P53 pathway activation by telomerase attrtion inX-DC primary fibroblats occurs in the absence of ribosome biogenesis failure and as consequence of DNA damage

BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence (AU)

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p53 pathway activation by telomere attrition in X-DC primary fibroblasts occurs in the absence of ribosome biogenesis failure and as a consequence of DNA damage.

BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.

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 (AU)

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Checkpoint kinase 1 modulates sensitivity to cisplatin after spindle checkpoint activation in SW620 cells.

Aneuploidy is a common feature of tumours that arise by errors in chromosome segregation during mitosis. The aim of this study was to evaluate possible signaling pathways involved in sensitization to chemotherapy in cells with chromosomal instability. We designed a screen using the fission yeast Squizossaccharomyces pombe, to isolate strains showing a phenotype of chromosome mis-segregation and higher sensitivity to the antitumoral drug Bleomycin. We examined differences in gene expression using a comparative analysis of genome-wide expression of the wild type strain and one of the mutants. The results revealed a set of genes involved in cell cycle control, including Mad3/BubR1 and Chk1. We then studied the levels of these two proteins in colorectal cancer human cell lines with different genomic content. Among these, SW620 cells showed higher BubR1 and Chk1 mRNA levels than control cells under normal conditions. Since Chk1 is required for both S and G2/M checkpoints, and the microtubule-destabilizing agent, nocodazole induces mitotic arrest, we attempted to investigate the potential anticancer effects of nocodazole in combination with cisplatin. These studies showed that SW620 cells undergo synergistic cell death after spindle checkpoint activation followed by cisplatin treatment, suggesting a role of Chk1 in this checkpoint, very likely dependent on BubR1 protein. Importantly, Chk1-depleted SW620 cells lost this synergistic effect. In summary, we propose that Chk1 could be a biomarker predictive of the efficacy of chemotherapy across different types of tumors with aneuploidy. These findings may be potentially very useful for the stratification of patients for treatment.

IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer.

Cisplatin-based chemotherapy is the paradigm of non-small-cell lung cancer (NSCLC) treatment; however, it also induces de novo DNA-hypermethylation, a process that may be involved in the development of drug-resistant phenotypes by inactivating genes required for drug-cytotoxicity. By using an expression microarray analysis, we aimed to identify those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Results revealed specific silencing by promoter hypermethylation of IGFBP-3 in CDDP resistant cells, whereas IGFBP-3 siRNA interference, induced resistance to CDDP in sensitive cells (P<0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (P<0.001). Thus, stage I patients, whose tumors harbor an unmethylated promoter, had a trend towards increased disease-free survival (DFS). We report that a loss of IGFBP-3 expression, mediated by promoter-hypermethylation, results in a reduction of tumor cell sensitivity to cisplatin in NSCLC. Basal methylation status of IGFBP-3 before treatment may be a clinical biomarker and a predictor of the chemotherapy outcome, helping to identify patients who are most likely to benefit from CDDP therapy alone or in combination with epigenetic treatment.

hCCR4/cNOT6 targets DNA-damage response proteins.

Radio and chemotherapy are the election options besides surgical resection, in cancer treatment. However, resistance to chemotherapy limits the effectiveness of therapy in the clinic. An improved knowledge of the mechanisms underlying the resistance to treatment would generate new therapeutic strategies. Genetic suppressor elements (GSEs) are short, biologically active, cDNA fragments that interfere with the function of their cognate gene. By selection of genetic suppressor elements (GSEs) conferring resistance to cisplatin, we identified the GSE11, that corresponds to the hCCR4/CNOT6 gene that mediates cellular sensitivity to the drug. Expression of GSE11-hCCR4 reduces hCCR4 protein levels in cells. Targeting hCCR4 with GSE11 or with siRNA, decreases sensitivity of mammalian cells to DNA-damaging agents. Overexpression of hCCR4 targets Chk2 following exposure to cisplatin, without interfering with the upstream ATM/ATR pathway, however histone gammaH2AX is strongly phosphorylated in these cells compared to control cells. Our results uncover a new function for a human protein involved in chemotherapy response. This finding introduces a new pharmacological target in the treatment of solid tumours.

MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer.

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.

FK506 sensitizes mammalian cells to high osmolarity by modulating p38 MAP kinase activation.

The immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA) have increased the survival rates in organ transplantation. Both drugs inhibit the protein phosphatase calcineurin (CaN) in activated T cells, exhibiting similar side-effects. Diabetes is observed more often in FK506 than CsA therapy, probably due to inhibition of new molecular targets other than CaN. We studied FK506 toxicity in mammalian cells. FK506, but not CsA, regulated p38 activation by osmotic stress, and decreased viability in osmostressed cells. In addition, FK506 treatment strongly increased the phosphorylation of the eukaryotic initiation factor-2alpha (eIF-2alpha) subunit. eIF-2alpha phosphorylation, p38 inhibition and cell lethality were relieved by addition of excess amino acids to the medium, suggesting that amino acid availability mediated FK506 toxicity. Therefore, these FK506-dependent responses could be relevant to the non-therapeutic effects of FK506 therapy.