Expression of human p53 requires synergistic activation of transcription from the p53 promoter by AP-1, NF-kappaB and Myc/Max.
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Transcriptional control of p53 expression participates in the generation of appropriate levels of active p53 in response to mitogenic stimulation. This prompted us to study the role of a putative AP-1 and a NF-kappaB motif in the human p53 promoter for transcriptional regulation. We show that mutation of the AP-1 or the NF-kappaB motif abolishes transcription from the human p53 promoter in HeLa, HepG2 and adenovirus type 5 E1-transformed 293 cells. In comparison, mutation of the previously characterized Myc/Max/USF binding site in the human p53 promoter reduces the transcription rate fivefold. The AP-1 motif in the human p53 promoter binds c-Fos and c-Jun and the NF-kappaB motif binds p50(NF-kappaB) and p65RelA. The cooperative nature of transcriptional activation by these factors was documented by repression of c-fos or NF-kappaB1 translation: Pretreatment of the cells with a c-fos or p50(NF-kappaB1) antisense oligonucleotide suppresses transcription from the human p53 promoter completely. In addition, we show that (a) the level of endogenous p53 mRNA and (b) transcription from the strictly p53-dependent human mdm2 promoter are reduced in the presence of c-fos, c-jun, p50(NF-kappaB1), p65RelA or c-myc antisense oligonucleotides, underscoring the importance of these transcription factors for the expression of functional p53. (+info)
Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.
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To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR. (+info)
Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.
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Constitutive NF-kappaB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different I kappaB inhibitors in adenocarcinoma cell lines. High constitutive NF-kappaB activity and low I kappaB-alpha expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-alpha or other NF-kappaB activating agents induced only weakly nuclear NF-kappaB activity without significant p100 processing and led to a very weak transcription of NF-kappaB-dependent reporter gene. Induction of NF-kappaB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-kappaB induction and NF-kappaB-dependent gene transcription following TNF-alpha stimulation. As a consequence of a competition between I kappaB-alpha and p100, cells expressing high levels of p100 respond poorly to NF-kappaB activating stimuli as TNF-alpha. (+info)
Evaluation of the renal effects of an antisense phosphorothioate oligodeoxynucleotide in monkeys.
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Antisense phosphorothioate oligodeoxynucleotides are therapeutic agents that provide target specificity resulting from Watson-Crick base pairing. However, there are nonspecific effects that in some instances result in toxicity. These compounds accumulate in the kidney and induce renal proximal tubular degeneration at high doses. The relationship between accumulation of phosphorothioate oligodeoxynucleotides in the kidney, indicators of renal toxicity, and histomorphology were investigated in rhesus monkeys. Monkeys received vehicle or an escalating dose regimen of 3, 10, 40, and 80 mg/kg of ISIS 2105 and were then evaluated for changes in clinical pathology indices, urinalysis parameters, and renal histopathology. Urinalysis revealed an increase in protein levels and a slight increase in blood content following the third 40 mg/kg dose and continuing through the 80 mg/kg doses, whereas other urinary markers of renal toxicity were unchanged. Creatinine clearance was slightly decreased in monkeys during the 80 mg/kg dose cycle. Granulation in the cytoplasm of proximal tubular epithelial cells was evident by microscopic examination of kidney and was present at all doses examined and increased with dose. Immunohistochemical staining localized the oligodeoxynucleotide within these granules. Histopathologic changes consisting of minimal to moderate tubular degeneration were present only at the higher doses of 40 and 80 mg/kg and at high tissue concentrations, and these changes occurred concurrent with functional alterations, whereas lower doses (< or = 10 mg/kg) did not affect a pathologic or functional change. (+info)
CpG oligodeoxynucleotides rescue BKS-2 immature B cell lymphoma from anti-IgM-mediated growth inhibition by up-regulation of egr-1.
