Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis.

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Identification of a new member of the MNP transcription factor family in differentiated HL60 cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells, MIP-1 alpha is expressed in transformed B cells and can also be induced during cellular activation of CD4+ T-cells and monocytes. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. In this communication we show evidence identifying a new member of the MNP transcription family, MNP-3, in PMA differentiated HL60 cells.

Elevated cellular polyamine levels enhance promoter activity in vivo.

Polyamines are small polycationic molecules that have been implicated in cell growth, differentiation and transformation. A possible mechanism for their involvement in these processes is via their influence on the expression of growth promoting genes. In this study we used an ornithine decarboxylase (ODC) overexpressing retroviral system to achieve high intracellular levels of polyamines in epidermal cells. We then looked at the effect this environment had on transcription from several promoter-reported gene constructs, including c-myc and ODC itself. In transient transfection studies elevated polyamines significantly enhanced the transcriptional activity of all promoters tested. In addition, reporter gene expression was induced approximately 3 fold in stable transformants containing an integrated c-myc or ODC promoter-reporter gene construct suggesting that elevated levels of polyamines, such as those found in transformed cells, have a stimulatory effect on gene expression.

MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.

Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.

Regulation of FGF-4 enhancer activity by transcription factor NF-Y.

Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.

The FGF-4 promoter is required for transformation and is active in both embryonal and somatic cells.

We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.

Changes in in vivo protein-DNA interactions occur at the c-myc P2 promoter during differentiation.

Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.

Vector methylation inhibits transcription from the SV40 early promoter.

Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.

Binding of transcription factor sp1 to exon-1 of C-myc is altered in burkitts-lymphoma.

In Burkitt's lymphoma (BL) a consistent chromosomal translocation involving the c-myc proto-oncogene locus on chromosome 8 and one of the immunoglobulin loci located on chromosomes 14, 22 or 2 results in the aberrant expression of c-myc. In six out of ten published BL sequences the translocated c-myc gene contains mutations and/or deletions in an Spl site in the first exon. We show by competition gel mobility shift assays and association constant comparison that the binding of Spl to this site is altered in four of these clones. The affinity of Spl for this site is increased 5-10 fold in Burkitt's lymphoma BL22 and decreased approximately 5 fold in BL2. Mutations at this site in cell lines Raji and Daudi also show a decrease in Sp1 binding when compared with the wild type sequence.

An avian 40 KDa nucleoprotein binds preferentially to a promoter sequence containing one single pair of methylated CpG.

In vitro transcription competition with oligonucleotides has shown that a down regulating factor can be displaced by a methylated oligonucleotide covering a specific region of the avian vitellogenin II gene promoter (Proc. Natl. Acad. Sci USA, (1990) 87, 3047-3051). Gel mobility shift and competition assays show that a protein binding preferentially to methylated DNA (MDBP-2) is present in fractionated hen and rooster nuclear extracts. The protein(s) bind to the methylated sequence 5' TTCACCTTmCGCTATG-AGGGGGATCATACTGG' 3' (nucleotide positions +2 to +32) of the vitellogenin II promoter and not to other methylated DNA sequences. Contact points of the MDBP-2 with DNA were studied by DNA binding interference experiments with partially depurinated and depyrimidinated oligonucleotides. The protein has an approximate molecular weight of 40 KDa and is mainly found in the liver and oviduct. Proteolytic clipping bandshift assays of the MDBP-2 from rooster and hen liver nuclear extracts indicate that the protein from the two sources are different. In vitro transcription experiments show that the addition of a purified nuclear fraction containing the addition of a purified nuclear dependent manner the transcription of vitellogenin II gene.

Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition.

We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.