Insertion of nuclear factor-kappaB binding sequence into plasmid DNA for increased transgene expression in colon carcinoma cells

To increase plasmid DNA (pDNA)-based transgene expression, 5, 10 or 20 repeats of nuclear factor kappaB (NF-kappaB) binding sequences were inserted upstream of the cytomegalovirus (CMV) promoter region of a conventional pDNA encoding firefly luciferase (pCMV-Luc) to obtain pCMV-kappaB5-Luc, pCMV-kappaB10-Luc and pCMV-kappaB20-Luc. Murine carcinoma colon 26 cells, in which NF-kappaB was constitutively activated, were co-transfected with a firefly luciferase-expressing pDNA and a renilla luciferase-expressing pDNA having no NF-kappaB binding sequences using cationic liposomes. The expression efficiency of pCMV-kappaB(n)-Luc was evaluated using the ratio of the luciferase activities. Increasing numbers of NF-kappaB binding sequences significantly increased transgene expression. The expression was increased by NF-kappaB activators and the effects were marked with pDNA having many NF-kappaB binding sequences. These results indicate that insertion of NF-kappaB binding sequences into pDNA is an effective approach to increase transgene expression in cancer cells in which NF-kappaB is activated.     

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages

The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.     

Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-alpha and IFN-gamma, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the 5' Bf promoter identified a region between -556 and -282 bp that mediated TNF-alpha responsiveness as well as the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. Site-directed mutagenesis of a NF-kappaB-binding element in this region (-433 to -423 bp) abrogated TNF-alpha responsiveness and decreased the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding to this NF-kappaB cis-binding element on TNF-alpha stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bf induction by TNF-alpha and reduced the synergistic induction by TNF-alpha and IFN-gamma. In addition, the proteasome inhibitor MG132, which blocks NF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-alpha and IFN-gamma. LPS was found to induce Bf promoter activity through the same NF-kappaB cis-binding site. These findings suggest that a NF-kappaB cis-binding site between -433 and -423 bp is required for TNF-alpha responsiveness and for TNF-alpha- and IFN-gamma-stimulated synergistic responsiveness of the Bf gene.     

Construction of strong mammalian promoters by random cis-acting element elongation

Synthetic oligonucleotides containing one of four kinds of cis-acting elements, binding sites for activating protein-1 (AP-1), nuclear factor kappaB (NF-kappaB), CArG binding factor A (CBF-A), and nuclear factor Y (NF-Y), were randomly ligated to construct DNA fragments. These fragments were inserted into the SalI site of a promoter probe vector; pGL3-TATASal, which is located immediately upstream of the TATA box sequence of the human heme oxygenase 1 gene and linked to the luciferase gene to construct 11 plasmid vectors. When these vectors were introduced into PC-3 cells of human prostate cancer, 6 out of the 11 transfectants showed a significantly higher luciferase activity than pGL3-TATASal. The two strongest promoters (clone 6 and clone 11) were investigated further Clone 6 turned out to be the strongest, showing a 3.0- and 8.4-fold activity in comparison to the two frequently used promoters--the cytomegalovirus (CMV) immediate early promoter and the simian virus 40 (SV40) early promoter respectively. Clone 11 was less active than clone 6, but still showed higher activity than the two promoters. When the plasmids were introduced into nine other cell lines, their activities varied but were still comparable to the two promoters. These results indicate that the method used here is simple and efficient for constructing strong promoters that are potentially useful for vectors in either gene therapy or recombinant vaccine.