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 À /nu À 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.

Cisplatin-based chemotherapy is the paradigm of non-smallcell 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 (Po0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (Po0.001). 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.

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.

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. ß