant rel/nfkb genes and activity in human cancer

“…Together, these ®ndings are consistent with a model in which the loss of the C-terminal transactivation domain of c-Rel may provide a selective growth advantage. c-Rel and other NF-kB factors have been shown to induce growth arrest or display a pro-apoptotic phenotype when expressed at high levels in some systems (Abbadie et al, 1993;Bash et al, 1997;Grimm et al, 1996;Huguet et al, 1997;Seitz et al, 1998;Sheehy and Schlissel, 1999;Zurovec et al, 1998). These ®ndings suggest that structural alterations in c-rel may be necessary in some cases for the manifestation of its oncogenic properties.…”

Characterization of a human REL-estrogen receptor fusion protein with a reverse conditional transforming activity in chicken spleen cells

“…Fusion of the hormone-binding domain of the human estrogen receptor (ER) to v-Rel (Boehmelt et al, 1992;Capobianco and Gilmore, 1993), chicken c-Rel (Zurovec et al, 1998), and mouse c-Rel (Grumont et al, 1999) has been shown to confer estrogendependent transcriptional activation activity onto these proteins. Therefore, in an effort to create a conditional human c-Rel (REL) protein, we fused human ER sequences at the C terminus of a highly transforming mutant of REL, RELD424-490 ( Figure 1a).…”

“…Whether the contrasting effects of estrogen on v-Rel-ER versus RELD424-490-ER are due to a mutation that has occurred in v-Rel (for example, a mutation that abolishes its ability to be regulated by estrogen-ER), are due to a natural sequence difference between v-Rel and human REL, or are due to a peculiarity of the fusion design is not clear. Of note, there is an aminoacid sequence in human REL (LTTAL, aa 163-167) that is similar to a consensus nuclear receptor interacting motif (LXXLL) that can be bound by ERa (Gee et al, 1999;Robyr et al, 2000); the corresponding sequence is different in v-Rel (YTLAL, aa 171-175), chicken c-Rel (YTLAL, aa 162-166), and mouse c-Rel (FTTAV, aa 163-167), all of which are regulated in the conventional fashion when expressed as ER fusion proteins (Capobianco and Gilmore, 1993;Zurovec et al, 1998;Grumont et al, 1999).…”