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Cross-linking of the IgM antigen receptor on an immature B cell lymphoma (BKS-2) induces growth arrest and apoptosis. This is accompanied by down-regulation of the immediate early genes, egr-1 and c-myc, and a reduction in NF-kappaB activity. Anti-IgM-induced growth arrest and apoptosis of this murine B cell lymphoma were prevented by oligodeoxynucleotides (ODN) containing the CpG motif, which are also known to be stimulatory for mature and immature B cells. The CpG but not non-CpG ODN rescued BKS-2 cells from anti-IgM-mediated growth inhibition by up-regulation of egr-1 and c-myc expression as well as by restoring NF-kappaB activity. Interestingly, changes in egr-1 expression occurred more rapidly than in c-myc expression. Also the c-myc levels remained high up to 6 h after addition of the anti-IgM, which was also the time until which the addition of CpG could be delayed without affecting its ability to provide complete protection. This CpG-induced rescue of B lymphoma cells was blocked by antisense egr-1 ODN, suggesting that the expression of egr-1 is important for the effects of CpG ODN on the growth and survival of BKS-2 cells. (+info)
Antisense oligonucleotides to class III beta-tubulin sensitize drug-resistant cells to Taxol.
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A major impediment to the successful use of Taxol in the treatment of cancer is the development of drug resistance. The major cellular target of Taxol is the microtubule that is comprised of alpha- and beta-tubulin heterodimers. Binding sites for Taxol have been delineated on the beta-tubulin subunit that has six isotypes. We have recently described increased expression of the brain-specific human class III beta-tubulin isotype, encoded by the Hbeta4 gene, in both Taxol-resistant ovarian tumours and non-small-cell lung cancer cell lines. To evaluate directly the role of the class III beta-tubulin isotype in mediating Taxol resistance, antisense phosphorothioate oligodeoxynucleotides (ODN) targeted against various regions of the Hbeta4 gene have been designed and examined for their efficacy in reducing Hbeta4 gene and protein expression. Taxol-resistant lung cancer cells, A549-T24, which are 17-fold resistant to Taxol and display a fourfold increase in Hbeta4 expression compared to the parental A549 cells, were treated with 1 microM antisense ODNs. Two ODNs, AS1 and AS3, were found to reduce mRNA expression by 40-50%, as determined by reverse transcription polymerase chain reaction. A concentration-dependent reduction in Hbeta4 mRNA expression was demonstrated with AS1 ODN. Immunofluorescence staining of cells treated with AS1 ODN revealed a decrease in class III protein expression which corresponded to a 39% increase in sensitivity to Taxol (P < 0.005). These findings support an important role for Hbeta4 (class III) beta-tubulin expression in Taxol resistance and have potential implications for the treatment of Taxol-resistant tumours. (+info)
Antisense oligonucleotides against the alpha-subunit of ENaC decrease lung epithelial cation-channel activity.
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Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS and an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the alpha-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels. (+info)
Respiratory uncoupling induces delta-aminolevulinate synthase expression through a nuclear respiratory factor-1-dependent mechanism in HeLa cells.
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Nuclear respiratory factor (NRF)-1 appears to be important for the expression of several respiratory genes, but there is no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of doxycycline, UCP-1 mRNA and protein were undetectable. In the presence of doxycycline, UCP-1 was expressed and oxygen consumption doubled. This rise in oxygen consumption was associated with an increase in NRF-1 mRNA. It was also associated with an increase in NRF-1 protein binding activity as determined by electrophoretic mobility shift assay using a functional NRF-1 binding site from the delta-aminolevulinate (ALA) synthase promoter. Respiratory uncoupling also caused a time-dependent increase in protein levels of ALA synthase, an early marker for mitochondrial biogenesis. ALA synthase induction by respiratory uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the region of the NRF-1 initiation codon; a scrambled oligonucleotide with the same base composition had no effect. Respiratory uncoupling increases oxygen consumption and lowers energy reserves. In HeLa cells, uncoupling also increases ALA synthase, an enzyme critical for mitochondrial respiration, but only if translatable mRNA for NRF-1 is available. These data suggest that the transcription factor NRF-1 plays a key role in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy. (+info)