Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

Generation of dyskeratosis congenita-like hematopoietic stem cells through the stable inhibition of DKC1.

Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

Structure of Dictyostelium discoideum telomeres. Analysis of possible replication mechanisms.

Telomeres are nucleo-protein structures that protect the ends of eukaryotic chromosomes. They are not completely synthesized during DNA replication and are elongated by specific mechanisms. The structure of the telomeres and the elongation mechanism have not been determined in Dictyostelium discoideum. This organism presents extrachromosomal palindromic elements containing two copies of the rDNA, also present at the end of the chromosomes. In this article the structure of the terminal region of the rDNA is shown to consist of repetitions of the A(G)n sequence where the number of Gs is variable. These repeats extend as a 3' single stranded region. The G-rich region is preceded by four tandem repetitions of two different DNA motifs. D. discoideum telomere reverse transcriptase homologous protein (TERTHP) presented RNase-sensitive enzymatic activity and was required to maintain telomere structure since terthp-mutant strains presented reorganizations of the DNA terminal regions. These modifications were different in several terthp-mutants and changed with their prolonged culture and subcloning. However, the terthp gene is not essential for D. discoideum proliferation. Telomeres could be maintained in terthp-mutant strains by homologous recombination mechanisms such as ALT (Alternative Lengthening of Telomeres) or HAATI (heterochromatin amplification-mediated and telomerase-independent). In agreement with this hypothesis, the expression of mRNAs coding for several proteins involved in homologous recombination was induced in terthp-mutant strains. Extrachromosomal rDNA could serve as substrate in these DNA homologous recombination reactions.

GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.

Mutations in XLF/NHEJ1/Cernunnos gene results in downregulation of telomerase genes expression and telomere shortening.

NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.

GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells.

Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.

Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

Development and validation of a rapid HPLC method for the quantification of GSE4 peptide in biodegradable PEI-PLGA nanoparticles.

In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100µg/ml (R(2)=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.

Expression of the genetic suppressor element 24.2 (GSE24.2) decreases DNA damage and oxidative stress in X-linked dyskeratosis congenita cells.

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.

Targeted cargo delivery in senescent cells using capped mesoporous silica nanoparticles.

Learning to let go with age: Intracellular controlled release of molecules within senescent cells was achieved using mesoporous silica nanoparticles (MSNs) capped with a galacto-oligosaccharide (GOS) to contain the cargo molecules (magenta spheres; see scheme). The GOS is a substrate of the senescent biomarker, senescence-associated ß-galactosidase (SA-ß-gal), and releases the cargo upon entry into SA-ß-gal expressing cells.

Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain.

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.

Cytotoxicity mechanisms of pyrazino[1,2-b]isoquinoline-4-ones and SAR studies.

The cytotoxicity showed by 1b, an interesting representant of the title compounds, for HT-29 human colon cancer cells (CI(50) value of 1.95 x 10(-7)M) has been related to the induced cell death at the G2 phase and not to DNA damage. This compound promotes the degradation of components of the G2/M checkpoint machinery, in particular cdc2, Cyclin B1 and Wee1, which represents a novel mechanism of cytotoxicity. Degradation of Wee1 seems to be mediated by proteasome activity but degradation of cdc2 has to occur through a different mechanism. The activity of 1b on G2 cell cycle components suggests that tumor cells that are arrested in G2/M by anticancer drugs like cisplatin could be targeted by compound 1b, increasing the apoptosis induction, and that their optimized analogs might be useful in the treatment of colon cancer through combination therapies with cisplatin or other anticancer drugs that affect the cytoskeleton integrity such as taxol and taxotere. SAR studies with compounds obtained by manipulation of the N(2) and C(4)-functional groups and the C(6)-chain of compound 1b have confirmed the importance of these structural features in the in vitro antitumor activity. Fused oxazolidine derivatives as compound 5 were inactive, and the lack of activity found in the replacement of the C(4)-lactam by a cyanoamine function, as in compounds 8-10, could be explained considering that their all-syn relative configuration makes them too stable to generate alkylating iminium species.

MKP1 repression is required for the chemosensitizing effects of NF-kappaB and PI3K inhibitors to cisplatin in non-small cell lung cancer.

