Paula J. Enrietto

High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro

To determine the physiological processes in which the transcription factor c-Rel may act, we have examined its pattern of expression in the avian embryo by in situ hybridization. These studies showed that c-rel is expressed ubiquitously at low levels and at high levels in isolated cells undergoing programmed cell death by apoptosis or autophagocytosis. To further establish a functional link between expression of c-rel and cell death, we examined the biological consequences of c-rel overexpression in vitro. In primary avian fibroblasts, overexpression of c-rel leads to transformation and dramatic life span extension. In contrast, bone marrow cells expressing high levels of c-rel undergo a process of programmed cell death displaying features of both apoptosis and autophagocytic cell death. Thus, these experiments suggest a critical role for c-rel not only in the control of cell proliferation, but also in the induction of cell death.

Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-ReIER

A conditional v-Rel estrogen receptor fusion protein, v-RelER, causes estrogen-dependent but otherwise unaltered v-rel-specific transformation of chicken bone marrow cells. Here, we demonstrate that such v-relER-transformed cells exhibit B lymphoid determinants in line with earlier studies on v-rel-transformed cells. However, following inactivation of v-RelER oncoprotein activity by administration of an estrogen antagonist, cells differentiate into antigen-presenting dendritic cells as judged by several morphological and functional criteria. Additionally, under yet different culture conditions, v-relER cells differentiate into cells resembling polymorphonuclear neutrophils. Our studies therefore suggest that the conditional v-RelER, and probably also the authentic v-Rel, transform a common progenitor for neutrophils and dendritic cells.

Isolation of a cDNA encoding a novel chicken chemokine homologous to mammalian macrophage inflammatory protein-1β ☆

Abstract A cDNA encoding a novel chicken chemokine homologous to mammalian chemokine macrophage inflammatory protein 1β (MIP-1β) was isolated and characterised. The cDNA encodes a protein which is 75–80% homologous to human and mouse MIP-1β. All conserved amino acids characteristic of the mammalian chemokine family have been evolutionarily preserved in chicken MIP-1β, suggesting similar protein folding patterns and functional properties.

Characterization of changes in gene expression associated with malignant transformation by the NF-κB family member, v-Rel

In this study, alterations in gene expression patterns have been examined in v-Rel-transformed avian bone marrow cells. Using a conditional v-Rel estrogen receptor chimera (v-RelER) which transforms cells in an estrogen-dependent manner, we constructed subtraction cDNA libraries from v-RelER-transformed bone marrow cells. Several different sequences were identified whose expression was altered upon hormone activation of v-RelER. These include two genes related to the MIP-1 chemokine family (mip-1β and a tca3 homologue), a cell surface antigen sca-2 and the transcription factor nfkb1. The expression of each gene was assayed in a number of wild-type and mutant v-Rel-expressing fibroblast and hematopoietic cells. All v-Rel-transformed hematopoietic cells tested express high levels of nfkb1 and sca-2. In fibroblasts, wild-type v-Rel induced expression of mip-1β and nfkb1, while nontransforming mutants of v-Rel failed to do so, suggesting a role for these two genes in v-Rel mediated transformation. Finally, these genes are expressed at high levels in cells overexpressing wild-type and truncated forms of c-Rel, implying that v-Rel transforms, in part, by induction of c-Rel target genes.

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

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-Rel-specific, transformation of avian fibroblasts in vitro. Our results demonstrate that c-RelER heterodimerizes with wild-type c-Rel and forms specific complexes with IκB-α. Estrogen causes translocation of c-RelER to the nucleus and stabilizes its binding to DNA. Hormone-activated c-RelER induces transcription of at least four cellular genes that are constitutively active in wild-type c-Rel-transformed fibroblasts. Two distinct cell populations were examined that differed with respect to their growth phenotypes. The growth of fibroblasts with moderate expression levels of c-RelER was stimulated by estrogen. In contrast, the addition of estrogen to cells with high c-RelER 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 effects 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 affect cell cycle control and/or cytokinesis. Furthermore, they also indicate that the biological properties of c-Rel in cell growth and differentiation will potentially differ depending on the level of expression.

A small deletion in the carboxy terminus of the viral myc gene renders the virus MC29 partially transformation defective in avian fibroblasts

In order to characterize the role of the carboxy terminus of the viral myc protein in the transformation of avian fibroblasts and macrophages, several Bal31 deletion mutants were created which removed varying portions of the carboxy terminus of the myc protein. Only one such mutant, S90.9, which had lost nine amino acids of myc retained biological activity when tested in macrophages and fibroblasts. This mutant transformed avian macrophages in a manner similar to that of wild-type, but appeared to be partially transformation defective in fibroblasts. Chicken embryo fibroblast cultures infected with S90.9 exhibited an intermediate phenotype morphologically when compared to wild-type-infected cells. When tested for growth in soft agar, the presence or absence of actin cables and fibronectin on the cell surface, and growth rate, S90.9-infected cells showed intermediate behavior when compared to wild-type or helper virus-infected fibroblasts. These experiments suggest that the carboxy terminus of the myc protein, which is highly basic in nature, is involved in the transformation of avian fibroblasts.