RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required. The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition. Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1. The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors. Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported. Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN and more than 98% similarity with the human c-JUN sequence. We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition.

In an extensive screen of a cDNA library prepared from serum‐stimulated mouse NIH 3T3 cells, we identified three distinct jun‐related clones. Two of them were carrying c‐jun and junB sequences respectively, whereas the sequence of the third group of clones (junD) was distinct from these two and from v‐jun. The amino acid sequences derived from these jun‐related clones are very well conserved in five distinct regions including the putative DNA binding domain. Truncated c‐Jun and JunD proteins containing the C‐terminus recognize the same DNA sequences which were defined as the PEA1/AP1 binding sequence or TPA response element (TRE). Furthermore, both can trans‐activate a promoter including the TRE, and this activation is further enhanced by c‐fos. Contrary to c‐jun and junB transcription, which are strongly stimulated by serum or TPA treatment of quiescent 3T3 cells, junD transcription is not significantly stimulated in these conditions. The tissue distribution and levels of expression of junD mRNA differ from that of c‐jun and junB mRNA. These observations suggest that each of these Jun‐related gene products has a distinct role in the control of gene activity and growth in the organism.

We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c‐fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c‐fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half‐life of fos B mRNA is in the order of 10‐15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c‐fos protein, forms a complex in vitro with c‐jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c‐jun and jun B proteins to an AP‐1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.

We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family. Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain. The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity. RelB does not bind with high affinity to NF-KB sites, but heterodimers between RelB and p50-NF-KB do bind to different NF-KtB-binding sites with a similar affinity to that shown by p50-NF-KB homodimers. However, RelB/p50-NF-KB heterodimers, in contrast to p50-NF-KB homodimers, transactivate transcription of a promoter containing a KB-binding site.Growth factors and other mitogens are capable of rapidly inducing a complex set of more than 100 genes in quiescent fibroblasts (1,13,31,32,38). These genes, which have been named immediate-early or early response genes, are transcriptionally activated within minutes after addition of growth factor or mitogen independently of de novo protein synthesis. Among these genes are several encoding bona fide or putative transcription factors such as members of the fos and jun families, zinc finger proteins (for reviews, see references 8, 22, and 33), or others containing undefined motifs like the Rel domain found in the proto-oncogene c-rel product (11,12,20) and the transcription factor NF-KB (7,16,28,44,47,49,51).The proto-oncogene c-rel (60) is the cellular homolog of the v-rel oncogene (53), isolated from reticuloendotheliosis virus strain T, a turkey-derived acutely transforming retrovirus (56) causing lymphoid leukemia. The c-rellv-rel genes are part of a rapidly growing family of transcription factors. One member of this family is the dorsal gene of Drosophila melanogaster, which is involved in establishing the dorsalventral axis (54) and is able to bind to NF-KB-like DNA sequences (23, 57). The other members are the transcription factor p50-NF-KB (also known as KBF1) with its precursor pl05-NF-KB (7,16,28,44), p50B(p49)-NF-KB with its precursor p97-NF-KB (6a, 51), and p65-NF-KB (47, 49). p50 and p65 form a heterodimer that is able to bind to NF-KB-binding sites found in many enhancers and promoters such as in the immunoglobulin kappa genes, interleukin-2, interleukin-6, ,-interferon, and others (for a review, see reference 35).All the members of the Rel family share a highly conserved domain of approximately 300 amino acids termed the Rel domain, which is considered to function as a DNAbinding and protein dimerization region with properties yet to be defined in detail. Additionally, all the members of this family show a regulated movement from the cytoplasm to the nucleus (for reviews, see references 17 and 36). In particular for the NF-KB complex it is known that p65 is able * Corresponding author.to interact with an inhibitor, IKB, which keeps it in the cytoplasm. After activation of cells, IKB is i...

Emerging literature suggests that metabolic pathways play an important role in the maintenance and progression of human cancers. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. In the studies reported here, we aimed to understand whether tumor cells require the activity of either human isoform of stearoyl-CoA-desaturase (SCD1 or SCD5) for survival. Inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition, when tumor cells are cultured in reduced (2%) serum conditions, but has little impact on cells cultured in 10% serum. Depletion of SCD5 had minimal effects on cell growth or apoptosis. Consistent with the observed dependence on SCD1, but not SCD5, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids, the product of the enzymatic reaction catalyzed by SCD1. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that desaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy. Mol Cancer Res; 9(11); 1551-61. Ó2011 AACR.