Treatment of non-small cell lung cancer (NSCLC) with cisplatin has a level of antitumor activity still modest. We have shown previously that MKP1/DUSP1 inhibits cisplatin-induced apoptosis in NSCLC cells and is overexpressed in tumors from most patients with stage I-II NSCLC. Here, using different NSCLC cell lines we found that MKP1 and NF-kappaB are differentially expressed. We studied whether targeting MKP1, NF-kappaB or both affects cisplatin-induced cell death. MKP1 is expressed in H460 and H727 cells. H727 and H1299 cells showed constitutive phosphorylation of Akt and increased NF-kappaB activity than did H460 cells. H460-MKP1-siRNA-expressing cells (but not H727-MKP1-siRNA or H1299-MKP1-siRNA cells) exhibit a marked increase in cisplatin response compared with parental cells. Treatment with the PI3K inhibitor LY294002 or the NF-kappaB inhibitor BAY11-7082 enhanced cisplatin antitumor activity in parental H1299 cells but only weakly affected responses of H727 and H460 cells. MKP1-siRNA expression enhanced the chemosensitization effect of LY294002 and BAY11-7082 on H727 and H460 cells. Additionally, NSCLC cell lines with higher NF-kappaB-constitutive activation were the most sensitive to PS-341 (Bortezomib), a non-specific NF-kappaB inhibitor. This finding suggests the proteasome as a suitable strategy in treating NSCLC tumors with high constitutive NF-kappaB activity. Altogether, these results showed that either an activated PI3K/Akt/NF-kappaB pathway and/or high MKP1 was linked to reduced sensitivity to cisplatin in NSCLC cells. Inhibition of NF-kappaB or PI3K potently enhanced cisplatin cytotoxicity in cells with endogenous or genetically induced low MKP1 levels. These findings support the potential improvement in cisplatin responses by co-targeting NF-kappaB or Akt and MKP1.

Pyrazino[1,2-b]isoquinolines: synthesis and study of their cytostatic and cytotoxic properties.

The in vitro antitumor potential of novel pyrazino[1,2-b]-isoquinoline-4-ones that contain a half portion of significant natural products was explored in three cancer cell lines: MDA-MB 231 human breast carcinoma, A-549 human lung carcinoma, and HT-29 human colon carcinoma. In general, these compounds show mid to low muM GI(50)s, but LC(50)s over 100 microM with the exceptions of compounds 3b and 31 that are moderately toxic in all cell lines, while compound 4a is highly toxic and selective for HT-29 cells with LC(50) values in the high nanomolar range. Experiments directed to elucidate possible mechanisms of action with compounds 3a, 29, and 31 showed that compound 3a is able to efficiently induce apoptosis triggered directly from the G2/M phase of cell cycle, while compounds 29 and 31 are potentially cytostatic agents that induce the G1/S arrest of cell cycle. All three compounds do not act through DNA damage, since they do not activate this signaling at the level of sensors, transducers, and executers. Furthermore, the apoptosis induction of 3a is not mediated by activation of pro-apoptotic kinases JNK and p38 or by activation of AKT.

Human recombinant erythropoietin does not promote cancer growth in presence of functional receptors expressed in cancer cells.

Human recombinant erythropoietin (hrEPO) therapy might be associated with tumor progression and death. This effect has been suggested to be secondary to rhEPO binding to its receptor (EPOR) expressed on cancer cells. However, there are several concerns about EPOR functionality when expressed on cancer cells. In this paper we have provided evidence that EPOR expressed in cancer cells could be implicated in proliferation events because a transfection of EPOR siRNA to EPOR-expressing bladder cancer cells resulted in a marked reduction in cell growth. However, these cell lines do not grow in the presence of hrEPO. Furthermore, bladder cancer patients that expressed EPOR in tumor samples had a reduced survival in absence of rhEPO treatment. Therefore, EPOR is implicated in bladder cancer growth but this effect appears to be independent from rhEPO supplementation. Reports which suggest that rhEPO promotes cancer growth due to the expression of EPOR in cancer cells must be observed with caution since in the presence of functional EPOR rhEPO does not promote growth.