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.

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.

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.

Pulmonary diffusing capacity in chronic dialysis patients.

Patients with end-stage renal disease treated by hemodialysis with bioincompatible membranes are exposed during the dialysis period to acute effects on lung microcirculation, which may result in pulmonary fibrosis and diffusion defects in long-standing dialysis. To investigate the occurrence of these possible chronic pulmonary alterations, we determined lung function in patients with chronic renal failure not undergoing hemodialysis and in patients who had been receiving regular hemodialysis both for short and long periods of time. Forty-three patients divided into three groups were studied: 17 patients before dialysis with a mean (SD) creatinine clearance of 14.1 (6.8) ml/min 11.73 m2, 10 patients receiving regular hemodialysis for a period of less than 12 months (mean 6.4 +/- 3.5 months), and 16 patients receiving regular hemodialysis for more than 5 years (mean 8.3 +/- 3.6 years). First-use bioincompatible cellulosic dialysis membranes were used in all the cases. The following parameters were recorded: forced vital capacity (FVC), forced expiratory volume in 1s (FEV1), total lung capacity (TLC), residual volume (RV), carbon monoxide transfer factor (TLCO), accessible lung volume (VA), carbon monoxide transfer factor/accessible lung volume (KCO- that is, TLCO/VA), and arterial blood gases. Patients receiving regular hemodialysis for more than 5 years showed significantly lower values of TLCO and KCO than patients before dialysis and patients receiving regular hemodialysis for less than 12 months. Seventy-five percent of patients on long-term hemodialysis had markedly reduced TLCO or KCO values (below 80% of the reference value) as compared with 17% of patients before dialysis and 10% of patients dialyzed for less than 12 months (P < 0.001). Differences among groups for the remaining parameters were not observed. In conclusion, patients undergoing long-term regular hemodialysis with a bioincompatible membrane showed a selective reduction in pulmonary diffusing capacity possibly due to chronic pulmonary fibrosis.

Opposite effects of the Hsp90 inhibitor Geldanamycin: induction of apoptosis in PC12, and differentiation in N2A cells.

The inhibitor of the Hsp90 chaperone Geldanamycin has been reported to have several cellular effects, such as inhibition of v-src activity or destabilization of Raf-1 among others. We show now that Geldanamycin treatment induces different phenotypes in different cell lines. In PC12 cells, it triggers apoptosis, whereas in the murine neuroblastoma N2A, it induces differentiation with neurite outgrowth. Geldanamycin effects cannot be mimicked by inhibition of the c-src protein tyrosine kinases, and nerve growth factor does not protect PC12 cells from apoptosis. Mitogen-activated protein kinase activities ERK and JNK are activated differently according to cell type: in PC12 cells JNK is activated, and its inhibition abolishes apoptosis, but not ERK; in N2A cells, both ERK and JNK are activated, but with peak activities at different times.

Oleoylanilide, a possible causative agent of toxic oil syndrome, interferes with the cytoskeleton in a neuronal cell line.

We have examined the effects of oleoylanilide, one of the main candidates in the etiology of the toxic oil syndrome, in the neuroblastoma cell line N2A. Oleoylanilide treatment causes two kinds of phenomena: alteration of the actin cytoskeleton, creating a brush-like protrusion of actin at the periphery of the cells, and reduction of the adhesiveness of these cells to laminin and fibronectin, two of the main components of the extracellular matrix in the central nervous system. These effects could be correlated with symptoms shown in the acute and chronic phases of the disease.

Apoptosis induced by protein kinase C inhibition in a neuroblastoma cell line.

The protein kinase C inhibitor bisindolylmaleimide GF109203X has a dual effect on the behavior of the neuroblastoma cell line Neuro-2A; when the inhibitor is added in conditions that induce differentiation (absence of serum), neurite outgrowth is potentiated in a dose-dependent manner. However, if the inhibitor is added in growth-promoting conditions (presence of serum), programmed cell death (apoptosis) is induced, as assessed by internucleosomal DNA cleavage and specific immunoassays. This effect is also seen with other specific protein kinase C inhibitors. Bcl2 gene overexpression protects Neuro-2A cells from apoptosis, as has been found in other systems. We also show that calpain I, a neutral Ca(2+)-activated proteinase, participates in this apoptotic pathway. Our results point to a key role of protein kinase C in the regulation of growth and differentiation in Neuro-2A cells.

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.