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The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.

[Background]: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. [Materials and Methods]: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. [Results]: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. [Discussion]: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.

Uso de agentes inductores GSE24.2 para la elaboración de composiciones farmacéuticas para el tratamiento de enfermedades que cursan con senescencia celular. La presente invención describe el uso de un compuesto inductor o activador de la telomerasa en la elaboración de un medicamento o composición farmacéutica para el tratamiento de una enfermedad o situación patológica, preferentemente humana, causada por un proceso de senescencia relacionado con la disminución de los telómeros o con una alteración de la actividad de la telomerasa. Esta composición farmacéutica puede ser útil para un tratamiento regeneración tisular, por ejemplo de tejidos epiteliales o de células hematopoyéticas, e incluso para la inmortalización de células eucariotas para su uso en investigación o procesos biotecnológicos.

Secuencia de nucleótidos y péptidos GSE 24.2 de disquerina que puede inducir actividad de la telomerasa, procedimiento para obtener la misma, composiciones terapéuticas y aplicaciones de la misma. Sector biotecnológico con aplicaciones relacionadas con la salud humana, y más específicamente compuestos biológicos - secuencias de nucleótidos, péptidos y células humanas transformadas - con aplicaciones terapéuticas para los seres humanos.

Secuencia de nucleótidos y péptidos GSE 24.2 de la disquerina inductores de la actividad telomerasa, procedimiento de obtención, composiciones terapéuticas y sus aplicaciones. La presente invención describe un compuesto inductor o activador de la actividad telomerasa basado en la secuencia de nucleótidos del fragmento GSE 24.2 de la disquerina o la secuencia proteínica o peptídica codificado por dicha secuencia de nucleótidos. Igualmente forman parte vectores que comprenden dicha secuencia y células transformadas con la misma, y composiciones farmacéuticas que contienen todos estos elementos. Estas composiciones pueden ser utilizadas en el tratamiento de enfermedades del siguiente grupo: envejecimiento o aceleración del envejecimiento, enfermedades neurodegenerativas y disqueratosis congénita.

This invention relates to the use of an inductor or activator compound GSE24.2 for producing a medicament or a pharmaceutical composition for treating a preferably human illness or pathological situation caused by a senescence process. Said pharmaceutical composition can be useful for a treatment for regenerating tissues, for example of epithelial tissues or haematopoietic cells, and also for the immortalisation of eucaryotic cells in order to use of same in biotechnological investigation or processes., Esta invención relaciona al uso de un inductor o de un compuesto activador GSE24.2 para producir un medicamento o a una composición farmacéutica para tratar una enfermedad preferiblemente humana o una situación patológica causada por un procedimiento de la senectud. El bote dicho de la composición farmacéutica sea útil para un tratamiento para regenerar los tejidos, por ejemplo de tejidos epiteliales o de células haematopoietic, y también para el immortalisation del uso de las células eucarióticas para de mismo en la investigación o procedimientos biotechnological.

Telomeres from most eukaryotes are composed of repeats of guanine-rich sequences whose main function is to preserve the end of the chromosomes. Telomeres are synthesised by a reverse transcriptase enzyme, telomerase (TERT), which forms part of a ribonucleoprotein complex containing also a RNA template molecule (TERC) and dyskerin. Exhaustion of telomeres during cell divisions triggers a DNA damage response that induces a senescence phenotype. The DNA damage machinery plays an essential role in maintaining the integrity of the genome and also detecting telomere shortening. However in some syndromes that involved mutations either in the telomerase complex genes or those involved in maintaining DNA secondary structure, such as the recQ helicase WRN, a higher frequency in the development of different types of malignancies is observed. We here describe the biology of some of these diseases, together with the molecular modifications in the telomerase complex genes and the impact of these alterations on the development of particular types of cancer. © 2009 Feseo.

DNA repair pathways enable tumour cells to survive DNA damage induced by external agents such as therapeutic treatments. Signalling cascades involved in these pathways comprise the DNA-dependent protein kinase (DNA-PK), Ataxia-telangiectasia mutated (ATM), ATM and Rad3 related (ATR) and checkpoint kinases I and 2 (Chk1/Chk2), among others. ATM and ATR phosphorylate, respectively, Chk2 and Chk1, leading to activation of checkpoints. Chk2 acts as a signal distributor, dispersing checkpoint signal to downstream targets such as p53, Cdc25A, Cdc25C, BRCA1 and E2F1. A role of Chk2 as a candidate tumour suppressor has been suggested based on both mouse genetics and somatic tumour studies. We will discuss here the possible role of this kinase in human carcinogenesis and the possibility to use it as a target to increment DNA damage in cancer cells in response to DNA-damaging therapies.

Dyskerin gene is mutated in patients with X-linked dyskeratosis congenita (X-DC), which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2-expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT was constitutively activated in GSE24-2 cells in a c-myc expression-dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the nuclease hypersensitive element-III (NHEIII) site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from patients with X-DC and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease. © 2008 by The American Society of Hematology.

Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu-/nu- mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC. © 2006 Nature Publishing Group All rights reserved.