NF-KB denotes a transcription factor which has been implicated in the induced expression of many genes and viruses (for reviews see references 2 and 27). NF-KB is presumed to exert its effect through binding to cis-acting, so-called KB elements present in the respective promotersenhancers. The original KB site was defined as an essential functional component of the intronic immunoglobulin K light-chain enhancer, and its decameric sequence is GGGAC TTTCC (the KB consensus sequence reads GGGRNNY YCC). Included among the NF-KB-responsive genes are many involved in immune responses and/or acute-phase reactions, such as the cytokines interleukin-2, interleukin-6, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha, as well as the interleukin-2 receptor a, the immunoglobulin K light chain, and angiotensinogen. In has been recognized to exist as a heterodimer of a p50 and a p65 subunit (47). p50 can form homodimers, but it is the heterodimer that is thought to be the primary transactivating complex owing to potentially transactivating sequences present in p65 (34,38).The genes encoding NF-KB have recently been cloned. p50 is part of a larger precursor protein of 105 kDa. We have cloned this gene, originally designated 243, as a mitogeninduced cDNA selected from a collection of such clones which harbor immediate-early response genes (6), and others have cloned this gene by using nucleic acid probes based on partial peptide sequences of purified p50 protein (13,24,31). The 105-kDa precursor protein consists of two main domains, an amino-terminal region of about 300 amino acids related to the amino-terminal part of the c-Rel protein (7) and a carboxy-terminal portion containing several so-called cell cycle or ankyrin repeat structures. The carboxy-terminal domain inhibits binding of the precursor protein to DNA. Upon removal of the repeat domain by proteolytic cleavage, the amino-terminal region corresponding to the p50 protein and containing all sequences related to c-Rel specifically binds KB DNA elements (37). The p65 gene also encodes a 38). c-Rel, p50, and p65 share extensive homology in their amino-terminal regions, the so-called Rel homology domain, but differ elsewhere. c-Rel and p65 do not contain the cell cycle (ankyrin) repeats. The finding that p50 and p65 are related to c-Rel and bind KB sites has led to the recent discovery that the c-rel proto-oncogene and the v-rel oncogene products are also capable of binding KB DNA. However, only c-Rel transactivates, whereas v-Rel inhibits KB-dependent transcription (5,19,23,36). In addition, a rel-related drosophila gene product, Dorsal, which is the essential morphogenic determinant for ventraldorsal polarity, binds to sites closely resembling KB sites and activates gene expression (20,45). These observations suggest a family of NF-KB-like complexes which together are responsible for the pleiotropic cellular effects originally ascribed to just one transcription factor complex. 685on May 7, 2018 by guest

The nfkb2 gene encodes the p100 precursor which produces the p52 protein after proteolytic cleavage of its COOH-terminal domain. Although the p52 product can act as an alternative subunit of NF-κB, the p100 precursor is believed to function as an inhibitor of Rel/NF-κB activity by cytoplasmic retention of Rel/NF-κB complexes, like other members of the IκB family. However, the physiological relevance of the p100 precursor as an IκB molecule has not been understood. To assess the role of the precursor in vivo, we generated, by gene targeting, mice lacking p100 but still containing a functional p52 protein. Mice with a homozygous deletion of the COOH-terminal ankyrin repeats of NF-κB2 (p100−/−) had marked gastric hyperplasia, resulting in early postnatal death. p100−/− animals also presented histopathological alterations of hematopoietic tissues, enlarged lymph nodes, increased lymphocyte proliferation in response to several stimuli, and enhanced cytokine production in activated T cells. Dramatic induction of nuclear κB–binding activity composed of p52-containing complexes was found in all tissues examined and also in stimulated lymphocytes. Thus, the p100 precursor is essential for the proper regulation of p52-containing Rel/NF-κB complexes in various cell types and its absence cannot be efficiently compensated for by other IκB proteins.