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1.  Development of K562 cell clones expressing β-globin mRNA carrying the β039 thalassaemia mutation for the screening of correctors of stop-codon mutations 
Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing β-thalassaemia.
doi:10.1042/BA20080266
PMCID: PMC3582994  PMID: 19216718
aminoglycoside antibiotics; K562 cell; locus control region; nonsense mutation; thalassaemia
2.  Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients 
American journal of hematology  2009;84(11):720-728.
In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations.
doi:10.1002/ajh.21539
PMCID: PMC3572903  PMID: 19810011
3.  Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3–1 cells 
Artificial DNA, PNA & XNA  2012;3(2):97-296.
One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
doi:10.4161/adna.21061
PMCID: PMC3429536  PMID: 22772035
NF-kappaB; transcription factor decoy; inflammation; Peptide Nucleic Acids; PNA-DNA chimeras
4.  Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus 
BMC Molecular Biology  2008;9:110.
Background
Alternative splicing of the locus AβH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) β-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.
Results
In the present study, we extended the functional characterization of the P1 promoter of the AβH-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression.
The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo.
A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.
Conclusion
Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.
doi:10.1186/1471-2199-9-110
PMCID: PMC2625362  PMID: 19087304
5.  Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human AβH-J-J Locus, Encoding Aspartyl-β-Hydroxylase, Junctin, and Junctate 
Molecular and Cellular Biology  2005;25(8):3261-3275.
Alternative splicing of the locus AβH-J-J generates three functionally distinct proteins: an enzyme, AβH (aspartyl-β-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation in eukaryotic cells. To understand the regulation of these two proteins, we identified and functionally characterized one of the two promoter sequences of the AβH-J-J locus. We demonstrate that the P2 promoter of the AβH-J-J locus contains (i) a minimal sequence localized within a region −159 bp from the transcription initiation site, which is sufficient to activate transcription of both mRNAs; (ii) sequences which bind known transcriptional factors such as those belonging to the myocyte enhancer factor 2 (MEF-2), MEF-3, and NF-κB protein families; and (iii) sequences bound by unknown proteins. The functional characterization of the minimal promoter in C2C12 cells and in the rat soleus muscle in vivo model indicates the existence of cis elements having positive and negative effects on transcription. In addition, our data demonstrate that in striated muscle cells the calcium-dependent transcription factor MEF-2 is crucial for the transcription activity directed by the P2 promoter. The transcription directed by the AβH-J-J P2 promoter is induced by high expression of MEF-2, further stimulated by calcineurin and Ca2+/calmodulin-dependent protein kinase I, and inhibited by histone deacetylase 4.
doi:10.1128/MCB.25.8.3261-3275.2005
PMCID: PMC1069596  PMID: 15798210

Results 1-5 (5)