59 research outputs found
Regulació del promotor del gen Dihidrofolat Reductasa en la proliferació cel·lular: relació amb la resistència generada pel metotrexat
[spa] Hemos determinado que la activación de la proteína Quinasa C y la movilización del calcio intracelular son vías bioquímicas involucradas en el desarrollo de la resistencia al metotrexato. Los inhibidores de la proteína Quinasa C son capaces de contrarrestar el incremento de la resistencia generado por el tratamiento con el éster de forbol TPA, y entre estos inhibidores, la Calfostina C también es efectiva en la reducción de la resistencia basal al metotrexato. Este resultado podría tener una aplicación práctica en la terapia con metotrexato ya que la administración conjunta de un inhibidor de la proteína Quinasa C podría actuar como modulador de la resistencia al tratamiento quimioterápico. Por otro lado, hemos profundizado en el estudio del promotor del gen dihidrofolato reductasa tanto en condiciones basales como en el proceso de proliferación y hemos determinado la gran importancia del factor de transcripción SP1 en la activación de la transcripción de este gen. Además, hemos establecido la existencia de un complejo transcripcional, en todas las fases del proceso de proliferación, entre la proteína supresora de tumores retinoblastoma y el factor de transcripción SP1 cuando este último se une al promotor del gen Dihidrofolato Reductasa.
Finalmente, hemos establecido una relación a nivel molecular entre la unión del factor de transcripción SP1 al promotor del gen Dihidrofolato Reductasa y el desarrollo de la resistencia al metotrexato
Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells
Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases
Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells
In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity
PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing
PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs
Regulation of Sp1 by cell cycle related proteins
Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NFκB inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NFκB had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NFκB, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression
Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology
Monogenic disorders are often the result of single point mutations in specific genes,leading to the production of non-functional proteins. Different blood disorders suchas ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, andHemophilia A and B are usually caused by point mutations. Gene editing toolsincluding TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correctmutations responsible for different diseases. However, alternative molecular tools suchas triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), notrelying on nuclease activity, have also demonstrated theirability to correct mutationsin the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins(PPRHs) technology, which can represent an alternative gene editing tool within thisfield. Repair-PPRHs are non-modified single-stranded DNA molecules formed by twopolypurine mirror repeat sequences linked by a five-thymidine bridge, followed by anextended sequence at one end of the molecule which is homologous to the DNAsequence to be repaired but containing the corrected nucleotide. The two polypurinearms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between thepurines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tractslocated relatively near the target mutation in the dsDNA in asequence-specific manner byWatson-Crick bonds, thus producing a triplex structure which stimulates recombination.This technology has been successfully employed to repair a collection of mutants ofthedhfrandaprtgenes within their endogenouslociin mammalian cells and could besuitable for the correction of mutations responsible for blood disorders
Polypurine Reverse Hoogsteen Hairpins as a tool for gene repair and editing
Podeu consultar el llibre complet a: http://hdl.handle.net/2445/12071
Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells
Walnuts have been gathering attention for their health-promoting properties. They are rich in polyphenols, mainly ellagitannins (ETs) that after consumption are hydrolyzed to release ellagic acid (EA). EA is further metabolized by microbiota to form urolithins, such as A and B, which are absorbed. ETs, EA and urolithins have shown to slow the proliferation and growth of different types of cancer cells but the mechanisms remain unclear. We investigate the role of urolithins in the regulatory mechanisms in prostate cancer, specifically those related to the androgen receptor (AR), which have been linked to the development of this type of cancer. In our study, urolithins down-regulated the mRNA and protein levels of both prostate specific antigen (PSA) and AR in LNCaP cells. The luciferase assay performed with a construct containing three androgen response elements (AREs) showed that urolithins inhibit AR-mediated PSA expression at the transcriptional level. Electrophoretic mobility shift assays revealed that urolithins decreased AR binding to its consensus response element. Additionally, urolithins induced apoptosis in LNCaP cells, and this effect correlated with a decrease in Bcl-2 protein levels. In summary, urolithins attenuate the function of the AR by repressing its expression, causing a down-regulation of PSA levels and inducing apoptosis. Our results suggest that a diet rich in ET-containing foods, such as walnuts, could contribute to the prevention of prostate cancer
Polypurine reverse-Hoogsteen (PPRH) oligonucleotides can form triplexes with their target sequences even under conditions where they fold into G-quadruplexes
Polypurine reverse-Hoogsteen (PPRH) oligonucleotides are non-modified DNA molecules composed of two mirror-symmetrical polypurine stretches linked by a five-thymidine loop. They can fold into reverse-Hoogsteen hairpins and bind to their polypyrimidine target sequence by Watson-Crick bonds forming a three-stranded structure. They have been successfully used to knockdown gene expression and to repair single-point mutations in cells. In this work, we provide an in vitro characterization (UV and fluorescence spectroscopy, gel electrophoresis and nuclease assays) of the structure and stability of two repair-PPRH oligonucleotides and of the complexes they form with their single-stranded targets. We show that one PPRH oligonucleotide forms a hairpin, while the other folds, in potassium, into a guanine-quadruplex (G4). However, the hairpin-prone oligonucleotide does not form a triplex with its single-stranded target, while the G4-prone oligonucleotide converts from a G4 into a reverse-Hoogsteen hairpin forming a triplex with its target sequence. Our work proves, in particular, that folding of a PPRH oligonucleotide into a G4 does not necessarily impair sequence-specific DNA recognition by triplex formation. It also illustrates an original example of DNA structural conversion of a G4 into a reverse-Hoogsteen hairpin driven by triplex formation; this kind of conversion might occur at particular loci of genomic DNA
Parallel Clamps and Polypurine Hairpins (PPRH) for Gene Silencing and Triplex-Affinity Capture: Design, Synthesis, and Use
Nucleic acid triplexes are formed when a DNA or RNA oligonucleotide binds to a polypurine-polypyrimidine-rich sequence. Triplexes have wide therapeutic applications such as gene silencing or site-specific mutagenesis. In addition, protocols based on triplex-affinity capture have been used for detecting nucleic acids in biosensing platforms. In this article, the design, synthesis, and use of parallel clamps and polypurine-reversed hairpins (PPRH) to bind to target polypyrimidine targets are described. The combination of the polypurine Watson-Crick strand with the triplex-forming strand in a single molecule produces highly stable triplexes allowing targeting of single- and double-stranded nucleic acid sequences. On the other hand, PPRHs are easily prepared and work at nanomolar range, like siRNAs, and at a lower concentration than that needed for antisense ODNs or TFOs. However, the stability of PPRHs is higher than that of siRNAs. In addition, PPRHs circumvent off-target effects and are non-immunogenic
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